Subscriber access provided by The University of British Columbia Library
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 33
Environmental Science & Technology
1
Fluoxetine exhibits pharmacological effects and trait-based
2
sensitivity in a marine worm
3
Cameron M. Hird1, Mauricio A. Urbina1,2, Ceri N. Lewis1, Jason R. Snape3, Tamara
4
S. Galloway1
5
1
6
Exeter, Exeter, EX4 4QD, United Kingdom
7
2
8
de Concepción, P.O. Box 460-C, Concepción, Chile
9
3
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
10 11
Corresponding author email:
[email protected], phone: 0(+44)1392 263436
12
13
14
15
16
17
18
19
1 ACS Paragon Plus Environment
Environmental Science & Technology
20
Page 2 of 33
Abstract:
21
Global production of pharmacologically active compounds exceeds 100,000
22
tonnes annually, a proportion of which enters aquatic environments through patient
23
use, improper medicine disposal and production. These compounds are designed to
24
have mode-of-action (MoA) effects on specific biological pathways, with potential to
25
impact non-target species. Here, we used MoA and trait-based approaches to
26
quantify uptake and biological effects of fluoxetine, a selective serotonin reuptake
27
inhibitor, in filter and deposit feeding marine worms (Hediste diversicolor). Worms
28
exposed to 10 µg L-1, accumulated fluoxetine with a body burden over 270 times
29
greater than exposure concentrations, resulting in ~10 % increased coelomic fluid
30
serotonin, a pharmacological effect. Observed effects included weight loss (up to 2
31
% at 500 µg L-1), decreased feeding rate (68% at 500 µg L-1) and altered metabolism
32
(oxygen consumption, ammonia excretion and O:N from 10 µg L-1). Bioconcentration
33
of fluoxetine was dependent on route of uptake, with filter feeding worms
34
experiencing up to 130 times greater body burden ratios and increased magnitudes
35
of effects than deposit feeders, a trait-based sensitivity likely as a consequence of
36
fluoxetine partitioning to sediment. This study highlights how novel approaches such
37
as MoA and trait-based methods can supplement environmental risk assessments of
38
pharmaceuticals.
39
40
41
42
2 ACS Paragon Plus Environment
Page 3 of 33
43
44
Environmental Science & Technology
Introduction: Over 4000 pharmaceutical products are available worldwide for medicinal and
45
veterinary purposes,1 resulting in annual production exceeding 100,000 tonnes of
46
pharmacologically active compounds. A proportion of these, along with their
47
metabolites, are released to the aquatic environment, primarily through sewage
48
effluent as a consequence of patient use, where they may impact non-target
49
organisms.2 Pharmaceuticals are designed to have specific biological effects and are
50
unlikely to exhibit acute lethality or general adverse effects as focused on by
51
traditional environmental risk assessments (ERAs), emphasizing the importance of
52
the reconsideration of traditional ecological risk assessment paradigms for these
53
compounds.3 It is ecologically relevant to focus on mode-of-action (MoA) sub-lethal
54
effects on conserved biological pathways vital to maintenance and physiological
55
functioning4 in addition to apical endpoints such as survival and growth to improve
56
our understanding of pharmacological actions and effects within ecotoxicology.
57
Fluoxetine hydrochloride is a selective serotonin reuptake inhibitor (SSRI)
58
used to treat psychiatric disorders, predominantly depression. It was one of the most
59
highly prescribed SSRIs in England in 2014 (>6.2 million prescriptions).5 An
60
environmental quality standard (EQS) value of interest for fluoxetine in the UK has
61
been proposed by the water framework directive (WFD) as 0.01 µg L-1.6 Gardner et
62
al.7 identified that effluent from over 50 % of UK sewage-treatment plants (STPs)
63
exceeded this, with a median concentration of 0.023 µg L-1. In the US, effluent
64
discharges as high as 0.54 µg L-1 have been recorded,8 with stream concentrations
65
reaching 0.043 µg L-1.9 Dilution in estuarine environments will be lower than in ocean
66
surface waters, hence it may be more applicable to focus on effluent values for these
3 ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 33
67
habitats when estuarine data is unavailable. Fluoxetine has a sorption coefficient (log
68
Koc) value of 4.72 at pH 8,10 indicating it may preferentially partition to sediment and
69
