Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: Bioaccumulation, physiological and histological changes

Journal Pre-proof Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: Bioaccumulation, physiological and histological changes

Karolina Nowakowska, Joanna Giebułtowicz, Maciej Kamaszewski, Antoni Adamski, Hubert Szudrowicz, Teresa Ostaszewska, Urszula Solarska-Dzięciołowska, Grzegorz NałęczJawecki, Piotr Wroczyński, Agata Drobniewska PII:

S1532-0456(19)30386-2

DOI:

https://doi.org/10.1016/j.cbpc.2019.108670

Reference:

CBC 108670

To appear in:

Comparative Biochemistry and Physiology, Part C

Received date:

6 August 2019

Revised date:

9 October 2019

Accepted date:

12 November 2019

Please cite this article as: K. Nowakowska, J. Giebułtowicz, M. Kamaszewski, et al., Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: Bioaccumulation, physiological and histological changes, Comparative Biochemistry and Physiology, Part C(2019), https://doi.org/10.1016/j.cbpc.2019.108670

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© 2019 Published by Elsevier.

Journal Pre-proof Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: bioaccumulation, physiological and histological changes

Karolina Nowakowskaa, b, Joanna Giebułtowicza*, Maciej Kamaszewskic, Antoni Adamskicd, Hubert Szudrowiczc, Teresa Ostaszewskac, Urszula Solarska-Dzięciołowskaa, Grzegorz

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Nałęcz-Jaweckib, Piotr Wroczyńskia, Agata Drobniewskab a

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– Department of Bioanalysis and Drugs Analysis, Faculty of Pharmacy, Medical University

– Department of Environmental Health Sciences, Faculty of Pharmacy, Medical University

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b

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of Warsaw, 1 Banacha Street, Warsaw, PL-02097

c

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of Warsaw, 1 Banacha Street, Warsaw, PL-02097

–Department of Ichthyology and Biotechnology in Aquaculture, Warsaw University of Life

– Institute of Biochemistry and Biophisics, Polish Academy of Science, 5a Pawinskiego

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d

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Sciences-SGGW, 8 Ciszewskiego Street, Warsaw, PL-02-786

Street, Warsaw, PL-02106

*Corresponding author E-mail: [email protected]

ABSTRACT Antidepressants have been detected in surface waters worldwide at ng–µg/L concentration. These compounds can exert adverse effects on fish even at low levels. But, all previous 1

Journal Pre-proof analyses have concentrated on adult fish. The aim of the study was to assess the effect of environmental concentrations of sertraline, paroxetine, fluoxetine and mianserin, and their mixtures on such unusual endpoints as physiological and histological changes of zebrafish (Danio rerio) larvae. We also determined the bioconcentration of the pharmaceuticals. Fish Embryo Toxicity test was used to analyse the influence on developmental progression. Histological sections were stained with hematoxylin and eosin. Proliferating cells in liver

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were determined immunohistochemically by detection of Proliferating Cell Nuclear Antigens. The bioconcentration factor was measured by liquid chromatography coupled to mass

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and at medium concentration as single compound.

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spectrometry. Pharmaceuticals were used at low, medium and high concentrations in mixtures

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Exposure to the analyzed pharmaceuticals increased the rate of abnormal embryo and larvae

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development, accelerated the hatching time and affected the total hatching rate. Three-times lower proliferation of hepatocytes was observed in larvae exposed to paroxetine, mianserin,

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sertraline and the mixture of the pharmaceuticals at the highest concentrations. The highest

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bioaccumulation factor (BCF) was obtained for sertraline. The BCF of the analyzed compounds was higher if the organisms were exposed to the mixtures than to single pharmaceuticals. To conclude, the exposure of zebrafish larvae to selected antidepressants and their mixtures may cause disturbances in the organogenesis of fish even at environmental concentrations.

KEYWORDS Antidepressants; Bioaccumulation; Fish Embryo acute Toxicity assay; Proliferating Cell Nuclear Antigen; SSRI;

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Journal Pre-proof 1

INTRODUCTION

Depression is a leading cause of disability worldwide, and is a major contributor to the overall global burden of disease. According to Global Health Estimates in 2015, 4.4% of global population were estimated to suffer from depression (WHO). Thus, antidepressants are among the eight most important drug classes used in medicine. After administration, antidepressants are excreted in urine and feces. Fluoxetine (FLX) and paroxetine (PAR) are eliminated mainly

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in urine (80% and 64% of the oral does), whereas sertraline (SER) in equal amount in urine and feces (44%). The pharmaceuticals frequently undergo liver metabolism thus are excreted

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as parent compound or metabolites in different ratio. For instance PAR is eliminated as parent

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drug with less than 2% in urine and less than 1% in feces, whereas SER in less than 0.2% in

