Pharmaceuticals are emerging as environmentally problematic compounds. As they are often not appropriately removed by sewage treatment plants, pharmaceutical compounds end up in surface water environments worldwide at concentrations in the ng to μg L−1 range. There is a need to further explore single compound and mixture effects using e.g. in vivo test model systems. We have investigated, for the first time, behavioral effects in larval zebrafish (Danio rerio) exposed to a binary mixture of an antidepressant drug (citalopram) and a synthetic opioid (tramadol). Citalopram and tramadol have a similar 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, tramadol and 1:1 binary mixture from fertilization until 144 h post fertilization. No effects on heart rate, spontaneous tail coiling, or death/malformations were observed in any treatment at tested concentrations. Behavior (hypoactivity in dark periods) was on the other hand affected, with lowest observed effect concentrations (LOECs) of 373 μg L−1 for citalopram, 320 μg L−1 for tramadol, and 473 μg L−1 for the 1:1 mixture. Behavioral EC50 was calculated to be 471 μg L−1 for citalopram, 411 μg L−1 for tramadol, and 713 μg L−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.

Selective serotonin reuptake inhibitors (SSRIs) are psychotropic pharmaceuticals used as antidepressants. SSRIs are commonly found in surface waters in populated areas across the globe. They exert their efect by blocking the serotonin re-uptake transporter in the presynaptic nerve ending. The present study examined whether behavioural efects to exposure to SSRI citalopram depend on personality and sex in the stickleback (Gasterosteus aculeatus). Three aspects of stickleback behaviour are examined: feeding behaviour, aggression, and boldness. We exposed sticklebacks to 350–380 ng/l citalopram for 3 weeks. Feeding and aggressive behaviour were recorded before and after exposure, whereas scototaxis behaviour was tested after exposure. The results show treatment efects in feeding and aggressive behaviour. Feeding is suppressed only in the male group (χ2=20.4, P<0.001) but not in the females (χ2=0.91, P=0.339). Aggressive behaviour was signifcantly afected by treatment (χ2=161.9, P<0.001), sex (χ2=86.3, P<0.001), and baseline value (χ2=58.8, P<0.001). Aggressiveness was suppressed by citalopram treatment. In addition, the fsh showed no change in aggression and feeding behaviour over time regardless of sex and treatment, which indicate personality traits. Only females are afected by treatment in the scototaxis test. The exposed females spent signifcantly (χ2=5.02, P=0.050) less time in the white zone than the female controls.

The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

Selective Serotonin Re-uptake Inhibitors (SSRIs) are a class of psychotropic drugs used to treat depression in both adolescents and pregnant or breast-feeding mothers as well as in the general population. Recent research on rodents points to persistent behavioural effects of pre- and perinatal exposure to SSRI which last into adulthood. To study effects of developmental exposure in fish, three-spine sticklebacks were exposed to 1.5 µg/l of the SSRI citalopram in the ambient water for 30 days, starting two days post-fertilisation. After 100 days of remediation in clean water the fish were put through an extensive test battery. Feeding behaviour was tested as the number of bites against a piece of food and found to be increased in the exposed fish. Aggression levels were measured as the number of bites against a mirror image during 10 minutes and was also found to be significantly increased in the exposed fish. Novel tank behaviour and locomotor activity was tested in an aquarium that had a horizontal line drawn half-way between the bottom and the surface. Neither the latency to the first transition to the upper half, nor the number of transitions or the total time spent in the upper half was affected by treatment. Locomotor activity was significantly reduced in the exposed fish. The light/dark preference was tested in an aquarium where the bottom and walls were black on one side and white on the other. The number of transitions to the white side was significantly reduced in the exposed fish but there was no effect on the latency to the first transition or the total time spent in the white half. The results in the current study indicate that developmental SSRI exposure causes persistent behavioural effects in fish and contribute to the existing knowledge about SSRIs as environmental pollutants.

