ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2019

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1614

Effect of elevated pCO2 andenvironmental oxazepam on thebehavior and physiology of teleostfish

LAURA E. VOSSEN

ISSN 1651-6206ISBN 978-91-513-0807-4urn:nbn:se:uu:diva-395993

Dissertation presented at Uppsala University to be publicly examined in Room B21,Biomedical Center, Husargatan 3, Uppsala, Friday, 13 December 2019 at 09:15 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin English. Faculty examiner: Professor Robert Gerlai (University of Toronto).

AbstractVossen, L. E. 2019. Effect of elevated pCO2 and environmental oxazepam on the behaviorand physiology of teleost fish. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1614. 63 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0807-4.

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior andphysiology of teleost fish: i) elevated concentrations of carbon dioxide (CO2) in the context ofocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in thecontext of pharmaceutical pollution.

Anthropogenic emissions of CO2 are lowering the pH of the oceans. Studies on coral reeffish exposed to CO2 concentrations projected for the year 2100 (~1000 μatm) reported alarmingbehavioral effects, of which attraction to predator odor was the most surprising. To explain thisbehavioral reversal, it was hypothesized that ion-regulatory adjustments to compensate for thedecrease in blood pH would result in altered transmembrane gradients of chloride ions, renderingthe major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), excitatory.We investigated whether zebrafish (Danio rerio), an often used model species, showed similarbehavioral disruptions in elevated CO2. Zebrafish behavior was however largely unaffected byan approximately month long exposure to ~1600 μatm CO2. We continued by investigatingthe reproductive, anxiety-related behavior and aggression in another model species, the three-spined stickleback (Gasterosteus aculeatus). However, also stickleback behavior and responsesto social subordination were not affected by CO2, in contrast to earlier findings. We concludedthat CO2 had no major effect on the behavior of zebrafish and three-spined stickleback.

In the second part of this thesis, I investigated behavioral effects of oxazepam, an anxiolyticpharmaceutical (benzodiazepine) acting on the GABA system. Studies on perch (Percafluviatilis) have shown that exposure to dilute concentrations of oxazepam (1.8 μg L-1), closeto those found outside the municipal sewage treatment plant in Uppsala (0.58 μg L-1), canincrease activity, decrease sociality and increase feeding rates. I show that similar oxazepamconcentrations can also affect zebrafish. Moreover, I show that females are more sensitive tooxazepam showing reduced anti-predator responses at 0.57 μg L-1 while in males this effect wasobserved first at 60 μg L-1. Furthermore, and in contrast to wild-caught zebrafish, laboratoryzebrafish did not show any effect of the oxazepam exposure. This finding has implications forthe use of laboratory zebrafish in ecotoxicological and pharmacological studies, as results mightnot translate to wild fish. Finally, I show that zebrafish can develop tolerance to the anxiolyticeffects of oxazepam during chronic (28 days) exposure. This is an important discovery thatcould mitigate the effects of this form of pharmaceutical pollution on wild fish.

Keywords: fish, GABA, CO2, ocean acidification, pharmaceutical pollution, oxazepam

Laura E. Vossen, Department of Neuroscience, Box 593, Uppsala University, SE-75124Uppsala, Sweden.

© Laura E. Vossen 2019

ISSN 1651-6206ISBN 978-91-513-0807-4urn:nbn:se:uu:diva-395993 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-395993)

“False facts are highly injurious to the progress of science, for they often long endure; but false views, if supported by some evidence, do little harm, as every one takes a salutary pleasure in proving their falseness; and when this is done, one path towards error is closed and the road to truth is often at the same time opened.” Charles Darwin, 1871. In The Descent of Man, and Selec-tion in Relation to Sex, Vol. II, Chapter XXI.

To Rikard, Kerstin and ‘Lasse’

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Vossen, L.E., Jutfelt, F., Cocco, A., Thörnqvist, P.O., Winberg, S. (2016) Zebrafish (Danio rerio) behaviour is largely unaffected by elevated pCO2. Conservation Physiology, 4(1):1-8

II Sundin, S., Vossen, L.E., Nilsson-Sköld, H., Jutfelt, F. (2017) No effect of elevated carbon dioxide on reproductive behaviors in the three-spined stickleback. Behavioral Ecology, 28(6):1482-1491

III Vossen, L.E., Sundin, S., Rudin, J., Thörnqvist, P.O., Birnir, B., Jut-felt, F., Winberg, S. (2019) No evidence that exposure to elevated pCO2 affects behaviour or monoamine neurotransmitters of three-spined stickleback. Manuscript

IV Vossen, L.E., Červený, D., Sen Sarma, O., Thörnqvist, P.O., Jutfelt, F., Fick, J., Brodin, T., Winberg, S. (2019) Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish. Science of the Total Environ-ment, in press

V Vossen, L.E., Červený, D., Österkrans, M., Thörnqvist, P.O., Jutfelt, F., Fick, J., Brodin, T., Winberg, S. (2019) Chronic exposure to oxa-zepam pollution produces tolerance to anxiolytic effects in zebrafish (Danio rerio). Submitted

Reprints were made with permission from the respective publishers.