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urine and 12-14% in feces (van Harten, 1993; Wishart et al., 2018). Human excretion is

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considered to be the most important source of the pharmaceuticals in wastewater treatment plants (WWTPs). Due to their chemical properties, antidepressants are incompletely removed

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during the sewage treatment processes and contaminate surface waters. Recently, several studies have shown the occurrence of antidepressants in the aquatic environment at ng–µg L−1

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concentrations. The highest concentrations was noted for FLX and PAR, up to 1.4 and 1.1 µg L−1, respectively (Martínez Bueno et al., 2007; Weinberger and Klaper, 2014). In untreated sewage, which are sometimes directly discharged to surface water the concentrations may reach even 39.73 µg L−1 for PAR and 3.47 µg L−1 for FLX (Salgado et al., 2011). Although pharmaceuticals may be considered to be the best known chemicals (in terms of their pharmacodynamics, kinetics and human toxicity), our understanding of the environmental fate of these substances, especially their bioaccumulation in aquatic organisms and the chronic toxicity of low levels, is scarce (van der Ven et al., 2006). Bioaccumulation is defined as uptake of a xenobiotic into an organism and is presented as the ratio of the 3

Journal Pre-proof xenobiotic concentration in the organism to the concentration in the environment (Puckowski et al., 2016). The bioaccumulation of pharmaceuticals has been reported both in laboratory studies (Chen et al., 2017; Grabicova et al., 2014; Nakamura et al., 2008) and in the field (Grabicova et al., 2017; Ramirez et al., 2009). However, only Steinbach et al. (2013) showed data on the bioaccumulation of a pharmaceutical (verapamil) on the early life stages of aquatic organisms (i.e. common carp (Cyprinus carpio). To the best of our knowledge, no research has been published concerning the bioaccumulation of SSRIs in the early stages of

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the fish life-cycle. Especially when not a single compounds are used, but their mixtures.

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Apart from the large amount of ecotoxicological data on antidepressants (Brooks,

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2014; Chen et al., 2017; Overmyer et al., 2010) little is known about how acute exposure to

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high environmental concentrations affects aquatic organisms, especially the most sensitive

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forms such as larvae. The Fish Embryo Toxicity (FET) test with D. rerio is a tool widely used to assess the toxicity of environmental contaminants (Sun et al., 2014). In the FET test, also

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other effects can also be observed, for example influence on developmental progression: morphological malformations, delayed development and pericardial edema. These endpoints

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haven’t been studied so far regarding antidepressants. Our study are in line with more and more popular scopes of environmental studies, where other endpoints than death or reproduction changes are used. Other examples of unusual endpoints are metabolic rate, ionoregulation, oxidative stress and metabolome changes as well as studied in our manuscript histochemical changes in organs and Proliferating Cell Nuclear Antigen (PCNA) level (McRae et al., 2018; Mishra et al., 2019). PCNA is a cofactor and sliding clamp for DNA polymerase, which indicate homeostasis state of hepatocytes (Georgescu et al., 2008). Implications of all mentioned unusual endpoints are not fully understood, but thanks to them we have more and more evidence that environmental concentration of some pharmaceuticals affects homeostasis of aquatic organisms. 4

Journal Pre-proof Since in the environment the organisms are exposed to several compounds, in our study we concentrate not only on single compounds, but mainly on the mixture of FLX, SER, PAR and mianserin (MIAN). The first aim of this study was to determine the bioaccumulation factor (BCF) of selected antidepressants in zebrafish larvae after 96 hpf (hours after fertilization). The BCF was calculated (for low, medium and high level of mixtures and medium level of single compounds) based on the accumulation of drugs in larval tissues using liquid chromatography coupled to mass spectrometry (LC-MS). The second aim was to

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determine the effects of these solutions on such unusual endpoints as physiological and

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histological responses of the larvae of D. rerio. Histological examination was performed

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using a light microscope on hematoxylin and eosin staining. The proliferation index was

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assessed after PCNA antibody application on paraffin sections of the larvae. As the control unexposed larvae were used. The concentrations used were selected as close to

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Klaper, 2014).

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environmental, but with remarkable impact on biota (Ford et al., 2018; Weinberger and

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Journal Pre-proof 2 2.1

MATERIALS AND METHODS Chemicals and reagents

FLX standard was purchased from Sigma–Aldrich (Poznan, Poland), while MIAN, PAR and SER were a gift from the Drug Research Institute in Warsaw, Poland. All the pharmaceutical standards were of high purity grade (>90%). Nortriptyline and doxepin (internal standards, ISs) were supplied by Sigma–Aldrich (Poznan, Poland). The individual standard stock

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solutions as well as internal standard solution were prepared on a weight basis in methanol at

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a concentration of 1 mg/mL and stored at −20°C. Working solutions were prepared ex

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tempore by dilution of the stock solutions with water. The IS working solution (500 ng/mL) was prepared ex tempore by dilution of the stock solutions with acetonitrile. The solvents,

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HPLC gradient grade methanol, acetonitrile (LiChrosolv) and formic acid 98% were provided

2.2

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system (Milli-Q water).