Selective serotonin re-uptake inhibitors are pharmaceuticals used to treat a range of psychological disorders. They are frequently found in surface waters in populated areas. In recent years, they have been shown to affect the behaviour of various aquatic organisms in a way that can have ecological effects. In this study, we exposed zebrafish of both sexes to nominally 0.00, 0.15 and 1.50 µg L−1 Escitalopram in flow-through tanks for three weeks. Subsequently, ten swimming behaviour parameters were quantified using high-resolution video tracking. There were noticeable gender differences in the behaviour responses to Escitalopram. Female fish exposed to 1.50 µg L−1 Escitalopram had a lower maximum swimming velocity, stopped less often and exhibited increased boldness (reduced thigmotaxis) compared to controls. Male fish exposed to 1.50 µg L−1 had a lower maximum swimming velocity compared to control fish. At the end of exposures, both length and weight of the females exposed to 1.50 µg L−1 Escitalopram were significantly less than the group of control fish. In addition, males exposed to 1.50 µg L−1 Escitalopram were significantly shorter than control fish. The behaviour, weight and body length of the fish exposed to nominally 0.15 µg L−1 was not significantly different from control fish in either sex. The results of this study demonstrate that Escitalopram can affect subtle but ecologically important aspects of fish behaviour and lends further credibility to the assumption that Escitalopram is an environmentally active pharmaceutical.

Sewage treatment plant effluents contain a complex mixture of pharmaceuticals, personal care products and industrial chemicals, thus exposing aquatic organisms. Still, the consequences of exposure to combinations of different classes of drugs is largely unknown. In this study, we expose adult zebrafish (Danio rerio) males and females to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-ethinyl estradiol (EE₂) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyse three non-reproductive behaviours of importance for population fitness.

Two weeks exposure to 0.1 and 0.5 ng/LEE₂ resulted in increased anxiety in males in the scototaxis (light/dark preference) test. Significantly longer latency periods before entering the white zone and fewer visits in the white zone were observed in males exposed to both 0.1 and 0.5 ng/LEE₂ compared to unexposed males. No significant effects of citalopram alone (0.1 and 0.5 µg/L) were observed in the scototaxis test. The combined exposures (0.1 ng/L EE₂ + 0.1 µg/L citalopram and 0.5 ng/L EE₂ + 0.5 µg/L citalopram) resulted in abolishment of the anxiogenic effects of EE₂, with significantly shorter latency period (low dose) and more transitions to white (high and low dose) than in fish exposed to EE₂ alone. No significant effects of either EE₂, citalopramor the combination of the two were observed in females. In the novel tank test, significantly more transitions to the upper half of the tank were observed in males exposed to 0.1 µg/L citalopram alone compared to unexposed males while males exposed to 0.1 ng/lEE₂ had significantly shorter latency period to enter the upper half. Exposure to the combination of the two low concentrations did, however, result in a significantly longer latency and fewer transitions to upper half compared to both control, EE₂- and citalopram-exposed males. These males also spent significantly less time in the upper half than the fish exposed to 0.1 ng/l EE₂ or 0.1 µg/l citalopram alone. No significant effects on novel tank behaviour were observed in females or males exposed to the higher concentrations. In the shoaling test, males exposed to 0.1 µg/L citalopram and females exposed to 0.5 ng/l EE₂ made significantly fewer transitions away from peers while males exposed to 0.1 µg/L citalopram + 0.1 ng/l EE₂ performed significantly more transitions than the fish exposed to 0.1 µg/L citalopram alone.

In conclusion, this study shows that very low concentrations ofEE_2, at or slightly above the predicted noeffect concentration (NOEC), affects anxiety in zebrafish males. Furthermore, citalopram, in spite of marginal effect of its own at such low levels, counteracts the response to EE₂. This study represents an initial effort to understand the effects on water-living organisms of the cocktails of anthropogenic substances contaminating aquatic environments.

Selective serotonin re-uptake inhibitors (SSRIs) are a class of anxiolytic and anti-depressant drugs. SSRIs act on the evolutionarily ancient serotonergic system which is virtually identical throughout the vertebrate phylum. Serotonin is involved in a wide range of processes ranging from neuronal and craniofacial embryonic development to regulation of behaviour. However, SSRIs are also emerging pollutants, mainly entering the environment via sewage treatment plants. Since the serotonergic system is virtually identical in humans and other animals, exposed animals will be affected in similar ways as humans and suspicions are rising that ecologically important behaviours may be affected in subtle ways. Using the three-spined stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio) as model organisms, this thesis focuses on the behavioural effects of SSRIs in fish. The SSRI used throughout this thesis is citalopram, which has been found in fish in coastal areas of the Baltic Sea and other parts of the world.