Papers not included in this thesis

Cocco, A., Rönnberg, A.M.C., Jin, Z., André, G.I., Vossen, L.E., Bhandage, A.K., Thörnqvist, P.O., Birnir, B. and Winberg, S. (2017) Characterization of the γ-aminobutyric acid signaling system by reverse transcription-quantitative polymerase chain reaction. Neuroscience, 343:300-321

Contents

Introduction ................................................................................................... 15Ocean acidification ................................................................................... 15

Previous research on acidification ....................................................... 15Effect of near-future pCO2 on fish ....................................................... 16Acid-base regulation ............................................................................ 18The GABA hypothesis ......................................................................... 20Applying organized skepticism to OA research .................................. 23

Pharmaceutical pollution .......................................................................... 25Prioritizing pharmaceuticals ................................................................ 25Psychoactive pharmaceuticals ............................................................. 26Oxazepam and the benzodiazepines .................................................... 28Effect of benzodiazepines on behavior ................................................ 29Effect of benzodiazepines on physiology ............................................ 31Mitigation ............................................................................................. 34

Aims of the thesis .......................................................................................... 35

Materials and methods .................................................................................. 36Study species ............................................................................................ 36

Zebrafish .............................................................................................. 36Three-spined stickleback ..................................................................... 37

Behavioral tests ......................................................................................... 38Open field test (OFT) ........................................................................... 38Light/ dark test (LDT) .......................................................................... 38Novel tank diving test (NTDT) ............................................................ 39Detour test ............................................................................................ 39

Marking individuals with elastomer tags .................................................. 40Microdissection of fish brain tissue .......................................................... 40Physiological measurements ..................................................................... 40

RT-qPCR .............................................................................................. 40HPLC with electrochemical detection ................................................. 41Radio-immunoassay (RIA) .................................................................. 41

Results ........................................................................................................... 43Paper I ....................................................................................................... 43Paper II ..................................................................................................... 43Paper III .................................................................................................... 43Paper IV .................................................................................................... 44Paper V ..................................................................................................... 44

Discussion ..................................................................................................... 46

Acknowledgements ....................................................................................... 49

References ..................................................................................................... 52

Abbreviations

ACTH adrenocorticotropic hormone CA carbonic anhydrase CO2 carbon dioxide CRH corticotropin releasing hormone DA dopamine DCC dextran coated charcoal DHBA 3,4-dihydroxybenzylamine hydrobromide DOPAC 3,4-dihydroxyphenylacetic acid DRN dorsal raphe nuclei EC50 half maximal effective concentration EE2 17α-ethinylestradiol GABA γ-amino butyric acid IP intraperitoneal HCl hydrochloric acid HCO3

- bicarbonate ion HPA axis hypothalamic pituitary adrenocortical axis HPI axis hypothalamic pituitary interrenal axis HPLC-EC high performance liquid chromatography with

electrochemical detection H+ hydrogen ion LDT light dark test LOEC lowest observable effect concentration LC50 median lethal dose MAO monoamine oxidase MRC mitochondria rich cell Na+ sodium ion NE norepinephrine NOx nitrogen oxides, i.e. nitric oxide (NO) and nitrogen diox-

ide (NO2) NTDT novel tank diving test OA ocean acidification OECD organisation for economic cooperation and development OFT open field test O2 oxygen

PBS phosphate-buffered saline solution PNA- MRC peanut lectin agglutinin negative mitochondria-rich cell POA preoptic area PVC pavement cell pCO2 partial pressure of CO2 pHe extracellular pH pHi intracellular pH SAM sympathetic adrenomedullary system SNRI serotonin norepinephrine reuptake inhibitor SO2 sulfur oxide SSRI selective serotonin reuptake inhibitor VMAT vesicular monoamine transporter WWTP waste-water treatment plant 5-HIAA 5-hydroxyindoleacetic acid 5-HT serotonin

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Introduction

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior and physiology of teleost fish: i) elevated concentrations of the greenhouse gas carbon dioxide (CO2) in the context of ocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in the context of pharmaceutical pollution. Both CO2 and oxazepam have been hypothesized to influence the GABA system of fish, albeit in different ways. In this general introduction, I will describe the two related research fields of ocean acidification and pharmaceutical pollution, and discuss the research articles linking CO2 and oxazepam exposure to behavioral and physiological alterations in fish.

Ocean acidification Human CO2 emissions are acidifying the world’s oceans. Over the past 800 000 years, the atmospheric partial pressure of CO2 (pCO2) had been in the range of 172-300 ppm (parts per million) (Zeebe and Ridgwell, 2011). As a result of human CO2 emissions, atmospheric pCO2 has now exceeded 400 ppm (US Department of Commerce, 2017). Ocean pH has decreased by 0.1 pH unit since the onset of the industrial revolution, and is expected to decline by another 0.6 units by the year 2300 (Zeebe and Ridgwell, 2011) (Figure 1). By the end of this century, atmospheric and ocean pCO2 is ex-pected to have reached approximately 1000 µatm in a ‘business as usual’ scenario (Orr, 2005).

