Behavioral Effects of Citalopram, Tramadol, And

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Bachour, R-L., Golovko, O., Kellner, M., Pohl, J. (2020) Behavioral effects of citalopram, tramadol, and binary mixture in zebrafish (Danio rerio) larvae Chemosphere, 238: 124587 https://doi.org/10.1016/j.chemosphere.2019.124587

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Behavioral effects of citalopram, tramadol, and binary mixture in zebraﬁsh (Danio rerio) larvae

* Raougina-Laouisa Bachour a, Oksana Golovko b, Martin Kellner c, Johannes Pohl a, a Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden b Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden c Department of Natural Sciences, Technology and Environmental Studies, Sodert€ orn€ University, Huddinge, Sweden highlights

Citalopram and tramadol binary mixture effects studied in zebraﬁsh (Danio rerio). Embryo-larvae were exposed from fertilization until six days post-fertilization. Heart rate, spontaneous tail coiling, and death/malformation incidence unaffected. 1 1 Anxiolytic effects in citalopram (EC50:471mgL ) and tramadol (EC50:411mgL ). 1 The mixture was less anxiolytic (EC50:713mgL ), indicating interaction effects. article info abstract

Article history: Pharmaceuticals are emerging as environmentally problematic compounds. As they are often not Received 4 July 2019 appropriately removed by sewage treatment plants, pharmaceutical compounds end up in surface water Received in revised form environments worldwide at concentrations in the ng to mgL 1 range. There is a need to further explore 2 August 2019 single compound and mixture effects using e.g. in vivo test model systems. We have investigated, for the Accepted 13 August 2019 first time, behavioral effects in larval zebrafish (Danio rerio) exposed to a binary mixture of an antide- Available online 13 August 2019 pressant drug (citalopram) and a synthetic opioid (tramadol). Citalopram and tramadol have a similar Handling Editor: David Volz mode of action (serotonin reuptake inhibition) and are known to produce drug-drug interactional effects resulting in serotonin syndrome (SS) in humans. Zebrafish embryo-larvae were exposed to citalopram, Keywords: tramadol and 1:1 binary mixture from fertilization until 144 h post-fertilization. No effects on heart rate, Zebrafish embryotoxicity spontaneous tail coiling, or death/malformations were observed in any treatment at tested concentra- 5-HT tions. Behavior (hypoactivity in dark periods) was on the other hand affected, with lowest observed effect SSRI concentrations (LOECs) of 373 mgL 1 for citalopram, 320 mgL 1 for tramadol, and 473 mgL 1 for the 1:1 Synthetic opioid 1 1 mixture. Behavioral EC50 was calculated to be 471 mgL for citalopram, 411 mgL for tramadol, and Serotonin syndrome 713 mgL 1 for the 1:1 mixture. The results of this study conclude that tramadol and citalopram produce hypoactivity in 144 hpf zebrafish larvae. Further, a 1:1 binary mixture of the two caused the same response, albeit at a higher concentration, possibly due to SS. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction Although most drugs are fully metabolized to inactive metabolites, some compounds are preserved in their active form and eliminated Pharmaceutical drug residues are regularly detected in the through the kidneys (Bergheim et al., 2012). As a consequence, surface water environment in the ng to mgL 1 concentration range minute concentrations of drugs are constantly released into the (Fick et al., 2009; Grabicova et al., 2017). Pharmaceutical com- aquatic environment (Lindberg et al., 2014). Numerous pharma- pounds end up in surface water mainly due to inadequate removal ceuticals may bioaccumulate in aquatic and semi-aquatic in- in sewage treatment plants (Lindberg et al., 2014; Luo et al., 2014). vertebrates, thereby exposing predators (including fish) to levels exceeding surface water concentrations (Richmond et al., 2018). The exposure to pharmaceuticals may result in ecological effects fl * Corresponding author. Box 7028, 750 07, Uppsala, Sweden. in uencing aquatic lifeforms since drug target sites are evolution- E-mail address: [email protected] (J. Pohl). arily conserved. For example, the zebrafish (Danio rerio) has been https://doi.org/10.1016/j.chemosphere.2019.124587 0045-6535/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2 R.-L. Bachour et al. / Chemosphere 238 (2020) 124587 proposed to have about 86% orthologs to human drug targets developed serotonergic system both in the central nervous system (Gunnarsson et al., 2008). Water-dwelling organisms continuously and peripheral system (Winberg et al., 1997). Zebrafish larvae exposed to anti-depressant and anti-anxiety drugs might be develop the 5-HT system around 96 h post-fertilization (hpf; affected by e.g. increased predation pressure (Klaminder et al., Brustein et al. (2003)). The zebrafish larvae also possess a distinct 2019). There is thus a need for research establishing drug-drug behavioral repertoire in response to stress stimuli (e.g. rapid interaction properties of pharmaceuticals mixtures in non-target changes in illumination; Padilla et al. (2011)). species (Backhaus, 2014; Gunnarsson et al., 2019). The present study aimed to identify behavioral and embryotoxic One pharmaceutical drug frequently found in surface waters is effects in embryo-larval zebrafish exposed to citalopram, tramadol, citalopram (Fick et al., 2009; Vasskog et al., 2006). Citalopram is a and a 1:1 binary mixture. To our knowledge, this study is the first to selective serotonin reuptake inhibitor (SSRI) type drug and is one of combine an SSRI (citalopram) and opioid (tramadol) in a zebrafish the most prescribed and used antidepressants in the world. The embryo-larvae behavioral test. The rationale for studying the drug has two racemic enantiomers, S-(þ) and R-() where S-(þ)is combination of the compounds stemmed from the hypothesis that the active enantiomer and R-() is inactive (Hyttel et al., 1992), tramadol co-exposure would potentiate citalopram embryo-larval although some studies have shown