تاثیرات ترکیبی غلظت های پایین ۱۷ آلفا-اتینل استرادیول و سیتالوپرام بر رفتار عدم تولید مثل در ماهی راه راهی بالغ (Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio

Aquatic organisms are exposed to a wide range of chemicals from agriculture, industry and municipal sewage, often in complex mixtures. High concentrations of pharmaceuticals are present in effluents from sewage treatment plants (STPs) (Fick et al., 2011; Nikolaou et al., 2007; Weigel et al., 2004). The bioavailability and intended biological activity of pharmaceuticals give them high potential to cause sublethal effects on non-target organisms. Fish, as vertebrates, share many common physiological features with humans and are therefore very likely to be affected by waterborne pharmaceuticals designed to affect human physiology (Gunnarsson et al., 2008). Earlier studies on the effects of within-class mixtures of EDCs on fish exist (Brian et al., 2007; Kortenkamp, 2007; Lin and Janz, 2006; Santos et al., 2006; Thorpe et al., 2003). However we lack knowledge about the effects of mixtures of pharmaceuticals with different modes of action. In the present study, we analyzed the effects of low levels of the endocrine disrupting chemical (EDC) EE2, close to the NOEC of 0.1 ng/L, and the SSRI citalopram, as well as a combination of the two, on non-reproductive behavior in zebrafish (Danio rerio). EE2 from human oral contraceptives is present in effluents from STPs in concentrations from less than 1 ng/L up to 300 ng/L (Hannah et al., 2009; Kolpin et al., 2002; Laurenson et al., 2014; Sun et al., 2013). The predicted no effect concentration (NOEC) of EE2 for waterliving organisms is as low as 0.1 ng/L (Caldwell et al., 2012). EE2 is environmentally persistent, bio-magnification has been observed (Aris et al., 2014) and is regarded as the EDC contributing the highest ecological risk in waste water (Laurenson et al., 2014; Sun et al., 2013). EDCs have the potential to interfere with the function of the hormone systems of all vertebrates (Guillette and Gunderson, 2001; Waring and Harris, 2005; Vos et al., 2000). Human fetal exposure to estrogenic compounds has been associated with depression (Wolstenholme et al., 2012) and distractibility, affected verbal skills, learning and memory, reduced masculine play (Xu et al., 2010). Behavioral variables shown to be affected by estrogenic compounds in rodents include aggression, anxiety, play behavior, attention, learning and memory and sexual behavior (Dugard et al., 2001; Ryan and Vandenbergh, 2006; Wolstenholme et al., 2012, 2011; Xu et al., 2010). Environmental-like levels of EE2 caused alterations in female sexual behavior in adult rats exposed during development (Della Seta et al., 2008) and juvenile rats showed an anxiety-like response in a novelty preference test after developmental exposure (Zaccaroni et al., 2016). Developmental exposure of EE2 has also showed to cause anxiety-related behavior, alter spatial memory, disturbed maternal behavior and a lack of discrimination between gonad-intact and castrated males in female mice (Ryan and Vandenbergh, 2006). Male mice developmentally exposed to low doses of EE2 showed an increase in sexual behavior and modifications of neuronal networks. The effects were also transgenerationally transmitted to the F4 generation (Derouiche et al., 2015). In fish EE2 has shown to cause reduced fertility and fecundity, feminization in male fish, skewed sex ratios and decreased egg and sperm production as well as behavioral changes (Aris et al., 2014). EE2 exposure has caused alterations in risky behavior in the threespine stickleback (Bell, 2004) and guppies (Heintz et al., 2015) as well as boldness in Siamese fighting fish (Dzieweczynski et al., 2014). We have previously found that EE2 increases anxious behavior in guppies (Poecilia reticulata) and zebrafish (Danio rerio) exposed as adults (Hallgren et al., 2011; Reyhanian et al., 2011) or during development (Volkova et al., 2015b; Volkova et al., 2012). Developmental exposure resulted in irreversible effects (Volkova et al., 2015b), which were shown to be transgenerationally transferred (Volkova et al., 2015a). Citalopram has been detected in STP effluents in concentrations ranging from 9.2 ng/L (Vasskog et al., 2006) to 720 ng/L (Wahlberg et al., 2008) and in surface waters between 4 ng/L (Giebułtowicz and Nałęcz-Jawecki, 2014) and 76 μg/L (Fick et al., 2009); more typical concentrations in polluted recipients are around 10–۱۵۰ ng/L (González Alonso et al., 2010; Grabicova et al., 2015; Metcalfe et al., 2010; Nödler et al., 2011). Several SSRIs are present in STP effluents, and the combined load of 7 major SSRIs and their metabolites was up to 3.2 μg/L downstream a Canadian STP (Brooks et al., 2005). The predicted water concentration needed to obtain human therapeutic levels in fish is 141 ng/L (Fick et al., 2010). Brain SSRI bioaccumulation has been observed in fish caught downstream an STP (Brooks et al., 2005), and citalopram has been found in the liver of perch (Perca fluviatilis) caught in the inner parts of the Stockholm archipelago (Woldegiorgis et al., 2006). SSRIs are psychoactive drugs prescribed for treating depression and other psychiatric disorders. SSRIs reduce the re-uptake of the neurotransmitter serotonin (5-hydroxy-tryptamine; 5-HT) into the pre-synaptic nerve terminal by inactivating 5-HT transporters (5-HTT), resulting in an increased concentration of extracellular 5-HT in the synapse. 5-HT is ubiquitous to all vertebrate groups and influences a wide range of behaviors and endocrine functions (Anon., 2010). In fish, SSRI have been shown to affect behaviors like feeding (Kellner et al., 2015), aggression (Winberg and Thörnqvist, 2016) and anxiety (Barbosa et al., 2012; Kellner et al., 2016; Olsén et al., 2014; Sackerman et al., 2010). Citalopram, an abundantly prescribed SSRI, has given an anxiolytic response in the novel tank test in zebrafish (Sackerman et al., 2010), guppies (Olsén et al., 2014) and three-spine sticklebacks (Kellner et al., 2016). In this study, adult zebrafish were exposed to low, environmentally relevant concentrations of two pharmaceuticals commonly found in the environment, EE2 and citalopram, and analyzed for impact on non-reproductive behavior. The aim of the study was to investigate if the behavioral effects previously found of the two substances could still be seen at very low concentrations. We also further wanted to investigate if the effects of the two compounds, the anxiolytic effects of citalopram and anxiogenic effects of EE2 would counteract and affect the behavioral outcome in the combinatory exposure. We utilized two tests assessing anxiety, the scototaxis test (Maximino et al., 2010) and the novel tank (NT) test (Egan et al., 2009), and one test analyzing social behavior by mean of shoal cohesion (Moretz et al., 2007). We studied both effects on single-substance exposures as well as a combination of the two.