Fluoxetine Exhibits Pharmacological Effects and Trait-Based

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Fluoxetine exhibits pharmacological effects and trait-based sensitivity in a marine worm Cameron McAuley Hird, Mauricio A Urbina, Ceri N. Lewis, Jason R. Snape, and Tamara S. Galloway Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03233 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 6, 2016

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Environmental Science & Technology

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Fluoxetine exhibits pharmacological effects and trait-based

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sensitivity in a marine worm

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Cameron M. Hird1, Mauricio A. Urbina1,2, Ceri N. Lewis1, Jason R. Snape3, Tamara

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S. Galloway1

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Exeter, Exeter, EX4 4QD, United Kingdom

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de Concepción, P.O. Box 460-C, Concepción, Chile

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Department of Biosciences, College of Life and Environmental Sciences, University of

Departamento de Zoología, Facultad de Ciencias Naturales y Oceanográficas, Universidad

AstraZeneca Global Environment, Alderley Park, Macclesfield, SK10 4TF, United Kingdom

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Corresponding author email: [email protected], phone: 0(+44)1392 263436

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Abstract:

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Global production of pharmacologically active compounds exceeds 100,000

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tonnes annually, a proportion of which enters aquatic environments through patient

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use, improper medicine disposal and production. These compounds are designed to

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have mode-of-action (MoA) effects on specific biological pathways, with potential to

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impact non-target species. Here, we used MoA and trait-based approaches to

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quantify uptake and biological effects of fluoxetine, a selective serotonin reuptake

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inhibitor, in filter and deposit feeding marine worms (Hediste diversicolor). Worms

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exposed to 10 µg L-1, accumulated fluoxetine with a body burden over 270 times

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greater than exposure concentrations, resulting in ~10 % increased coelomic fluid

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serotonin, a pharmacological effect. Observed effects included weight loss (up to 2

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% at 500 µg L-1), decreased feeding rate (68% at 500 µg L-1) and altered metabolism

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(oxygen consumption, ammonia excretion and O:N from 10 µg L-1). Bioconcentration

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of fluoxetine was dependent on route of uptake, with filter feeding worms

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experiencing up to 130 times greater body burden ratios and increased magnitudes

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of effects than deposit feeders, a trait-based sensitivity likely as a consequence of

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fluoxetine partitioning to sediment. This study highlights how novel approaches such

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as MoA and trait-based methods can supplement environmental risk assessments of

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pharmaceuticals.

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Introduction: Over 4000 pharmaceutical products are available worldwide for medicinal and

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veterinary purposes,1 resulting in annual production exceeding 100,000 tonnes of

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pharmacologically active compounds. A proportion of these, along with their

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metabolites, are released to the aquatic environment, primarily through sewage

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effluent as a consequence of patient use, where they may impact non-target

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organisms.2 Pharmaceuticals are designed to have specific biological effects and are

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unlikely to exhibit acute lethality or general adverse effects as focused on by

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traditional environmental risk assessments (ERAs), emphasizing the importance of

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the reconsideration of traditional ecological risk assessment paradigms for these

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compounds.3 It is ecologically relevant to focus on mode-of-action (MoA) sub-lethal

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effects on conserved biological pathways vital to maintenance and physiological

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functioning4 in addition to apical endpoints such as survival and growth to improve

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our understanding of pharmacological actions and effects within ecotoxicology.

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Fluoxetine hydrochloride is a selective serotonin reuptake inhibitor (SSRI)

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used to treat psychiatric disorders, predominantly depression. It was one of the most

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highly prescribed SSRIs in England in 2014 (>6.2 million prescriptions).5 An

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environmental quality standard (EQS) value of interest for fluoxetine in the UK has

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been proposed by the water framework directive (WFD) as 0.01 µg L-1.6 Gardner et

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al.7 identified that effluent from over 50 % of UK sewage-treatment plants (STPs)

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exceeded this, with a median concentration of 0.023 µg L-1. In the US, effluent

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discharges as high as 0.54 µg L-1 have been recorded,8 with stream concentrations

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reaching 0.043 µg L-1.9 Dilution in estuarine environments will be lower than in ocean

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surface waters, hence it may be more applicable to focus on effluent values for these



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habitats when estuarine data is unavailable. Fluoxetine has a sorption coefficient (log

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Koc) value of 4.72 at pH 8,10 indicating it may preferentially partition to sediment and

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dissolved organic matter (DOM). Despite this, measured environmental

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concentrations in sediment are scarce, although fluoxetine has been detected in river