that the R-enantiomer may behavioral effects, in accordance with studies showing such have an inhibiting effect on the S-enantiomer (Zhong et al., 2009). outcome in rodents (e.g. Fox et al. (2009)). In order to capture SS- The antidepressant effect arises through a mechanism inhibiting like effects, we recorded endpoints including the incidence of synaptic serotonin (5-hydroxytryptamine (5-HT)) reuptake, giving lethality and malformations, spontaneous tail coiling, heart rate, rise to increased 5-HT concentrations in the central nervous system and swimming activity in response to alternating dark and light pre-synaptic clefts (Bezchlibnyk-Butler et al., 2000). When cit- periods in a 6 d static zebrafish embryo-larvae exposure study. alopram is consumed by humans, about 12% of the parent compound leaves the body unmetabolized (Milne and Goa, 1991). The 2. Materials & methods remainder is metabolized into citalopram-N-oxide, citalopram propionic acid and the active metabolite desmethylcitalopram 2.1. Chemicals and test solutions (Hyttel et al., 1992). Desmethylcitalopram is then further degraded to didesmethylcitalopram (Sangkuhl et al., 2011). Relatively high The chemicals used in the present study were citalopram citalopram concentrations (~8 mgL 1) have been detected down- hydrobromide (CAS number: 59729-32-7, purity 98%) and tra- stream from pharmaceutical production sites in India (Fick et al., madol hydrochloride (CAS number: 36282-47-0, purity 99%), and 13 2009). isotope-labeled internal standards of tramadol ( C, D3) and cit- Tramadol is a commonly used synthetic opioid medication for alopram (D-4). All chemicals were purchased from Sigma Aldrich pain treatment (Rougemont-Bücking et al., 2017). The effect causes (Sweden). Test solutions of citalopram, tramadol and 1:1 cit- analgesia (pain relief) when tramadol is converted into the active alopram:tramadol mixture were prepared by dissolving the metabolite O-desmethyltramadol which acts as an agonist to the m- respective compound in carbon-filtered tap water (used as control opioid receptor (Vazzana et al., 2015). However, it is reported that treatment) by stirring and vortexing at room temperature (22 C). tramadol may also be used for depression and anxiety treatment The pharmaceutical concentrations used for the exposure studies via its 5-HT and norephedrine reuptake inhibition properties were determined by prior concentration-response range screen- (Miotto et al., 2017). Tramadol consists of two chiral centers with ings. Citalopram was serially diluted 1:4 corresponding to five two enantiomers, the S,S-enantiomer and the R,R-enantiomer. The nominal test concentrations (8000, 2000, 500, 125 and 31 mgL 1). S,S-enantiomer has a strong effect on the norephedrine and 5-HT Tramadol was likewise serially diluted 1:4 corresponding to five reuptake by inhibition while the R,R-enantiomer has 5-HT reup- nominal test concentrations (6250, 1563, 391, 98 and 24 mgL 1). A take properties also derived from inhibition (Miotto et al., 2017). binary mixture solution with a ratio of 1:1 citalopram:tramadol The unchanged excretion of the tramadol parent compound in was prepared with at a nominal maximum concentration of urine is about 15e35% (Kasprzyk-Hordern et al., 2009). Tramadol is 10000 mgL 1 (combination of 5000 mgL 1 citalopram and relatively stable in the aquatic environment, with concentrations of 5000 mgL 1 tramadol) and serially diluted 1:4, yielding in total four up to 50 mgL 1 measured in Cameroon river sediments (Kusari nominal test concentrations (10000, 2500, 625, 156 and 39 mgL 1). et al., 2016). Water quality parameters were monitored for each test solution When citalopram and tramadol are co-administered in humans, before and after each test, and did not differ significantly from the a drug-drug interaction termed serotonin syndrome (SS) may occur control treatment (pH: 8.36 ± 0.04, conductivity: 450 ± 30 mS/cm, (Park et al., 2014). SS is manifested when a single reuptake inhibitor dissolved O2:90± 5%). or when several inhibitors interact (Boyer and Shannon, 2005). The mechanistic underpinnings of SS involve excessive 5-HT concen- 2.2. Chemical analysis trations in the synaptic cleft, causing an imbalance in the binding of 5-HT to the 5-HT1A- and 5-HT2A receptor (Beakley et al., 2015). The The measured concentrations at the start of the embryo expo- exhibited symptoms of SS are related to neuromuscular activity, sure (t ¼ 0 h) are represented by a mean of triplicate samples and tachycardia (increased resting heart rate), tremors, and mental the measured concentrations at the end of the embryo exposure conditions including confusion and agitation (Boyer and Shannon, (t ¼ 144 h) are based on single samples. No samples from t ¼ 144 2005). Co-administering these compounds should be avoided as were analyzed in the mixture study. The chemical analysis method the resulting SS syndrome may ultimately be fatal (Rougemont- was based on a previously published study (Rostvall et al., 2018). Bücking et al., 2017). Drug-drug interactions leading to clinical SS For chemical analysis, the samples were filtered using a regener- in mammal studies have been reproduced in fish studies, sug- ated cellulose syringe filter (0.22 mm pores). One milliliter of the gesting fish could be sensitive to 5-HT-linked adverse effects from filtered extract was placed in an autosampler vial with 10 ng of the 13 co-exposure of several SSRI and mechanistically related com- isotope-labeled internal standards of tramadol ( C, D3) and cit- pounds (Stewart et al., 2013). Zebrafish embryo models are alopram (D-4) per aliquot of sample. The samples were analyzed increasingly used within the framework of biomedical science, using liquid chromatography tandem-mass spectrometry (LC-MS/ including research on behavioral toxicology (Noyes et al., 2018). MS) with an LC system from Thermo Fisher Scientific and a triple- Zebrafish are genetically similar to mammals and possess a highly stage quadrupole MS/MS TSQ Quantiva (Thermo Fisher Scientific, R.-L. Bachour et al. / Chemosphere 238 (2020) 124587 3