Previous research on acidification Acidification in fish was studied in the 1960’s to 1980’s in the context of acidification of lakes and streams by deposition of SO2, NOx and HCl in western Europe (Morris et al., 1989). Much of our knowledge on acid-base regulation in fish stems from this research field, but the case of elevated CO2 nevertheless differs in three important aspects: i) effects of elevated CO2 have been almost exclusively studied in marine teleost species, that are con-sidered to be more sensitive to pH changes because oceans are less variable in pH (Ishimatsu et al., 2005); ii) in lake acidification studies, fish were ex-posed to much lower pH compared to the relatively small differences in pH

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in studies manipulating pCO2 (Morris et al., 1989); iii) OA studies on fish have focused on behavioral alterations, while lake acidification studies have paid more attention to plasma ion composition.

In the 1990’s, aquaculture research paid some attention to the problem of elevated pCO2 in aquaculture settings with high fish density, but these stud-ies used much higher CO2 concentrations (15 000 – 55 000 µatm; (Fivelstad et al., 2003)), while pCO2 due to anthropogenic emission is not expected to increase above 2 000 µatm, due to depletion of fossil fuels (Gattuso and Hansson, 2011). Stunning fish with water saturated with CO2 (typically to a pH of 5.0) is furthermore a common euthanasia method in aquaculture. At these concentrations fish display ‘aversive reactions’ including attempting to escape the tank and uninterrupted circular swimming behaviors (Gräns et al., 2015; Marx et al., 1997; Seth et al., 2013). The time it takes for fish to loose equilibrium seems to differ between species, in Arctic charr this takes 2-4 minutes (Gräns et al., 2015), while in eels it took on average 109 minutes until swimming movements ceased (Marx et al., 1997).

Figure 1. Atmospheric pCO2 (in ppm) at Mauna Loa Observatory and seawater pCO2 (in µatm) at Aloha station have increased, while seawater pH at Aloha station has decreased since monitoring started. Seasonal fluctuations in atmospheric pCO2 can be observed. The location of the research stations in the Pacific Ocean north of Hawaii is indicated in the map in the upper left of the graph. From (Feely, 2008). Reprinted with permission from the publisher.

Effect of near-future pCO2 on fish The first study reporting behavioral disruptions after exposure to near-future pCO2 was published in 2009 by Philip Munday and his research team

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(Munday et al., 2009). This study reported that larval orange clownfish (Am-phiprion percula) became attracted to the pungent smell of a swamp tree (Melaleuca nervosa), a smell the control exposed group avoided, and the group exposed to lower pH (pH 7.8 instead of 8.15) was furthermore indif-ferent to the smell of parents or non-parents, whereas control individuals preferred the smell of non-parents (Munday et al., 2009). A subsequent study reported that CO2 exposed settlement-stage larvae (day 11) spent >99% of their time in predator odor, when given the choice between seawater and predator odor of two different predator species in an Atema choice flume (Dixson et al., 2010). Soon thereafter, Göran Nilsson from Oslo University proposed a neural mechanism explaining these paradoxical effects of CO2 on behavior (Nilsson et al., 2012), namely that changes in ion concentrations associated with the physiological response to the initial acidosis render the major inhibitory neurotransmitter in vertebrate brain, γ-aminobutyric acid (GABA), excitatory. In the following sections I will explain this “GABA hypothesis”, as well as the experimental support for it.

Figure 2. The number of research articles per year in the field of ocean acidification as a whole (height of the red bars) and the number of specifically on fish (in blue). Estimates were obtained by searching the Web of Science Core Collection for re-search articles, reviews and book chapters specifying the topic as [“ocean acidifica-tion”] or [“ocean acidification” AND “fish OR teleost”] on 23 August 2019.

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OA and Fish

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Acid-base regulation Acid-base balance is tightly regulated in fish and other vertebrates, and is highly efficient (Evans et al., 2005). While terrestrial air breathers increase ventilation in response to an increase in pCO2, water breathers already have very low arterial pCO2 because CO2 easily dissolves in water. Instead, fish primarily rely on physicochemical buffering and direct transfer of acid-base equivalents between the animal and the external environment to compensate for pH disturbances (Perry and Gilmour, 2006). (Hyperventilation may con-tribute to some extend, but is limited by the ‘osmoregulatory compromise’, i.e. too much ventilation would adversely increase the capacity for ion and water movement (Ern and Esbaugh, 2016; Esbaugh, 2018; Esbaugh et al., 2016; Perry and Gilmour, 2006).