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sediment at 0.019 µg g-1.9 This concentration is higher than those reported to elicit

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biological effects in vertebrate and invertebrate marine species.11

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Fluoxetine persists in the environment through continuous discharge from

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STPs,2,7 displaying minimal biological degradation during sewage treatment, with

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removal predominantly occurring through sorption to sewage sludge.10,12

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Consequently, fluoxetine has potential to accumulate in sediments when release

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rates exceed removal. Thus it is important to focus attention on estuarine species

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within benthic habitats that may experience greater fluoxetine exposure. The

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bioaccumulation potential of fluoxetine is illustrated by a pseudo-bioconcentration

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factor (ratio between internal and exposure concentrations) of 1347 in the freshwater

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mussel Elliptio complantana.13

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In mammals, fluoxetine works by binding to the serotonin reuptake transporter

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(SERT – SLC6A4), blocking serotonin (5-HT) reuptake at the synaptic cleft.9

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Serotonin plays a role in processes vital to human neurology and physiology14

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including gastrointestinal function, appetite satiation, mood and behaviour. Since the

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SERT signalling pathway is highly conserved across all taxa,15 pharmaceuticals

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targeting this pathway will likely have effects in non-target species, including

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invertebrates.16 Read-across hypothesis17 predicts fluoxetine to have a similar MoA

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in species where the SERT mechanism is conserved, although adverse outcomes

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may vary. Orthologs to mammalian SERT SLC6A4 are ubiquitous in metazoa for

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which sequence information is available.15 4 ACS Paragon Plus Environment

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Several studies have evaluated effects of fluoxetine on fish and invertebrates,

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showing among others; behavioural, reproductive and metabolic effects spanning

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aqueous concentrations several orders of magnitude around environmental

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concentrations.11 However, effect concentrations are variable with conflicting levels

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of sensitivity and a lack of robust chemical or molecular evidence to establish well-

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defined responses at given concentrations.11 Research has focussed primarily on

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pelagic organisms whereas, based on the prediction that fluoxetine may partition to

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sediments, it seems relevant to consider organisms with benthic associations, which

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often play dominant roles in ecosystem processes. This study aimed to test the hypothesis that fluoxetine is bioavailable to

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marine worms and elicits pharmacological and biological responses. A secondary

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hypothesis was that fluoxetine would partition to sediment, altering its bioavailability

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compared to aqueous exposure; consequently feeding mechanism would alter its

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uptake and effects. These hypotheses were tested by exposing individuals of the

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polychaete worm Hediste diversicolor (Harbour ragworm) to a range of fluoxetine

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concentrations and measuring uptake, behavioural, physiological and

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pharmacological effects. This study is the first to measure the effects of fluoxetine on

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serotonin levels in a marine invertebrate. Ragworms are one of the most abundant

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species in temperate estuarine sediments, playing dominant roles in bioturbation and

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nutrient cycling. H. diversicolor can switch feeding mechanism from deposit to filter

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feeding by producing mucous nets to capture organic material at burrow entrances.18

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This characteristic provides a unique opportunity to test the impact of feeding on

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uptake and effects of fluoxetine in a single species.

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Methods:

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Animal Husbandry:

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Non-mature H. diversicolor (mean ± S.E.: wet weight: 147.83 ± 8.36 mg; body

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length: 60.14 ± 2.75 mm) were collected from Exton (Devon, UK), a site un-impacted

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by major contaminants,19 and transported to the University of Exeter. Worms were

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transferred to 12 L holding tanks for acclimation (~7 days), containing sieved (< 1

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mm) sediment (5 cm depth, natural Exe mud – characterised by Langston et al.19)

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with overlying artificial seawater (ASW) (12 °C, 22 ‰, 12/12 h light / dark

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photoperiod). Water changes (50 %) were made daily followed by feeding ad libitum

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with crushed trout pellets.