United States). An Acquity UPLC BEH-C18 column (Waters, 100 mm performed at 144 hpf to establish behavioral effects (swimming x 2.1 i.d., 1.7 mm particle size from Waters Corporation, United activity) in response to shifting dark and light conditions. Each Kingdom) was used as an analytical column. Injection volume was plate was put in the Zebrabox tracking system (ViewPoint, France), 10 mL for all samples. A heated electrospray ionization (HESI) was and filmed for 75 min. The protocol consisted of an initial 15 min 0% used to ionize the target compounds. The spray voltage was set to light condition (acclimatization period), and six alternating 10 min static: positive ion (V) 3500.00. Nitrogen (purity >99.999%) was periods of 100% light and 0% light (dark conditions). The swimming used as a sheath gas (50 arbitrary units), auxiliary gas (15 arbitrary activity was determined as the total swimming distance (mm) per units) and sweep gas (2 arbitrary units). The vaporizer was heated larva per min, as well as total swimming distance (mm) per larva to 400 C and the capillary to 325 C. Two selected reaction moni- during light and dark conditions. Dead and observably malformed toring (SRM) transitions were monitored for all analytes. Data were larvae at 144 hpf (e.g. lack of swim bladder inflation) were omitted evaluated using TraceFinder™ 3.3 software (Thermo Fisher Scien- from the swimming activity data analysis. tific, United States).