Upon exposure of teleost fish to elevated pCO2, respiratory acidosis oc-curs within minutes, due to equilibration of arterial pCO2 with water pCO2, resulting in a state known as hypercapnia or hypercarbia. Extracellular pH (pHe) initially decreases as a function of the pCO2 and the intrinsic buffer value of the blood (Brauner and Baker, 2009; Melzner et al., 2009). Follow-ing respiratory acidosis (acidification of the blood), plasma pH recovers within 24-96 hours (Brauner and Baker, 2009), through active accumulation of bicarbonate (HCO3

-) in the extracellular space, which is matched by an equimolar decrease in chloride (Cl-) concentration. This is thought to be mediated by a Cl-/HCO3

- anion exchanger (belonging to the SLC26 or SLC4 gene family) located in the apical membrane of gill epithelial cells, so-called mitochondria rich cells (MRCs; more specifically PNA- MRCs), also termed ionocytes or chloride cells (Perry and Gilmour, 2006) (Figure 3). Simultane-ously, H+ is excreted and Na+ taken up, either by an apical membrane elec-troneutral Na+/H+ exchanger (NHE), and/ or via a V-type H+-ATPase cou-pled energetically to apical membrane Na+ channels (ENaCs) (Perry and Gilmour, 2006). The zinc metalloenzyme carbonic anhydrase (CA), present in the cytosol of gill cells, mediates the production of the substrates H+ and HCO3

- by catalyzing the hydration of CO2 (Gilmour and Perry, 2009). At least two mechanisms are available to regulate the rates of branchial

acid-base equivalent fluxes. First, the transporters and exchangers can be transcriptionally regulated during acid-base disturbances (Perry and Gilmour, 2006). Second, the surface area of the apical membranes of the MRCs that is exposed to the water can be adjusted by physical covering by adjacent pavement cells (PVCs), a process known as gill remodeling (Perry and Gilmour, 2006). Studies investigating transcriptional regulation of trans-porters and exchangers under OA through analyses of gene expression, en-zyme activity and protein abundance have reported mixed results, which suggests that marine fish maintain sufficient abundance of acid excretion proteins in their MRCs to cope with near-future pCO2 concentrations

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(Esbaugh, 2018). Gill remodeling has to my best knowledge, not been stud-ied under OA exposures.

Studies measuring blood CO2 chemistry following OA relevant CO2 con-centrations have indeed reported increases in plasma HCO3

- concentrations in response to ~1000 µatm CO2 for 24 hours or 14 days (Esbaugh et al., 2016, 2012). In the Antarctic fish Notothenia rossii, plasma [HCO3

-] was increased even after a 29-36 day acclimation to 1900 µatm CO2 (Strobel et al., 2012). Only one study, on spiny damselfish (Acanthochromis polyacan-thus), measured [HCO3

-] in brain tissue. It reported a significant increase in brain [HCO3

-] after a 4 day acclimation to 1900 µatm CO2 (Heuer et al., 2016). Plasma [Cl-] has only been measured in one study after 24 hours, 72 hours and 14 days of acclimation to ~1000 µatm, but was not reduced com-pared to controls, which could be due to the difficulty of detecting a 2mM change against a 160-170 mM background (Esbaugh et al., 2016).

Figure 3. A schematic representation of the model for acid-base regulation in the rainbow trout gill, as proposed by Perry and Gilmour (2006). The PNA+ mitochon-dria rich cells (MRCs) act as base-excreting cells by possessing apical membrane Cl− /HCO3

− exchangers and basolateral V-type H+-ATPase. The PNA− MRCs func-tion as acid-excreting cells by possessing apical membrane Na+/H+ exchangers and/ or Na+ channels linked to a V-type H+-ATPase, coupled with basolateral Cl−

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/HCO3− exchangers or Na+/HCO3

− co-transporters. Transporters known to be tran-scriptionally activated by acid–base disturbances are indicated. In addition a scheme for regulating the rate of Cl−/HCO3

− exchange by PNA+ MRCs is presented whereby adjacent pavements cells (PVCs) can expand or retract to alter the surface of the apical membrane exposed to the water. Similarly, experimental evidence ex-ists to support the occurrence of changes in the exposed apical surface area (SA) of the PNA− MRC cell type under acidotic conditions. Energy-consuming transporters are indicated by filled circles. CAc refers to cytosolic carbonic anhydrase. Note that whereas the figure depicts one cell of each type, the three cell types differ in abun-dance in the fish gill, with PVCs accounting for ∼80% of all cells in the branchial epithelium, and PNA+ and PNA− MRCs each accounting for ∼10% of all cells. Re-printed with permission from the publisher.

The GABA hypothesis The observation that orange clownfish became attracted to predator odor (Dixson et al., 2010) and hence behaved in an opposite way, sparked the idea that this could be mediated by a reversal in the function of the underlying neurotransmitters (Göran Nilsson, personal communication). GABA, being the major inhibitory neurotransmitter in vertebrate brain, was a likely candi-date. The ‘break on behavior’ had become a ‘gas pedal’. Nilsson had studied the GABA system of fish before, finding a role for this transmitter in hypox-ia tolerance and metabolic depression (Nilson and Lutz, 1993). Whether a reduced or even reversed function of GABA would be expected to give rise to an anxiolytic behavioral effect (such as reduced predator avoidance or even predator attraction), is an important question that I will address in the discussion section of this thesis. Here, I will first explain the proposed phys-iological mechanisms that constitute the hypothesis.