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by Merck (Darmstadt). Ultrapure water was obtained from a Millipore water purification

The Fish Embryo Toxicity Test

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The experiment was performed in the Department of Ichthyology and Biotechnology in Aquaculture, Warsaw University of Life Sciences. Fertilized eggs were obtained from the Zebrafish Core Facility in the International Institute of Molecular and Cell Biology in Warsaw. Synthetic medium hard water used in the test was prepared according to ISO 73463(ISO_7346-3:1996, 1996). The average water pH during the experiment was 7.1±0.1. To prevent hypoxia or under oxidation, water was left undisturbed for 24 h after sterilization before each test.

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Journal Pre-proof FET (Fish EmbryoToxicity) test was conducted according to OECD guideline 236 (OECD_236, 2013). The method was modified by using 6-well plates. Newly fertilized zebrafish AB/TL strains eggs were transferred to water with xenobiotics (10 eggs per well). SER, PAR, FLX, MIAN were used at medium concentration (nominal 10 µg/L) and their mixtures at low concentration (nominal 5 µg/L, MIX5), medium (nominal 10 µg/L, MIX10) and high (nominal 25 µg/L, MIX25). To minimize the uncertainty of measurement the concentrations were chosen as the lowest where the analyte enrichment was not needed. The

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measured concentration was 69%, 52%, 64% and 43% lower than the nominal (mainly due to

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plastic binding properties) for SER, PAR, FLX, MIAN respectively and was stable (decrease

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lower than 15%) within 48 h.

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We made three independent experiments. All experiments were performed in five

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replicates. Two replicates (n = 20) were used for bioaccumulation studies and another two (n = 20) for immunohistochemistry and histological tests. The plates were incubated at 27±0.5°C

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for 96 h. Every 24 h (i.e. 24 hpf, 48 hpf, 72 hpf, and 96 hpf), the number of living unhatched embryos, hatched larvae, and dead and deformed individuals (larvae) in wells were counted

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using a binocular (Nikon SMZ 2000, Tokio, Japan) with a camera (PixelLink PL-A662). All dead embryos were removed to prevent contamination of wells. Histopatological and immunohictochemical examination as well as determination of bioconcentration factors were made after 96 hpf. 2.3

LC-MS/MS analysis

Zebrafish larvae were collected after 96 hpf to determine BCF. Living larvae were combined from 2 wells (n=20) and mixed with the ISs (50 L), water (50 L) and acetonitrile (100 L), and homogenized using a glass homogenizer. Samples were cooled in a freezer (at −20°C) for 10 min and centrifuged (5 min at 10,000 × g). 7

Journal Pre-proof The supernatant (150 L) was mixed with 375 L of water and transferred to an autosampler vial. The water samples were centrifuged (10 min at 10,000 × g), mixed with the ISs (9:1, v/v) and transferred to vials. No clean up procedure was applied. Instrumental analyses were performed using Agilent 1260 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA) connected in series to a QTRAP®4000 (AB SCIEX, Framingham, Massachusetts, USA) equipped with a Turbo Ion Spray source that was

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operated in positive mode. The curtain gas, ion source gas 1, ion source gas 2 and collision gas (all high purity nitrogen) were set at 35 psi, 60 psi, 40 psi and “medium” instrument units,

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respectively, and the ion spray voltage and source temperature were set at 5,000 V and 600°C,

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respectively. Chromatographic separation was achieved with a Kinetex RP-18 column (100

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mm × 4.6 mm, particle size 2.6 µm) supplied by Phenomenex (Torrance, CA, USA). The

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column was maintained at 40°C at a flow rate of 0.5 mL/min. The mobile phases consisted of HPLC grade water with 0.2% formic acid as eluent A and acetonitrile with 0.2% formic acid

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as eluent B. The gradient (%B) was as follows: 0 min, 10%; 1 min, 10%; 8 min, 90%, 9 min, 90%. The injection volume was 10 µL. The target compounds were analyzed in Multiple

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Reaction Monitoring mode (Table 1). The validation was performed according to the European Medicines Agency guidelines (EMEA/CHMP, 2006). Such parameters as linearity, precision and accuracy were assessed. 2.4

Histology and immunohistochemistry

To analyze the teratogenic effects to developing embryos, freshly hatched larvae (n = 60) were collected after 96 hpf and anesthetized with MS-222 (tricaine methanesulfonate, 3amino benzoic acid ethyl ester, Sigma-Aldrich, Poznan, Poland) and fixed in 4% buffered formalin. The samples were embedded in paraffin and cross-sectioned to 5 µm thickness using a Leica RM2025 microtome (Leica Microsystems, Germany). The obtained histological 8