dissolved organic matter (DOM). Despite this, measured environmental
70
concentrations in sediment are scarce, although fluoxetine has been detected in river
71
sediment at 0.019 µg g-1.9 This concentration is higher than those reported to elicit
72
biological effects in vertebrate and invertebrate marine species.11
73
Fluoxetine persists in the environment through continuous discharge from
74
STPs,2,7 displaying minimal biological degradation during sewage treatment, with
75
removal predominantly occurring through sorption to sewage sludge.10,12
76
Consequently, fluoxetine has potential to accumulate in sediments when release
77
rates exceed removal. Thus it is important to focus attention on estuarine species
78
within benthic habitats that may experience greater fluoxetine exposure. The
79
bioaccumulation potential of fluoxetine is illustrated by a pseudo-bioconcentration
80
factor (ratio between internal and exposure concentrations) of 1347 in the freshwater
81
mussel Elliptio complantana.13
82
In mammals, fluoxetine works by binding to the serotonin reuptake transporter
83
(SERT – SLC6A4), blocking serotonin (5-HT) reuptake at the synaptic cleft.9
84
Serotonin plays a role in processes vital to human neurology and physiology14
85
including gastrointestinal function, appetite satiation, mood and behaviour. Since the
86
SERT signalling pathway is highly conserved across all taxa,15 pharmaceuticals
87
targeting this pathway will likely have effects in non-target species, including
88
invertebrates.16 Read-across hypothesis17 predicts fluoxetine to have a similar MoA
89
in species where the SERT mechanism is conserved, although adverse outcomes
90
may vary. Orthologs to mammalian SERT SLC6A4 are ubiquitous in metazoa for
91
which sequence information is available.15 4 ACS Paragon Plus Environment
Page 5 of 33
92
Environmental Science & Technology
Several studies have evaluated effects of fluoxetine on fish and invertebrates,
93
showing among others; behavioural, reproductive and metabolic effects spanning
94
aqueous concentrations several orders of magnitude around environmental
95
concentrations.11 However, effect concentrations are variable with conflicting levels
96
of sensitivity and a lack of robust chemical or molecular evidence to establish well-
97
defined responses at given concentrations.11 Research has focussed primarily on
98
pelagic organisms whereas, based on the prediction that fluoxetine may partition to
99
sediments, it seems relevant to consider organisms with benthic associations, which
100 101
often play dominant roles in ecosystem processes. This study aimed to test the hypothesis that fluoxetine is bioavailable to
102
marine worms and elicits pharmacological and biological responses. A secondary
103
hypothesis was that fluoxetine would partition to sediment, altering its bioavailability
104
compared to aqueous exposure; consequently feeding mechanism would alter its
105
uptake and effects. These hypotheses were tested by exposing individuals of the
106
polychaete worm Hediste diversicolor (Harbour ragworm) to a range of fluoxetine
107
concentrations and measuring uptake, behavioural, physiological and
108
pharmacological effects. This study is the first to measure the effects of fluoxetine on
109
serotonin levels in a marine invertebrate. Ragworms are one of the most abundant
110
species in temperate estuarine sediments, playing dominant roles in bioturbation and
111
nutrient cycling. H. diversicolor can switch feeding mechanism from deposit to filter
112
feeding by producing mucous nets to capture organic material at burrow entrances.18
113
This characteristic provides a unique opportunity to test the impact of feeding on
114
uptake and effects of fluoxetine in a single species.
115
5 ACS Paragon Plus Environment
Environmental Science & Technology
116
Methods:
117
Animal Husbandry:
Page 6 of 33
118
Non-mature H. diversicolor (mean ± S.E.: wet weight: 147.83 ± 8.36 mg; body
119
length: 60.14 ± 2.75 mm) were collected from Exton (Devon, UK), a site un-impacted
120
by major contaminants,19 and transported to the University of Exeter. Worms were
121
transferred to 12 L holding tanks for acclimation (~7 days), containing sieved (< 1
122
mm) sediment (5 cm depth, natural Exe mud – characterised by Langston et al.19)
123
with overlying artificial seawater (ASW) (12 °C, 22 ‰, 12/12 h light / dark
124
photoperiod). Water changes (50 %) were made daily followed by feeding ad libitum
125
with crushed trout pellets.