Nilsson et al. proposed that the increase in [HCO3-] and the equimolar de-

crease in [Cl-] associated with the physiological response to the initial acido-sis, would change the ion concentration gradients over the plasma membrane of neurons (Nilsson et al., 2012). Normally, extracellular [Cl-] is higher than intracellular [Cl-], such that Cl- ions flow into the cell when GABA binds to the GABAA receptor and the central ion pore of the receptor opens (Figure 4 upper left). The inflow of negative Cl- ions lowers the membrane potential, hyperpolarizing the neuron. Nilsson proposed that under OA, the decrease in extracellular [Cl-] leads to a reversal of the ion concentration gradient, such that intracellular [Cl-] is higher than extracellular [Cl-] and Cl- flows out of the cell when the GABAA receptor opens, resulting in excitation of the neu-ron (Figure 7 lower left).

By calculating the reversal potential of the GABAA receptor under control and 1900 µatm CO2, Heuer and Grosell deduced that this reversed ion flow is at least theoretically possible (Heuer and Grosell, 2014). There is however

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still a lack of experimental evidence for reversed Cl- flows, nor is there elec-trophysiological evidence that GABA application results in depolarization in neurons of CO2 exposed fish. Techniques are available for rodents but have not yet been tested on fish. For example, ion-selective self-referencing mi-croelectrodes can be made sensitive to Cl- (Luxardi et al., 2015; Reid and Zhao, 2011). The electrophysiological patch-clamp technique can be used to measure membrane potentials in response to application of GABA in neu-ronal slices from fish that were acclimatized to elevated pCO2.

Support for the GABA hypothesis comes from two lines of evidence: i) treatment with the GABAA receptor antagonist gabazine (and agonist mus-cimol) and ii) gene expression studies on components of the GABA system under control and elevated pCO2. Gabazine is a selective GABAA receptor antagonist, meaning that it binds to the GABA recognition site on the GABAA receptor, preventing GABA from binding and therefore keeping the receptor in a closed state (Figure 4 upper middle). Under control pCO2, gab-azine has anxiogenic properties and can lead to seizures in the animal be-cause it reduces synaptic inhibition by GABA (Figure 4 upper middle). Un-der elevated pCO2, gabazine prevents the reversed flow of Cl- ions and hence restores behavior to normal (Figure 4 lower middle). Two studies have fur-thermore applied the selective GABAA receptor agonist muscimol, (Hamilton et al., 2014) and Paper III. Muscimol also binds to the GABA binding site but opens the central pore in the GABAA receptor, hence it has sedative and anxiolytic effects on behavior under control pCO2 (Figure 4 upper right). Under elevated pCO2, muscimol would be expected to lead to an even stronger reversed flux of Cl-, hence CO2-induced behavioral effects would be expected to be enlarged (Figure 4 lower right).

Most studies concluded that gabazine treatment restored CO2 induced be-havioral disruptions, including impaired learning, retinal function, behavior-al lateralization, responses to predator odor and activity levels (Chivers et al., 2014; Chung et al., 2014; Lai et al., 2015; Lopes et al., 2016; Munday et al., 2016; Nilsson et al., 2012; Regan et al., 2016). Only one study concluded otherwise, finding a CO2-induced increase in scototaxis (dark preference), that was not restored by gabazine treatment (Hamilton et al., 2014). The dose used in most studies was based on a rodent study (Nelson et al., 2002), namely a 30-minute immersion treatment with a 4 mg L-1 gabazine concen-tration (Chivers et al., 2014; Chung et al., 2014; Hamilton et al., 2014; Lai et al., 2015; Nilsson et al., 2012; Ou et al., 2015), with one exception that used a 4 mg L-1 followed by a 8 mg L-1 gabazine concentration (Regan et al., 2016). Paper III provides dose-response data for gabazine and muscimol in three-spined stickleback.

It has been noted by several authors that in these OA studies, gabazine of-ten had no effect on animals exposed to control pCO2 (Esbaugh, 2018; Tresguerres and Hamilton, 2017). For example, larval clownfish spent equal amounts of time in predator cue when treated with gabazine (Nilsson et al.,

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2012), while gabazine treatment would be expected to reduce the time in predator cue. Gabazine treatment also did not affect the number of lines crossed (a measure for activity) (Chivers et al., 2014; Regan et al., 2016), while this drug would be expected to alter activity levels, although depend-ing on species and context, both increased and decreased activity may be expected. Yet another study reports that muscimol treatment did not affect the time spent close to a novel object (Hamilton et al., 2014), while this an-xiolytic agent would be expected to increase the time spent close to the novel object. One study did not include data on the control exposed fish at all (Chung et al., 2014).