2.4. Statistical analysis 2.3. Embryotoxicity- and behavioral tests The R software (R Core team, 2019) with the RStudio version The zebrafish embryotoxicity test (ZFET) assay used in this study 1.1.463 interface (RStudio Team, 2019) was used for statistical was based on the methodology described in Pohl et al. (2019). Adult computing and plotting. Statistical analysis of the endpoints heart zebrafish were kept under 12 h dark and light interval conditions at rate, spontaneous movements and behavior were done by ANOVA 26 C. Groups (n ¼ 10) of adult zebrafish were moved to aquariums and Dunnett's post hoc test. Fisher's test was used to analyze lethal equipped with stainless steel net cages with carbon-filtered tap (coagulation) and sublethal (malformations) effects which were water. During the next morning (09.00e09:30 a.m.), the stainless- categorized into one group (affected). The R package ggplot2 was steel net cages containing zebrafish were moved to aquariums filled used to produce plots (Wickham, 2016). Swimming activity in with fresh carbon-filtered tap water where they could spawn. Eggs response to dark conditions was normalized against control mean were collected from the aquarium with the highest proportion of and modeled in a 4 parameter logistic regression (the Hill equa- fertilized eggs for each test. Embryos (n ¼ 32 per treatment group, tion), before plotting and estimation of effect concentrations using distributed on two plates) were exposed to each test solution the drc R package (Ritz et al., 2015). The significance level was set at at ~ three hpf and placed in two 96-well round bottom plates with a 0.05 for all parametric- and nonparametric tests. 200 mL volume until 144 hpf. The plates were covered with Parafilm M (Bemis Company, United States) throughout the six d exposure (12:12 h light cycle, 26 ± 1 C air temperature). The eggs were 3. Results & discussion exposed statically (i.e. test solutions were not renewed over time) from fertilization until 144 hpf.. The present study was performed to investigate embryotoxicity- Incidences of lethal and sublethal effects were observed by and neurobehavioral endpoints in zebrafish exposed to citalopram, stereo microscopy at 24, 48 and 144 hpf. For instance, coagulation tramadol and a 1:1 binary mixture thereof. The endpoints studied was a lethal endpoint and pericardial- and yolk-sac edemas were included spontaneous tail coiling frequency, heart rate, the inci- considered as sublethal endpoints. At 24 hpf the embryos were dence of lethality/malformations, and behavioral effects (swim- filmed for 1 min with a Canon EOS 500D to determine spontaneous ming activity during alternating dark and light conditions). tail coilings per min. The heart rate (heartbeats per min) was Measured citalopram and tramadol concentrations are shown in recorded at 48 hpf by visual counting. The last observation was Table 1.

Table 1 Nominal and measured treatment concentrations of test solutions before (t ¼ 0 h) and after (t ¼ 144 h) the six d static exposure (mgL 1, LOQ ¼ limit of quantiﬁcation, NA ¼ not analyzed).