Effects on behaviour were investigated using several different tests measuring stress response, feeding behaviour, aggression and locomotor activity. Anxiolytic effects of 0.1 μg/l, 1.5 μg/l 15 μg/l were investigated as well as effects of 0.15 μg/l and 1.5 μg/l on feeding behaviour. Because serotonin is involved in the development of the nervous system, the effects of developmental exposure to 1.5 μg/l was studied after 100 days of remediation. Finally, because SSRIs rarely occur alone in natural waters, the effects on zebrafish of citalopram in a cocktail scenario, with the anxiogenic compound 17α-ethinyl estradiol (EE₂ ) was also investigated. Citalopram was found to have anxiolytic effects on the three-spined stickleback at 0.1 μg/l, 1.5 μg/l and 15 μg/l.

Citalopram also suppressed feeding behaviour within a week of exposure and at concentrations as low as 0.15 μg/l. Developmental exposure to 1.5 μg/l for 30 days was found to increase aggression and feeding behaviour and to reduce locomotor activity. The changes were persistent and remained in adult fish. In the cocktail scenario, citalopram in single-substance exposure had anxiolytic effects on one parameter in the novel tank test at 0.1 μg/l. Citalopram enhanced the anxiogenic effects of EE₂ in the novel tank test, but in the scototaxis test citalopram appeared to counteract the effects of EE₂. It is concluded that citalopram has the potential to affect behaviour in fish at concentrations that have been found in close proximity of sewage treatment plants.

Det serotonerga systemet är i stort sett identiskt hos människor och övriga vertebrater. Serotonin är inblandat i ett stort antal kroppsliga funktioner, bland annat stressreaktioner, reglering av födobeteende och aggression. Vidare är serotonin med och reglerar nervsystemets tillväxt under embryonalutvecklingen. Selektiva serotoninåterupptagshämmare (SSRI) är en grupp antidepressiva och lugnande läkemedel vars användning har ökat snabbt på senare år då de är effektiva och har få allvarliga bieffekter. SSRI verkar på det serotonerga systemet, genom att blockera återupptaget av serotonin i den presynaptiska nervänden. SSRI har tilldragit sig en viss uppmärksamhet som potentiella miljöhot då de visats kunna påverka ekologiskt relevanta beteenden hos fisk och andra akvatiska organismer vid relativt låga koncentrationer i miljön samtidigt som de bryts ned dåligt i avloppsreningsverk. Avhandlingen fokuserar på ekologiskt relevanta beteendeeffekter av SSRI på fisk, med storspigg (Gasterosteus aculeatus) och zebrafisk (Danio rerio) som modellorganismer. Citalopram har använts som försökssubstans då det anses vara den SSRI som har minst antal sidoeffekter på till exempel det dopaminerga systemet. Citalopram förekommer i utloppsvatten från reningsverk i alla industrialiserade länder och har även hittats i abborre i Östersjön.

Effekter av exponering för SSRI har påvisats med hjälp av olika beteendetest. Skototaxi-test och novel tank diving test mäter stressresponsen genom att kvantifiera preferensen för närhet till botten och mörka omgivningar. Ätbeteende har mätts som antal utfall mot en matbit under en given tidsperiod och aggression har mätts genom att räkna antal bett mot en spegel. Anxiolytiska effekter undersöktes vid koncentrationer på 0,1 µg/l, 15 µg/l och 1,5 µg/l. Effekter på ätbeteende undersöktes vid 0,15 µg/l och 1,5 µg/l. Eftersom serotonin är inblandat i embryonalutvecklingen testades de beteendemässiga effekterna av exponering för 1,5 µg/l under utvecklingen. Då citalopram sällan förekommer ensamt i miljön testades ett cocktailscenario där zebrafisk samtidigt exponerades för citalopram och den anxiogena substansen 17α-etinylestradiol (EE₂).