Figure 4. A graphical representation of the GABA hypothesis (Hamilton et al., 2014). Under control pCO2 (upper left), Cl- ions flow into the neuron upon GABA binding. Under elevated pCO2 (lower left), extracellular Cl- concentrations are reduced due to compensatory changes in acid-base regulation, which results in outflow of Cl- upon GABA binding. Gabazine (middle panels) binds to the GABA binding site, preventing GABA from opening the receptor, hence the neuron will not be inhibited under control pCO2 (leading to its anxiogenic effects; upper middle), but under elevated pCO2 (lower middle) gabazine will prevent the reversed flow of Cl- hence restore to normal. Muscimol (right panels) has a higher potency than GABA and will increase the inward flow of Cl- under control pCO2 (upper middle) leading to sedation of the animal, while it can aggravate the behavioral effects of exposure to elevated pCO2 by increasing the reversed flow of Cl- ions out of the neuron. Reprinted with permission from the publisher.

Two gene expression studies have been carried out to evaluate the effect of CO2 exposure on the GABA system. One study found an upregulation in the

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GABAA receptor subunit genes α12, α3, α4 and α6b in CO2 exposed three-spined stickleback (Gasterosteus aculeatus), while no changes were detected in the expression of genes related to GABAA receptor turnover, GABA me-tabolism or ion transport (Lai et al., 2016). In contrast, a transgenerational study on spiny damselfish (Acanthocrhomis polyacanthus) found no support for differential expression of GABAA receptor subunits between CO2 treat-ments (Schunter et al., 2016). Instead this study detected an overexpression of Al9a1, a protein involved in an alternative GABA synthesis pathway, in offspring of CO2-tolerant parents.

Tresguerres and Hamilton (2017) raised the important point that changes in [Cl-] and [HCO3

-] in brain intra- and extracellular fluids would also affect the function of other neurobiological receptors and processes, including gly-cine receptors, certain K+ channels, CO2-sensing peripheral neurons, in addi-tion to influencing neuronal network dynamics (Tresguerres and Hamilton, 2017). The field of OA has mainly focused on the role of GABA signaling while other processes have not received as much attention.

Applying organized skepticism to OA research The broader research field of OA has received criticism for claiming that OA presents an ocean calamity, i.e. perpetuating a world view in which a host of ecological syndromes resulting from human-driven pressures is leading to the collapse of oceanic ecosystems (Duarte et al., 2014). Assuming that any human pressure will have a negative effect on the ocean is a form of ‘white hat bias’, “a bias leading to distortion of information in the service of what may be perceived to be righteous ends” (Cope and Allison, 2009; Hendriks and Duarte, 2010). Also confirmation bias is a well-established bias present throughout the scientific literature, i.e. “the human tendency to, in cases of unequivocal or weak support, interpret information in a way that confirms one’s preexisting beliefs or hypotheses” (Duarte et al., 2014). These biases can distort information in a way that leads resources away from ‘real threats’ and towards ‘perceived threats’ to the ocean.

A way out of these biases is to apply organized skepticism to OA research (Browman, 2016; Duarte et al., 2014). In two special issues on OA in ICES Journal of Marine Science in 2016 and 2017, Browman (2016) raises a num-ber of important issues specific to OA research. First, short-term CO2 ma-nipulation experiments need to be more aware of the ‘inferential leap’ that a short-term challenge experiment can adequately mimic the effect of a long-term driver. Second, spatio-temporal variation in pCO2 has been documented extensively for natural habitats and needs to be incorporated in experiments. Third, inter-individual differences in tolerance in CO2 challenge experiments should be investigated rather than ignored. Forth, the acclimation, epigenet-ics and evolution of CO2 tolerance should be studied.

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In addition to these more general considerations, a number of methodo-logical concerns have been raised, some of which are specific to CO2 expo-sure experiments conducted on fish. In early exposure experiments, pCO2 was calculated from pH and alkalinity rather than measured directly using a spectrophotometer probe, which is a more accurate method (Moran, 2014). In addition, some experimental designs suffer from pseudoreplication, where the same mixing tank (that mixes seawater with CO2 gas) feeds into several experimental tanks (where the fish are housed), with only one mixing tank per CO2 level (Cornwall and Hurd, 2016). Some experiments testing larval fish for odor preferences made use of so-called ‘Atema’ flumes, which con-tain no collimator (e.g. honey comb structure), a structure that is inserted upstream of the choice chamber to ensure laminar flow in the presence of moving fish (Jutfelt et al., 2017). Some studies measured behavioral activity by manually estimating distance moved on a patch of coral reef outside of the field station without mention of gridlines to guide this estimation, and recording two fish simultaneously: “(…) Five aspects of activity and behav-ior were estimated: total distance moved (cm) during the observation period, mean distance ventured (cm) from the reef, maximum distance ventured (cm), height above substratum (categorized as percent of the time spent within the bottom, middle, or top one-third of the patch), and boldness (…)”(Ferrari et al., 2011; Munday et al., 2010). Hence, there is a need to replicate these early studies with improved methodology.