Journal Pre-proof sections were stained using the standard procedure with hematoxylin-eosin (H/E) staining. To visualize the proliferating cells in liver, immunohistochemical detection of PCNA was used. The obtained histological slides were deparaffinized in xylene and rehydrated in a gradient of ethanol. Endogenous peroxidase was blocked using 3% hydrogen peroxide. The histological slides were rinsed in Tris buffer (pH 8.0, Sigma-Aldrich, Poznan, Poland) and incubated with a primary antibody (Monoclonal mouse primary antibody anti-PCNA, clone PC10; cat no. DAKO M0879; DAKO). Slides were incubated for 1 h at room temperature. Visualization

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was performed using a DAKO EnVision + System – HRP (DAKO), according to the

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manufacturer’s instructions. The cell nuclei were counterstained using Harris hematoxylin.

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Then, slides were dehydrated, rinsed in xylene and mounted in DPX Mountant for histology

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(Sigma-Aldrich, Poznan, Poland). For the negative control test, slides not incubated with antibodies were used. Microscopic observations were performed using a Nikon Eclipse 90i

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microscope and the pictures were taken using a Nikon Digital Sight DS–U1 camera (Nikon

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Corporation, Tokyo, Japan). In order to assess the effect of the xenobiotics on liver function, the number of proliferating nuclei in the liver parenchyma was counted in 10 sections from

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each larvae and expressed as per 1,000 μm2 surface of the organ. Histomorphometric measurements and pictures were analyzed using NIS – Elements AR 2.10 software (Nikon Corporation, Tokyo, Japan). 2.5

Calculations and Statistical Analysis

Statistical comparisons were performed using a one-way analysis of variance (ANOVA) followed by an NIR post hoc test (p = 0.05) using Statistica 12.0 software (StatSoft Inc.). The results were presented as means and standard deviations. The degree to which bioconcentration occurs is expressed as BCF. The BCF was calculated as the ratio of the

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Journal Pre-proof chemical concentration in the organism (CB [mg/kg]) and the chemical concentration in water (CWB [mg/L]) according to the equation: BCF = CB/CWB

3 3.1

[L/kg]

RESULTS Survival rate and morphology

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The study was carried on 50 larvae in three independent experiments (in total 150 larvae per

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modification). Exposure to MIX25 decreased the survival of embryos and larvae after 96 hpf (85.8% vs. 94.2%) (p < 0.05) (Fig.1). Two of the analyzed pharmaceuticals, PAR and FLX,

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significantly (p < 0.05) accelerated the hatching time (based on the results after 72 hpf and 96

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hpf), and after 72 hpf 86.7% and 53.3% larvae were hatched, respectively. However, after 96

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hpf, the total hatching rate was lower for FLX, SER and MIAN compared to the control (p <

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0.05). No differences were observed for PAR and MIXs. In the merged group of MIX10 and MIX25 a percentage of abnormal hatching larvae

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development twice as high as that in other samples was found (p < 0.05). The most frequent teratogenic effects observed in all analyzed groups was scoliosis (Fig. 2). But, in the larvae exposed to FLX and MIAN, most severe abnormalities were observed including malformations in trunk or tail regions (these abnormalities were observed only in M10 and F10 groups) as well as pericardial edema (several cases). The pericardial edema was also observed in larvae exposed to SER and MIX25 (rarely). Only one pericardial edema was observed in the control larvae. 3.2

Histopathology and Immunohistochemistry

The study was carried on 20 larvae in three independent experiments (in total 60 larvae per modification). PCNA tests revealed that a significantly (p < 0.05) lower proliferation (310

Journal Pre-proof times) of hepatocytes occurred in larvae exposed to PAR, MIAN, SER and MIX25 (Fig 3). No effect on PCNA was detected in FLX or in the mixture of pharmaceuticals at low and medium concentrations. The results may suggest some protective effect of FLX on the suppression of proliferation caused by PAR, SER and MIAN. 3.3

Bioconcentration factors

No antidepressants were detected in any of the non-exposed control embryos. Since no

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feeding was performed during the experiment, antidepressants found in organisms were taken

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up directly from the water. Based on the concentration of target compounds in water and in tissues, bioconcentration factors were calculated for all embryo groups. The BCF of the

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analyzed compounds was higher when the tested organisms were exposed to mixtures than to

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single pharmaceuticals (Fig 4). For instance, BCF in MIX5 (nominal concentration 5 µg/L)

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calculated for each compound was similar to BCF calculated for MIAN, FLX, PAR and SER (nominal concentration 10 µg/L) separately. The embryos exposed to a mixture of

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antidepressants at a concentration of 10 µg/L (nominal) showed approximately 50% higher

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BCF than those exposed to single compounds at the same concentration. A similar pattern was observed for all SSRIs and MIAN. Comparing the ability of the analyzed antidepressants to bioconcentrate, the BCF for FLX (94–170 L/kg), PAR (190–290 L/kg) and MIAN (340–470 L/kg) is significantly lower than that for SER (1,130–2,280 L/kg).