Citalopram befanns ha anxiolytisk verkan på storspigg samt undertrycka ätbeteendet. Effekter på ätbeteendet uppstod inom en vecka efter exponering och vid den minsta testade dosen vilken var 0,15 µg/l. Storspigg som exponerats under embryonalutvecklingen var mer aggressiva, hade lägre lokomotoraktivitetoch gjorde fler utfall mot mat då de testades 100 dagar efter att exponeringen avslutats. Samtidigt exponering för citalopram och den anxiogena substansen 17α-etinylestradiol (EE₂) gav tvetydiga resultat. Citalopram ensamt hade ingen signifikant påverkan på beteendet i detta försök. I skototaxitestet motverkade citalopram den anxiogena effekten av EE₂ medan det förstärkte den anxiogena effekten i novel tank. Sammanfattningsvis har citalopram effekter på ekologiskt relevanta beteenden hos fisk i koncentrationer som förekommer i ytvatten. Det har också permanenta effekter på beteende om exponeringen sker under embryonalutvecklingen. Dessa resultat gör det sannolikt att citalopram och andra SSRI har ekologiska effekter i påverkade vattendrag.

Citalopram is an antidepressant drug, which acts by inhibiting the re-uptake of serotonin from the synaptic cleft into the pre-synaptic nerve ending. It is one of the most common drugs used in treatment of depression, it is highly lipophilic and frequently found in sewage treatment plant effluents and surface waters around the world. Citalopram and other selective serotonin re-uptake inhibitors have, at concentrations that occur in nature, been shown to have behavioural as well as physiological effects on fish and other animals. This study is the result of several different experiments, intended to analyse different aspects of behavioural effects of chronic citalopram exposure in fish. Our model species the three-spine stickleback is common in the entire northern hemisphere and is considered to be a good environmental sentinel species. Female three-spine sticklebacks were exposed to 0, 1.5 and 15μg/l nominal concentrations of citalopram for 21 days and subjected to the novel tank (NT) diving test. In the NT test, the fish exposed to 1.5μg/l, but not the 15μg/l fish made a significantly higher number of transitions to the upper half and stayed there for significantly longer time than the fish exposed to 0μg/l. The 15μg/l group, however, displayed a significantly lower number of freeze bouts and a shorter total freezing time. The test for locomotor activity included in the NT test showed that fish treated with 1.5 and 15μg/l displayed a significantly higher swimming activity than control fish both 5-7 and 15-17min after the start of the experiment. In the next experiment we compared fish exposed to 1.5μg/l and 0.15μg/l to pure water controls with regard to shoaling intensity and found no effect of treatment. In the final experiment the propensity of fish treated with 1.5μg/l to approach an unknown object and aggressive behaviour was investigated using the Novel Object test and a mirror test, respectively. The exposed fish ventured close to the unknown object significantly more often and stayed there for significantly longer time than unexposed fish. The aggression test yielded no statistically significant effects. It is concluded that citalopram changes the behaviour of the three-spine stickleback in a way that is likely to have ecological consequences and that it must not be considered an environmentally safe pharmaceutical.

Selective Serotonin Re-uptake Inhibitors (SSRI) are mood-altering, psychotropic drugs commonly used in the treatment of depression and other psychological illnesses. Many of them are poorly degraded in sewage treatment plants and enter the environment unaltered. In laboratory studies, they have been demonstrated to affect a wide range of behaviours in aquatic organisms. In this study we investigated the effect of a three-week exposure to 0.15 and 1.5 μg/l of the SSRI citalopram dissolved in the ambient water on the feeding behaviour in three-spine stickleback (Gasterosteus aculeatus). Feeding, measured as the number of attacks performed on a piece of frozen bloodworms during a 10-min period, was reduced by 30–40% in fish exposed to both 0.15 and 1.5 μg/l citalopram. The effects of the environmentally relevant concentration 0.15 μg/l on feeding, an important fitness characteristic, suggests that the ecological significance of environmental SSRI exposure may be pronounced.