One of the most commonly used behavioural tests in CO2 exposure stud-ies is the detour test, used to assess left or right turning preferences of fish. An individual fish is released in a T-shaped or double T-shaped chamber, and if needed, the animal is gently encouraged by the researcher to move through the runway and make left or right turns. Changes in population-level lateralisation behaviour have been interpreted as a CO2-induced disruption of cognitive function that might negatively influence fish survival (Domenici et al., 2012; Jutfelt et al., 2013; Lai et al., 2015). Reported effects have been in different directions and altered different aspects of the distribution of turning preferences. The first study on the topic reported a decrease in the proportion of individuals with a strong left or right bias (Domenici et al., 2012), while later studies found changes in the mean turning preference of the population towards a more right-biased turning preference (Lopes et al., 2016; Vossen et al., 2016; Welch et al., 2014) or more left-biased turning preference (Do-menici et al., 2014; Näslund et al., 2015). A recent study has raised questions regarding the within-individual repeatability of the detour test. Repeated testing of individually tagged animals revealed that repeatability across test-ing occasions was very low in all four teleost species included in the study (0.01>R>0.08), indicating that this test might not accurately reflect eye pref-erence (Roche et al., 2019).

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Pharmaceutical pollution Human and veterinary pharmaceuticals and their degradation products are leaking out into environmental matrices. Since many pharmaceuticals are only partially metabolized by the body (Huggett et al., 2003) and conven-tional sewage treatment does not include specific steps to remove pharma-ceuticals (Prasse et al., 2015), surface and coastal waters in Europe and the US typically contain pharmaceutical concentrations in the range of ng to µg L-1 (Batt et al., 2016; Björlenius et al., 2018; Hughes et al., 2012; Loos et al., 2009). Contaminated manure and sewage sludge may also reach the soil when it is used as a fertilizer on agricultural fields (Boxall, 2004; Jörgensen and Halling-Sörensen, 1999). In addition to these widespread emissions, pharmaceutical production sites, although localized to specific regions in the world, have been shown to discharge high concentrations of pharmaceuticals into the environment. Antibiotic concentrations in the treated effluent from one of the world’s largest ‘bulk drug’ production sites in Hyderabad, India, reached into the mg L-1 range (Fick et al., 2009), in some cases exceeding human therapeutic blood concentrations. This finding raised serious con-cerns about the development of antibiotic resistance in bacterial populations in receiving waters (Larsson, 2014), which has given the research field of pharmaceutical pollution considerable impetus (Brooks, 2018).

The concern about pharmaceutical pollution by wastewater treatment plants (WWTPs) gained broad public interest with the discovery that WWTP effluent is estrogenic to fish, inducing the synthesis of vitellogenin in male roach (Rutilus rutilus) (Sumpter and Jobling, 1995). Vitellogenin is a precur-sor protein of egg yolk normally only found in reproducing females, its ex-pression being under multihormonal control, with 17β-estradiol playing a dominant role. Subsequent histological examination of roach caught up-stream and downstream of WWTPs in the UK revealed a much larger pro-portion of intersex individuals (i.e. fish possessing both female and male gonadal characteristics) in downstream fish (Jobling et al., 1998). The au-thors pointed towards the dilute concentrations (in the order of tens of ng L-

1) of synthetic estradiol from birth control pills (17α-ethinylestradiol, EE2) that are present in WWTP effluent (Jobling et al., 1998; Sumpter and Jobling, 1995). A whole-lake experiment exposing fathead minnows (Pimephales promelas) to 5-6 ng L-1 EE2 for 7 years reported that the popu-lation nearly went extinct (Kidd et al., 2007), while in wild roach effective population sizes were not related to estrogen exposure (Hamilton et al., 2014).

Prioritizing pharmaceuticals With over 3000 active pharmaceutical ingredients on the market, several authors have advocated to focus research efforts on the drugs that pose the

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greatest risk to wildlife (Fick et al., 2010; Huggett et al., 2003; Johnson et al., 2017). Opinions differ regarding what particular rational strategy should be used to prioritize pharmaceuticals. Fick et al. (2010) used human thera-peutic concentrations to calculate the surface water concentration expected to induce a pharmacological effect in fish for 500 pharmaceuticals (Fick et al., 2010). The authors built upon a model developed in rainbow trout to predict the fish plasma bioconcentration factor from drug lipophilicity (Fitzsimmons et al., 2001) and then calculated the critical environmental concentration (CEC) as the ratio of the human therapeutic plasma concentra-tion divided by the predicted fish bioconcentration factor. There are several limitations of this approach; human and fish pharmacological effect concen-trations are assumed to be equal, the bioconcentration factors predicted by the model can be orders of magnitude wrong, drug metabolism and excretion is not taken into account, nor is bioaccumulation (i.e. trophic transfer) (Fick et al., 2010). Nevertheless this approach may be the ‘best of a bad job’ when experimental data on fish plasma (or tissue) concentrations is lacking. Ap-plying machine learning to the prediction of bioconcentration factors may also reduce estimation errors (Miller et al., 2019). Although surface water concentrations of the 500 investigated pharmaceuticals were always lower than the predicted CEC, environmental concentrations of diclofenac and ibuprofen (Loos et al., 2009) were less than a factor thousand removed from their predicted CEC, leaving only a narrow safety margin (Fick et al., 2010, p. 201). EE2 had a CEC of 0.37 ng L-1 (Fick et al., 2010) in line with reports of sublethal effects close to this concentration (Kidd et al., 2007), adding further validity to this model.