126
127
Exposure Routes:
128
1. Aqueous
129
Worms were transferred to 400 mL aerated glass beakers (n = 8 per beaker)
130
containing 300 mL ASW (12 °C, 22 ‰) and five 6 cm long glass tubes (diameter 8
131
mm) as artificial burrows. Beakers were blacked-out and a lid added to simulate
132
burrow darkness. Conditions were maintained for a total pre-exposure period of 72 h,
133
first feed taking place at 48 h. Filter feeding was induced by addition of 200 µL
134
Isochrysis sp. algae paste (ZM Systems, ~4 billion cells mL-1) and was characterised
135
by production of mucous nets at burrow entrances for particulate capture.18
136
After the 72 h pre-exposure period, worms (n = 72 per treatment) were
137
randomly allocated to individual 100 mL aerated glass beakers containing 75 mL
138
fluoxetine-spiked ASW and an artificial burrow for 72 h exposure (conditions 6 ACS Paragon Plus Environment
Page 7 of 33
Environmental Science & Technology
139
maintained as previously outlined). Feeding by addition of 25 µL Isochrysis sp. algae
140
paste occurred at the beginning of exposure. Fluoxetine-spiked ASW was prepared
141
by addition of fluoxetine hydrochloride (European Pharmacopoeia, CAS:56296-78-7)
142
to ASW (12 °C, 22 ‰), accounting for 36 atomic mass units (amu) for the
143
hydrochloride salt, to concentrations of: 0 (control), 10, 100, 500 µg L-1 (no solvent
144
required). Measured dosing concentrations are presented in Table 1. Experimental
145
parameters monitored throughout included: dissolved oxygen, pH, temperature,
146
salinity (Mettler Toledo SevenGo Duo with InLab 738 and InLab Expert Pro) and
147
ammonia; all remaining within quality control limits (O2: 100 ± 2 %; pH: 8.1 ± 0.1;
148
temperature: 12 ± 0.5 °C; salinity: 22 ± 1.0 ‰, ammonia: 0.05) and homogeneity of variance (Levene’s: p > 0.05). In
281
all cases except for metabolism, treatments were compared within feeding types 12 ACS Paragon Plus Environment
Page 13 of 33
Environmental Science & Technology
282
using ANOVA, followed by Tukey’s pairwise comparisons. Ammonia excretion and
283
metabolic rates and O:N data were compared via general linear model (factors:
284
concentration, time and their interaction). Regression analysis was performed
285
between weight change and serotonin concentration for both feeding types.
286
Significance was assumed at p < 0.05.
287
288
Results:
289
Fluoxetine Uptake and Pharmacological Effects
290
No mortalities were recorded. Fluoxetine was detected in worm samples (post
291
72 h exposure) in all treatments for both feeding types (Table 1), while control
292
samples remained below the limit of detection (LOD, 0.01 µg g-1). The highest
293
fluoxetine uptake occurred in filter feeding worms (mean ± s.e.: 24.1 ± 3.62 µg g-1)
294
exposed to the highest concentration (500 µg L-1). Similarly, the highest uptake in
295
deposit feeders (4.03 ± 0.40 µg mL-1) occurred at the highest concentration (2.5 µg
296
g-1), an uptake ~6 times lower than in filter feeders, despite exposure concentrations
297
being 5 times higher. Nor-fluoxetine in deposit feeders was only observed at the
298
highest fluoxetine concentration (2.46 µg g-1 ± 0.28), although the LOD was high (0.1
299
µg g-1). Body burden: exposure concentration ratios at 72 h (BB72) were determined
300
(Table 1). The highest ratios occurred at the lowest fluoxetine concentration (BB72 =
301
273.5, 10 µg L-1) for filter and the highest fluoxetine concentration for deposit feeders
302
(BB72 = 2.3, 2.5 µg g-1).
303 304
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
13 ACS Paragon Plus Environment
Environmental Science & Technology
Page 14 of 33
305
(ANOVA: F3,60 = 9.14, p < 0.001) feeders (Fig. 1a). The lowest observed effect
306
concentration (LOEC) in filter feeders was 10 µg L-1 fluoxetine (serotonin: 325.61 ±
307
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
308
feeders. Standardisation of serotonin by lipid content made no significant difference
309
to observed results (data not shown).
310
311 312
Weight and Metabolism Fluoxetine had a significant effect on weight in filter (ANOVA: F3,284 = 19.91, p
313
< 0.001) and deposit (ANOVA: F3,284 = 5.64, p < 0.01) feeders (Fig. 1b), showing
314
linear decreases in % weight change with increasing fluoxetine. In filter feeders, the
315
LOEC (10 µg L-1) resulted in weight loss (-1.98 ± 1.58 %), compared to weight gain
316
in control worms (5.98 ± 1.38 %). No weight loss was observed in deposit feeders,
317
however, weight gain was reduced. At the LOEC (0.25 µg g-1), weight change was
318
2.45 ± 0.85 % compared to control conditions: 4.98 ± 1.07 %. Regression analysis
319
between serotonin and weight change showed serotonin concentration was a
320
significant predictor of weight change in both filter (R2adj = 0.654, F63 = 59.48, p