4

DISCUSSION

The implications of low-concentration exposure of aquatic fauna to environmental discharge of pharmaceuticals are largely unknown. However, some adverse effects have already been described. Henry and Black (2008) found that long-term exposure to antidepressants could adversely affect the reproductive potential and population dynamics of various fish species. FLX impaired sexual maturation of western mosquitofish (Gambusia affinis), which may 11

Journal Pre-proof reduce the fertility of fish (Henry and Black, 2008). FLX may also reduce the number of zebrafish eggs spawned (Lister et al., 2009). To date, no detailed studies have been conducted on the influence of antidepressants and their mixtures on early stages of fish development. This kind of exposure can have far-reaching significance in terms of the explanation of many developmental abnormalities in adults. Some studies on rat embryos indicate that SSRIs are particularly dangerous to the early stages of mammalian development, for example bradycardia or heart block (Ababneh et al., 2012). Moreover, until now, exposure studies of

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antidepressants on fish have mainly focused on changes in their behavior. The drugs affect

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behavior in humans, so effects on behavior are likely to be the primary effect of the drug on

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fish (Sumpter et al., 2014).

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This study was the first of its kind to examine the impact of antidepressants at environmentally relevant concentration on embryogenesis using zebrafish as a model system.

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We have shown that 96-h zebrafish exposure to mixture of antidepressants affect the survival

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rate of embryos. We also observed differences in proliferation of liver cells and morphological changes among zebrafish embryos. Morphological malformations

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4.1

Analysis of hatched zebrafish embryos exposed to aqueous solutions of FLX and MIAN have shown abnormalities in trunk or tail regions (including spinal curvatures) and pericardial edema. The latter abnormality has also been observed in fish exposed to SER and MIX25. Many authors indicate that the presence of xenobiotics, including drugs, in the aquatic environment may cause a teratogenic effect such as adverse effects on the spontaneous tail coilings in embryos, cognitive impairment, and motor disturbances (Scholz, 2013; Selderslaghs et al., 2013). These deformations may be caused by disturbance to nervous system development. According to Selderslaghs et al. (2013), changes in the functionality of

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Journal Pre-proof components of the neural circuitry of the zebrafish early locomotor network (spinal cord and hindbrain) are observed most frequently. 4.2

Histopathological analysis and PCNA

PCNA in current study was determined in liver, since the liver is the organ where pharmaceuticals frequently occur in the highest concentrations. In the studies described by Brooks et al. (2005), the highest concentrations of antidepressants in adult fish exposed to

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their mixture were in the liver and the lowest in muscles. So, morphological and functional changes might occur in the liver earlier than in other organs.

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Homeostasis in the liver is maintained due to the balance between the state of proliferation,

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and apoptosis of hepatocytes. PCNA tests revealed that a significantly lower proliferation

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occurred in larvae exposed to PAR, MIAN, SER and MIX25. One of the possible reason of

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the phenomenon is disturbance of the homeostasis as a toxic effect of environmental concentration of antidepressants. PCNA participates in replication and repair of DNA. Thus,

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decrease in hepatocyte proliferation rate may indicate liver damage and reduced repair potential. Decrease of PCNA observed also Cheung et al. (2012) on liver of Japanese medaka

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embryos, where liver injury was caused by hypoxia. One of the mechanism of liver injury caused by antidepressants can be enhanced mitochondrial oxidative stress in the liver (Ruddell et al., 2008), which prompt organ damage and consequently a decrease in the proliferative index of the examined fish. Thus, high concentrations of PAR, MIAN, SER and their mixtures can cause liver damage and lower the rate of hepatocyte renewal, as was shown previously for fluoxetine in a rat model (Zlatković et al., 2014). The second possible explanation of lower PCNA in larvae exposed to PAR, MIAN, SER and MIX25 is differences in the maturation stage. PCNA is expressed in all cells during early development and is down-regulated during morphogenesis and differentiation (Naryzhny and Lee, 2007). Kamaszewski et al. (2014) found the highest, statistically significant proliferative 13

Journal Pre-proof index of hepatocytes in newly hatched Russian sturgeon larvae (Acipenser gueldenstaedtii). During the first 7 days, a statistically significant decrease in proliferative activity was observed in comparison with the day of hatching, and this was maintained until the beginning of exogenous feeding and juvenile fish stage. Thus, reduction of the proliferative activity of fish hepatocytes in some experimental groups may be the result of differences in the maturation stage of the examined larvae, i.e. in development processes occurring during ontogenesis. As an example, some larvae with lower PCNA were more advanced in

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development processes as a result of earlier breeding, in which a zebrafish is stretched in time.