Treatment Nominal concentration Measured concentration Measured in % of Measured concentration LOQ (mgL1) at t ¼ 0 h (mean, mgL1) nominal concentration at t ¼ 144 h (mgL1) (mgL1)

Citalopram 0

3.1. Embryotoxicity of citalopram, tramadol, and mixture there were no significant effects on embryo heart rate compared to the controls (Supplementary Data Fig. S1). Earlier studies have There were no significant effects in any recorded embryotoxicity shown the effects of 5-HT on zebrafish heart rate via the occurrence endpoint (i.e heart rate, spontaneous tail coiling, and incidence of of 5-HT in the heart cells (Stoyek et al., 2017). Exposure to other death/malformations) in zebrafish embryo-larvae exposed to cit- SSRI pharmaceuticals such as fluoxetine has been shown to cause a alopram, tramadol, or the mixture (Supplementary Data Fig. S1). heart rate reduction, meaning the SSRI also affected the 5-HT re- Early spontaneous tail-coilings appear before 24 hpf as the axons uptake in the heart (Stoyek et al., 2017). On the other hand, start to proliferate in the embryonic somites, and the tail-coiling increasing levels of 5-HT due to deprenyl exposure has been frequency at 24 hpf can be used for neurotoxicity assessment associated with increased heart rate in nine d post-fertilization (Basnet et al., 2017; Saint-Amant and Drapeau, 1998). As observed zebrafish larvae in a previous study (Sallinen et al., 2009). The in this study, embryo spontaneous tail movements at 24 hpf were present study could however not detect heart rate alterations not affected (Supplementary Data Fig. S1). This may indicate a low caused by citalopram, tramadol, or the mixture 48 hpf zebrafish sensitivity when studying the endpoint in zebrafish embryos embryos. This may be due to the fact that the 5-HT system is not exposed to the compounds citalopram, tramadol and the mixture at active until 96 hpf in zebrafish embryos (Brustein et al., 2003). concentrations about 20e5000 mgL 1. The functionality of the 5-HT At 144 hpf the proportion affected (dead and malformed) em- system in zebrafish embryo is initiated around 96 hpf (Brustein bryos were summarized. The results showed no significant effect et al., 2003). Hence, serotonergic compounds would not directly on the embryos exposed to citalopram, tramadol or the mixture affect the undeveloped brain, or other organs, at 24 hpf. compared to the controls (Supplementary Data Fig. S1). Citalopram 1 At 48 hpf the heart rate was visually measured for all drugs LC50-values of 38.4 mg L in zebrafish embryo-larvae (Steele et al., compositions at different concentrations. The results indicated that 2018) and 9.1 mg L 1 in Japanese medaka (Oryzias latipes) larvae

Fig. 1. Distance moved (mean ± SD) over time of zebraﬁsh embryos exposed to (A) citalopram, (B) tramadol and a (C) 1:1 binary mixture during light and dark conditions at 144 hpf. The left panel shows the total distance moved per min (mean) and to the right, the mean total distance moved per min (mean ± SD). One-way ANOVA and Dunnett's post hoc test was used for statistical analysis, where total distance moved for each larva was considered an individual replicate (**: p < 0.01, ***: p < 0.001). R.-L. Bachour et al. / Chemosphere 238 (2020) 124587 5

(Chiffre et al., 2016) have been reported previously, ~2 to 7 fold higher than the highest concentration used in the present study. Zebraﬁsh embryos have been exposed to tramadol in earlier studies with concentrations between 10 and 200 mgL 1 resulting in delayed hatching, but no signiﬁcant embryo mortality (Sehonova et al., 2016).

3.2. Behavioral effects at 144 hpf

Larval zebrafish behavior (swimming activity) in response to alternating light- and dark periods was assessed at 144 hpf. It is known that zebrafish larvae display a higher activity during dark conditions compared to light conditions, presumably due to a stress response triggered by the sudden shift from light to total darkness (Burgess and Granato, 2007; MacPhail et al., 2009). Several psychoactive compounds can cause decreased swimming activity (hypoactivity) in response to stress cues, which may affect fish species adversely since hypoactivity may result in increased predation risk (Saaristo et al., 2018). In the present study, hypoactivity during dark conditions was recorded in larvae exposed to citalopram, tramadol and the 1:1 binary mixture in comparison with unexposed control, demonstrating anxiolytic properties of these treatments with LOECs between ~300 and 400 mgL 1 (Fig. 1). When comparing the treatments in concentration-response curves (4 parameter logistic regression) it was revealed that the binary mixture was less potent (~50% higher EC50) in terms of hypoactivity during dark conditions than citalopram and tramadol respectively (Fig. 2).