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Exposure Routes:

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1. Aqueous

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Worms were transferred to 400 mL aerated glass beakers (n = 8 per beaker)

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containing 300 mL ASW (12 °C, 22 ‰) and five 6 cm long glass tubes (diameter 8

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mm) as artificial burrows. Beakers were blacked-out and a lid added to simulate

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burrow darkness. Conditions were maintained for a total pre-exposure period of 72 h,

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first feed taking place at 48 h. Filter feeding was induced by addition of 200 µL

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Isochrysis sp. algae paste (ZM Systems, ~4 billion cells mL-1) and was characterised

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by production of mucous nets at burrow entrances for particulate capture.18

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After the 72 h pre-exposure period, worms (n = 72 per treatment) were

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randomly allocated to individual 100 mL aerated glass beakers containing 75 mL

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fluoxetine-spiked ASW and an artificial burrow for 72 h exposure (conditions 6 ACS Paragon Plus Environment

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maintained as previously outlined). Feeding by addition of 25 µL Isochrysis sp. algae

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paste occurred at the beginning of exposure. Fluoxetine-spiked ASW was prepared

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by addition of fluoxetine hydrochloride (European Pharmacopoeia, CAS:56296-78-7)

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to ASW (12 °C, 22 ‰), accounting for 36 atomic mass units (amu) for the

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hydrochloride salt, to concentrations of: 0 (control), 10, 100, 500 µg L-1 (no solvent

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required). Measured dosing concentrations are presented in Table 1. Experimental

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parameters monitored throughout included: dissolved oxygen, pH, temperature,

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salinity (Mettler Toledo SevenGo Duo with InLab 738 and InLab Expert Pro) and

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ammonia; all remaining within quality control limits (O2: 100 ± 2 %; pH: 8.1 ± 0.1;

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temperature: 12 ± 0.5 °C; salinity: 22 ± 1.0 ‰, ammonia: 0.05) and homogeneity of variance (Levene’s: p > 0.05). In

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all cases except for metabolism, treatments were compared within feeding types 12 ACS Paragon Plus Environment

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using ANOVA, followed by Tukey’s pairwise comparisons. Ammonia excretion and

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metabolic rates and O:N data were compared via general linear model (factors:

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concentration, time and their interaction). Regression analysis was performed

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between weight change and serotonin concentration for both feeding types.

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Significance was assumed at p < 0.05.

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Results:

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Fluoxetine Uptake and Pharmacological Effects

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No mortalities were recorded. Fluoxetine was detected in worm samples (post

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72 h exposure) in all treatments for both feeding types (Table 1), while control

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samples remained below the limit of detection (LOD, 0.01 µg g-1). The highest

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fluoxetine uptake occurred in filter feeding worms (mean ± s.e.: 24.1 ± 3.62 µg g-1)

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exposed to the highest concentration (500 µg L-1). Similarly, the highest uptake in

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deposit feeders (4.03 ± 0.40 µg mL-1) occurred at the highest concentration (2.5 µg

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g-1), an uptake ~6 times lower than in filter feeders, despite exposure concentrations

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being 5 times higher. Nor-fluoxetine in deposit feeders was only observed at the

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highest fluoxetine concentration (2.46 µg g-1 ± 0.28), although the LOD was high (0.1

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µg g-1). Body burden: exposure concentration ratios at 72 h (BB72) were determined

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(Table 1). The highest ratios occurred at the lowest fluoxetine concentration (BB72 =

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273.5, 10 µg L-1) for filter and the highest fluoxetine concentration for deposit feeders

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(BB72 = 2.3, 2.5 µg g-1).

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There were dose-dependent linear increases in serotonin with increasing fluoxetine concentration in both filter (ANOVA: F3,60 = 12.98, p < 0.001) and deposit



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(ANOVA: F3,60 = 9.14, p < 0.001) feeders (Fig. 1a). The lowest observed effect

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concentration (LOEC) in filter feeders was 10 µg L-1 fluoxetine (serotonin: 325.61 ±

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10.19 ng mL-1 g-1), compared to 0.25 µg g-1 (304.81 ± 6.58 ng mL-1 g-1) in deposit

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feeders. Standardisation of serotonin by lipid content made no significant difference

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to observed results (data not shown).

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Weight and Metabolism Fluoxetine had a significant effect on weight in filter (ANOVA: F3,284 = 19.91, p

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< 0.001) and deposit (ANOVA: F3,284 = 5.64, p < 0.01) feeders (Fig. 1b), showing

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linear decreases in % weight change with increasing fluoxetine. In filter feeders, the

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LOEC (10 µg L-1) resulted in weight loss (-1.98 ± 1.58 %), compared to weight gain

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in control worms (5.98 ± 1.38 %). No weight loss was observed in deposit feeders,

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however, weight gain was reduced. At the LOEC (0.25 µg g-1), weight change was

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2.45 ± 0.85 % compared to control conditions: 4.98 ± 1.07 %. Regression analysis

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between serotonin and weight change showed serotonin concentration was a

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significant predictor of weight change in both filter (R2adj = 0.654, F63 = 59.48, p

Fluoxetine Exhibits Pharmacological Effects and Trait-Based

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