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The third explanation of lower PCNA of some larvae is disturbance of embryogenesis. For

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instance, in zebrafish embryos exposed to MIAN and SER, lower proliferative activity of

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hepatocytes as well as longer hatching time were observed. However, this phenomenon

4.3

Bioconcentration Factor

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requires further analysis.

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In the studies described by Ramirez et al. (2009), SER, FLX and norfluoxetine were the drugs found at the highest concentrations in fish exposed to drug mixtures, including antidepressants

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found in surface waters and wastewater. These drugs accumulated in the highest concentrations both in fish fillets (muscles) (max concentration of SER 11 ng/g) and in their livers (max concentration of SER and FLX 545 and 80 ng/g) (Ramirez et al., 2009). Brooks et al. (2005) detected FLX and SER in fish tissues of three different species collected from an effluent-dominant stream. The concentration in fish brains and livers was 10-fold higher than in muscles. This supports data on the higher bioconcentration of lipophilic compounds in tissues containing higher lipid content. However, for ionogenic compounds for example antidepressants, bioconcentration behavior is not well understood (Chen et al., 2017) and lipid-, phospholipid- and protein-water partitioning should be considered. Zebrafish eggs have a low lipid content, 4.8 – 7.3% (Hachicho et al., 2015); however, the lipid content percentage 14

Journal Pre-proof in low temperature fish is much higher (29% in case of whitefish, (Mueller et al., 2017)). Our preliminary analysis on the distribution of pharmaceuticals in the common bleak (Alburnus alburnus) collected in the river Vistula indicated high concentrations of the compounds in fish ovaries (data not published). Brooks et al. (2005) found, at much higher levels, more lipophilic and more active metabolites: desmethylfluoxetine and desmethylsertraline. In the present study, those derivatives have not been analyzed as no metabolism was assumed in fish larvae. Brain tissue analysis performed by Schultz et al. (2011) showed different patterns of

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antidepressant uptake. In this case again, drugs such as SER and FLX were present at the

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highest concentrations. In addition, the bioconcentration of SER was increased in the brains of

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fish exposed to the mixture of drugs, unlike for exposure only to SER. This supports our

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findings of higher uptake of drugs by fish larvae incubated in the mixtures. The uptake of chemicals into eggs is not easy, as a first barrier, the chorion consists

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mainly of glycoproteins (Hachicho et al., 2015). The effects of the tested drugs on the larvae

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development and time of hatching observed in our experiments indicate that the drugs could penetrate through the chorion.

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Most of the published research concerns bioconcentration of SER and FLX, where BCF values range from 65 to 6,200 L/kg and 42–906 L/kg, respectively (Chen et al., 2017; Grabicova et al., 2017; Grabicova et al., 2014; Koba et al., 2018; Lajeunesse et al., 2011). PAR has rarely been detected in fish tissues. According to Lajeunesse et al. (2011), its concentration in brown trout liver and brain was close to that for SER and FLX, i.e. 365 and 198 L/kg, respectively. To the best of knowledge, the bioconcentration of MIAN has not yet been studied. Chen et al. (2017) found low BCF for FLX and SER in adult zebrafish liver: 42 and 65 L/kg, respectively. Two-fold higher BCF values measured for SER in ovaries indicated the higher bioconcentration potential of fish ovaries. For SER, Chen et al. (2017) also found a very high depuration rate, suggesting fast elimination of this drug. The 15

Journal Pre-proof bioconcentration factors calculated for SER in fish exposed to municipal effluents is 85 and 180 L/kg in liver and brain, respectively (Grabicova et al., 2014). Similar BCF values were reported by Lajeunesse et al. (2011): 264 and 191 L/kg in brown trout liver and brain. Grabicova et al. (2017) analyzed brown trout exposed to effluents and detected SER in livers, kidneys and brains. The BCF values reached 1,500 and 2,400 L/kg for the kidney and liver, respectively. The highest BCF value, 6,200 L/kg for SER in fish liver, has been recorded by Koba et al. (2018). The BCF of SER obtained in our study (1,130-2,280 L/kg) was close to

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the average literature value.