3.2.1. Citalopram The swimming activity during dark conditions in larvae exposed Fig. 2. Concentration-response curve with the swimming distance in darkness (% m 1 fi relative control) plotted against log measured concentration in gL for citalopram, to citalopram signi cantly decreased and the lowest observed ef- tramadol and the 1:1 binary mixture (calculated by 4 parameter logistic regression, i.e. 1 fect concentration (LOEC) was 373 mgL (Fig. 1). Citalopram has the Hill equation). the pharmacological effect of antagonizing serotonin reuptake transporters (SERT) in studied fish and thereby provoking a multitude of behavioral effects (Gould et al., 2007; Sehonova et al., context of aquatic behavioral toxicology, with examples including 2018). Previous studies on zebrafish larval behavior have reported studies on crayfish displaying increased shelter dwelling after 1 effects at 20 mgL 1 (Steele et al., 2018). Japanese medaka larvae are treatment with environmental relevant (1 mgL ) concentrations reportedly affected at concentrations of 10 mg citalopram L 1 cit- (Buric et al., 2018; Lozek et al., 2019). Clinical side-effects of tra- alopram (Chiffre et al., 2016). Crayfish exposed to citalopram madol exposure include drowsiness and confusion, possibly (1 mgL 1), exhibit anxiolytic behaviors (Buric et al., 2018). Cit- explaining anxiolytic outcomes in non-target organisms (Langley alopram has also been shown to exert behavioral effects in adult et al., 2010). However, it may also be suspected that other mecha- three-spine stickleback (Gasterosteus aculeatus; Kellner et al. nisms, e.g. mitochondrial dysfunction leading to disturbed energy (2015)), guppy (Poecilia wingei; Olsen et al. (2014)) and zebrafish metabolism, could also be involved in decreasing swimming ac- (Nielsen et al., 2018), at concentrations well below the LC50 re- tivity (Zhuo et al., 2012). ported for each respective species. Anxiolytic responses in wild- caught tree-spine stickleback exposed to citalopram alone at con- 3.2.3. Mixture m 1 centrations of up to 15 gL have also been reported (Kellner et al., A decrease in swimming activity was observed in zebrafish 2016). Furthermore, citalopram has been shown to interfere with embryos exposed to the 1:1 binary mixture with a LOEC of fi sh embryonic development concerning behavior (Kellner et al., 473 mgL 1 (Fig. 1). By modelling the concentration-response curves 2018) and bone development (Fraher et al., 2016). of the swimming activity in darkness in % relative control of the According to the literature, citalopram has yet not been found in three tests we could observe that the 1:1 mixture was ~50% less the environment at concentrations approximating the effect con- 1 potent (EC50:713mgL ± 251 SE) than tramadol (EC50: centration observed in the present study (Fick et al., 2009; 1 1 411 mgL ± 118 SE) and citalopram (EC50:471mgL ± 177 SE) Grabicova et al., 2017). However, under real-life conditions, cit- individually (Fig. 2). alopram typically occurs in combination with a host of other SSRI To our knowledge, the present work is the first where a mixture and their metabolites, some of which retain SSRI activity, and the of an SSRI drug and tramadol were investigated to identify total SSRI concentration can be much higher than single compound behavioral effects in larval zebrafish possibly linked to SS. Adult measurements will show (Vasskog et al., 2008). zebrafish has earlier been used to demonstrate possible signs of SS (top dwelling) after exposure to a fluoxetine (SSRI) and lysergic acid 3.2.2. Tramadol diethylamid combination (Stewart et al., 2013). In the present The results of the present study indicated a significant anxiolytic study, we could not detect any indications of synergism following effect on zebrafish embryos exposed to tramadol and a LOEC of citalopram and tramadol co-exposure. On the other hand, the 320 mgL 1 (Fig.1). Tramadol has previously been investigated in the decreased potency of the 1:1 binary mixture could perhaps 6 R.-L. Bachour et al. / Chemosphere 238 (2020) 124587 illustrate signs of SS mental symptoms including e.g. agitation or Fraher, D., Hodge, J.M., Collier, F.M., McMillan, J.S., Kennedy, R.L., Ellis, M., increased anxiety (Maximino et al., 2013). However, this interpre- Nicholson, G.C., Walder, K., Dodd, S., Berk, M., Pasco, J.A., Williams, L.J., Gibert, Y., 2016. Citalopram and sertraline exposure compromises embryonic bone tation is speculative and more mechanistic research is needed to development. Mol. Psychiatry 21, 656e664. https://doi.org/10.1038/mp.2015. provide evidence. 135. Gould, G.G., Brooks, B.W., Frazer, A., 2007. [3H] citalopram binding to serotonin transporter sites in minnow brains. Basic Clin. Pharmacol. Toxicol. 101, 4. Conclusions 203e210. https://doi.org/10.1111/j.1742-7843.2007.00100.x. Grabicova, K., Grabic, R., Fedorova, G., Fick, J., Cerveny, D., Kolarova, J., Turek, J., Uncovering mixture effects of SSRIs and opioids on zebrafish Zlabek, V., Randak, T., 2017. Bioaccumulation of psychoactive pharmaceuticals in fish in an effluent dominated stream. Water Res. 124, 654e662. https://doi. models can inform about the risk of polluting the environment org/10.1016/j.watres.2017.08.018. with human pharmaceuticals affecting the 5-HT system. This study Gunnarsson, L., Jauhiainen, A., Kristiansson, E., Nerman, O., Larsson, D.G.J., 2008. was therefore performed to understand whether citalopram, tra- Evolutionary conservation of human drug targets in organisms used for envi- e fi ronmental risk assessments. Environ. Sci. Technol. 42, 5807 5813. https://doi. madol, and a mixture thereof would affect developing zebra sh org/10.1021/es8005173. embryo spontaneous tail coiling, heart rate, death/malformation Gunnarsson, L., Snape, J.R., Verbruggen, B., Owen, S.F., Kristiansson, E., Margiotta- € incidence, and larval behavior (swimming activity in light and dark Casaluci, L., Osterlund, T., Hutchinson, K., Leverett, D., Marks, B., Tyler, C.R., 2019. Pharmacology beyond the patient e the environmental risks of human drugs. conditions). The recorded endpoints tail coiling, heart rate and Environ. Int. 129, 320e332. https://doi.org/10.1016/j.envint.2019.04.075. incidence of death/malformations were not affected by any treat- Hyttel, J., Bøgesø, K.P., Perregaard, J., Sanchez, C., 1992. The pharmacological effect of ment. Larval behavior at 144 hpf was however affected by cit- citalopram resides in the (S)-(þ)-enantiomer. J. Neural Transm. 88, 157e160. alopram (LOEC: 373 mgL 1), tramadol (LOEC: 320 mgL 1), and https://doi.org/10.1007/BF01244820. 1 Kasprzyk-Hordern, B., Dinsdale, R.M., Guwy, A.J., 2009. The removal of pharma- mixture (LOEC: 473 mgL ). The mixture effect was seemingly ceuticals, personal care products, endocrine disruptors and illicit drugs during antagonistic, as the EC50 of larval hypoactivity in dark conditions wastewater treatment and its impact on the quality of receiving waters. Water m 1 ± Res. 43, 363e380. https://doi.org/10.1016/j.watres.2008.10.047. was higher in 1:1 binary mixture (713 gL 177 SE) than cit- € € m 1 ± Kellner, M., Porseryd, T., Porsch-Hallstrom, I., Hansen, S.H., Olsen, K.H., 2015. alopram (EC50:471gL 177 SE) and tramadol (EC50: Environmentally relevant concentrations of citalopram partially inhibit feeding 411 mgL 1 ± 118 SE), respectively. Since both compounds exert in the three-spine stickleback (Gasterosteus aculeatus). Aquat. 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