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FLX has been detected in goldfish collected from wetlands that receive treated

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municipal wastewater (Muir et al., 2017). The calculated BCF was 386 and 906 L/kg in caged

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and wild goldfish, respectively. A similar BCF has been reported by Lajeunesse et al. (2011): 345 and 138 L/kg in brown trout liver and brain. These data were 3–5 fold higher than BCF

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presented in this study. This discrepancy may be due to the different pHs of the water

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samples. For lipophilic compounds, the BCF correlates well with octanol-water partition coefficients (logP). For ionized chemicals, the bioaccumulation potential depends on the pH-

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dependent coefficient (log D). The BCF values of FLX measured for Japanese medaka are highly dependent on pH: 8.8, 30 and 260 L/kg at pH 7, 8 and 9, respectively (Nakamura et al., 2008). In our experiments, the pH of the water was close to 7, so the BCF value was expected to be lower than in freshwaters with pH around 8. However, on comparing all tested antidepressant BCF values, no relationships could be found with the log D calculated for pH=7 (Giebultowicz and Nalecz-Jawecki, 2014). This suggests that not only passive transport, but also other mechanisms of uptake should be considered. The biological activity of a compound depends on its concentration in an organism at a target location. Thus, an increase in the BCF may result in an increase in the toxic action. According to the blood plasma model, this toxicity depends on the concentration of the drug 16

Journal Pre-proof in the blood plasma and is similar for fish and mammals. The median (10th – 90th percentile) therapeutic serum concentrations of SER, FLX, PAR and MIAN are 20 (5.8 – 56), 139 (43 – 359), 43 (9.5 – 142) and 38 (11 – 101) ng/mL, respectively (Reis et al., 2009). Given the BCF values and assuming the same sensitivity of fish and mammals, the biological effects of the tested drugs, especially SER and MIAN, may be expected at sub- to low micrograms per liter concentrations in water. These orders of magnitude of concentrations can be found in the natural environment.

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Aquatic organisms are exposed not to one compound, but to mixtures of several. Since

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SSRIs share the same pharmacological mode of action, a concentration addition model may

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be applied in the estimation of the activity of mixtures. According to Henry and Black (2007),

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concentration addition is an adequate model of the toxicity of SSRI mixtures and indicates that individual SSRI components may act through a similar mechanism of toxic action.

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Conversely, Schultz et al. (2011) found that the effects of single compound exposures neither

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carried over, nor became additive when adult male fathead minnows (Pimephales promelas) were exposed for 21 days to a mixture of FLX and SER. This means that the estimation of the

examined.

5

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toxicological effect of a mixture of the compounds can be hard to predict and should be

CONCLUSIONS

Antidepressants and their mixtures, even at environmental concentrations, exert adverse effects on the early stages of fish development. This suggests that the pharmaceuticals may interfere with the proper functioning of some organs in adult fish. In our studies, SER showed the highest bioconcentration in zebrafish larval tissues. The lowest was demonstrated by FLX. It can, therefore, be expected that SER will have the highest impact on organogenesis and changes in adult fish. The research types that can provide more 17

Journal Pre-proof information on the influence of antidepressants on aquatic organisms are, among others, histological and immunohistochemical studies. In our study, a decrease in hepatocyte proliferation was noticed not only among larvae exposed to SER but also in the mixture of all antidepressants at the highest concentration as well as to single drugs such as PAR and MIAN. Antidepressants increased the rate of abnormal embryo and larvae development, accelerated the hatching time and also affected the total hatching rate. Since only one concentration of single pharmaceutical were examined the verification whether the observed

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changes are dose related is needed. We have also shown that the effect of mixtures are

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different than the one of single compounds and hard to predict regarding unusual endpoints of

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the exposure study.

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Many factors affect toxin bioavailability, uptake, distribution and elimination by adult fish. Thus, our results cannot be extrapolated to these organisms. However, some risk

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assessment can be performed. Our study highlights the need to study the pharmacodynamics

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of SSRI in fish and to link this with observed teratogenic effects.

18

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Table 1. MS/MS optimized conditions for analyzed compounds and their internal standards Quantitative DPa CEb CXPc Qualitative CEb CXPc product product [V] [V] [V] [V] [V] ion ion

Doxepin (IS)

280.1

107.0

71

33

6

235.1

25

14

Fluoxetine

310.3

44.1

56

37

6

148.1

13

12

Mianserin

265.1

208.0

96

31

12

118.0

43

8

Nortriptyline (IS)

264.1

233.1

71

23

14

91.1

35

6

Paroxetine

330.2

192.1

51

31

14

70.1

49

4

Sertraline

306.0

158.9

51

35

12

275.0

19

16

b

DP – declustering potential

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re

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CXP – collision cell exit potential

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c

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CE – collision energy

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a

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Compound

[M+H]+ ion

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Journal Pre-proof Figure captions: Fig 1. Survival rate (A) and percentage of larvae hatched after 72 hpf (B) and 96 hpf (C). Embryos were expose to single antidepressants at nominal concentration of 10 µg/L or its mixture at nominal concentrations 5 µg/L (Mix5), 10 µg/L (Mix 10) and 25 µg/L (Mix 25). The results are the mean of five replicates in three independent experiments (n = 150). a,b,c the letters denote homogeneous groups according to NIR Fisher test with p = 0.05; Fig. 2. Abnormal fetal development of embryos expose to single antidepressants at nominal

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concentration of 10 µg/L or its mixture at nominal concentrations 5 µg/L (Mix5), 10 µg/L (Mix 10) and 25 µg/L (Mix 25). A) Rate [%] after 96hpf. The results are the mean

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of five replicates in three independent experiments (n = 150). a,b,c the letters denote

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homogeneous groups according to NIR Fisher test with p = 0.05. The normal morphology (B, control group) and examples of abnormalities: C) scoliosis (mixture, 10

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µg/L), D) tail malformation (fluoxetine), E) pericardial edema (fluoxetine).

Fig. 3. Proliferating cell nuclear antigen (PCNA) test in zebrafish liver. Normal hepatocyte

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proliferative activity was observed in fluoxetine (A), Mix5 (B) and Mix 10. Lower proliferation was found for paroxetine (C), mianserin (D), sertraline and Mix 25; black

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arrow – PCNA positive nuclei, E) Quantitative assessment of PCNA in zebrafish liver. Results are expressed as means (20 larvae in three independent experiment, n = 60) and standard deviations. Lowercase letters denote homogeneous groups according to NIR Fisher test with p = 0.05. Abbreviations: L – liver; I – Intestine; YS – yolk sac, S – syncytium, Mix5 – mixture of fluoxetine, sertraline, mianserin and paroxetine, 5 µg/L, Mix 10 – mixture, 10 µg/L, Mix 25 – mixture, 25 µg/L.

Fig. 4. Bioconcentration factors (BCFs) of pharmaceuticals tested separately and in mixtures: Mix 5, Mix 10 and Mix 25 containing 5, 10 and 25 µg/L of each compound, respectively. Results are expressed as means (n = 60) and standard deviations. None of the analyzed pharmaceutical was detected in samples from the control group.

20

50

0 ab

B d

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bc ab ix

25

10

ab abc

ab

ab

21 a

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M

ix

5

abc

M

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ab

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100 c

M

abc

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on t Fl uo rol xe Pa tin e ro xe Se tine rt ra M line ia ns er in

C

Survival rate after 96 hpf[%] 100 bc

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on t Fl uo rol xe Pa tin e ro xe Se tine rt ra M line ia ns er in M ix 5 M ix 10 M ix 25

C

Hatching rate after 72 hpf [%]

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A a

50

0

22

25

ab

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ix

10

ab

M

ix

5

a

M

a

ix

b

M

a

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100 b

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on t Fl uo rol xe Pa tin e ro xe Se tine rt ra M line ia ns er in

C

Hatching rate after 96 hpf [%]

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C ab

50

0

ix

M

ix

0

23

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25

10

5

a

M

ix

15

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a

M

a

na

5

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. on t Fl uo rol xe Pa tin e ro xe Se tine rt ra M line ia ns er in

C

Abnormalities rate after 96 hpf [%]

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Fig 2

A a

10 a

a a

a

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B

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C

24

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D

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E

25

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Fig 3

26

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re

-p

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of

Journal Pre-proof

27

on t Fl uo rol xe Pa tin e ro xe Se tine rt ra M line ia ns er in M ix 5 M ix 10 M ix 25

C

PCNA positive nuclei/1000 m2 2.0 b

1.5

1.0

0.5

E

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2.5

a

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a b

b

b

a a

0.0

28

10

5

50

0

100

250

Fluoxetine Pa ro xe

25

10

5

0

of

tin e

M ix

M ix

ix

M

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-p

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150

et in e

25

200

Fl uo x

ix

M

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ix

M

ix

M

BCF[L/kg] BCF[L/kg]

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Fig 4.

Paroxetine

400

300

200

100

29

10

5

ra lin e

25

Sertraline

3000

ix

M

ix

M

5

of

rin

25

10

ix

M

ia ns e

M

ro

-p

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2000

Se rt

ix

1000

M

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ix

0

M

M

BCF[L/kg]

BCF[L/kg]

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Mianserin

600

400

200

0

30

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6

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Journal Pre-proof Highlights

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Effects of low concentrations of antidepressants on zebrafish larvae Teratogenic effect of antidepressants on morphology of zebrafish larvae Alterations in proliferating cell nuclear antigen level comparing to control Bioaccumulation study of single and mixture of antidepressants Higher bioaccumulation in larvae exposed to a mixture than to a single compound

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    

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Figure 1

Figure 2

Figure 3

Figure 4