Johnson et al. (2017) systematically reviewed the scientific and grey liter-ature for established ecotoxicological effect concentrations (LOEC, EC50, LC50, lethal and sublethal toxicity) and compared these to river water con-centrations from the UK (Johnson et al., 2017). Sixty chemicals were inves-tigated including metals, pesticides, pharmaceuticals, surfactant, nanoparti-cles and other persistent organic pollutants. Risk ratio was calculated as the median river water concentration divided by the median of the effect concen-trations. This study concluded that most pharmaceuticals pose little risk compared to the other chemicals investigated, with the exception of EE2. Although the risk ratio can be criticized for paying too little attention to sub-lethal effects (which likely disappear when median concentrations are tak-en), the graphical presentation of environmental and ecotoxicity concentra-tions along a concentration axis can be a helpful tool to spot where the these overlap.

Psychoactive pharmaceuticals After an initial ‘transient obsession’ with EE2 (to paraphrase (Johnson et al., 2017)) other highly prescribed pharmaceuticals gained interest, in particular

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the selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine (Prozac) (Brooks, 2014; Brooks et al., 2003). Reproductive endpoints devel-oped in the context of EE2 appeared largely insensitive to chronic fluoxetine exposure (Foran et al., 2004) but see (Schultz et al., 2011; Weinberger II and Klaper, 2014). However several independent research teams have reported disruptions in anti-predator behaviors in chronic exposure (2-4 weeks) to fluoxetine. Larval fathead minnows (Pimephales promelas) exposed as em-bryos to fluoxetine (25, 125 or 250 µg L-1) showed reduced C-start escape behavior (Painter et al., 2009), and adults of the same species swam closer to a ‘mock predator’ (an image of a black circle on a white background) (Weinberger II and Klaper, 2014). Similarly, fluoxetine exposed (0.03 or 0.5 µg L-1) guppies (Poecilia reticulata) took longer time to escape a mock predator (this time represented by a fish shaped dark paper that was moved towards the tank), and this delay was more pronounced in individual vs. group tested fish (Pelli and Connaughton, 2015). Zebrafish (Danio rerio) daily injected with 10 mg kg-1 fluoxetine for two weeks (~ 4-6 µg) spent more time in the white compartment of a scototaxis arena (Maximino et al., 2011).

Effects reported after exposure to fluoxetine concentrations in ng L-1 range have been less consistent. Wild-caught musquitofish exposed to 25 or 226 ng L-1 fluoxetine entered the strike zone of a predator (a live dragonfly nymph) sooner, but no difference was observed between trials with and without the predator present (Martin et al., 2017). The number of musqui-tofish performing a C-start and C-start velocity (in response to the drop of a cylindrical metal probe into the tank) did not differ between fluoxetine treatments (8 or 97 ng L-1), but fluoxetine exposed fish spent longer time freezing after the event, except for females exposed to the high fluoxetine concentration (Martin et al., 2017). In the light/ dark test, fluoxetine exposed musquitofish had a shorter latency to enter the white side of a scototaxis arena, but females showed this behavioral disruption at a lower fluoxetine concentration (61 ng L-1) than males (352 ng L-1) (Martin et al., 2019). Gup-py courtship displays were largely unaffected by chronic fluoxetine (61 ng L-

1 and 350 ng L-1 for 28 days), with the exception of exposed males conduct-ing more sneak copulations (Fursdon et al., 2019). At these very low concen-trations, also effects that could be interpreted as amplified anti-predator re-sponses have been reported. Wild-caught guppies exposed to very low con-centrations of fluoxetine (4 or 16 ng L-1 for 4 weeks) remained stationary for longer, spent longer time under plant cover and were less active following a simulated bird attack (Saaristo et al., 2017).

In addition to fluoxetine, other antidepressants have been shown to dis-rupt anti-predator behaviors in similar ways, including the SSRIs sertraline (Valenti Jr. et al., 2012), citalopram (Kellner et al., 2016), and the serotonin-norephinephrine reuptake inhibitor (SNRI) venlafaxine (Parrott and Metcalfe, 2018, 2017). Also gestational exposure to EE2 has also been re-

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ported to influence anti-predator behaviors of guppies by increasing bottom dwelling in the novel tank diving test (Volkova et al., 2012).

Oxazepam and the benzodiazepines Oxazepam came into focus as a micropollutant when it was discovered that laboratory exposure at concentrations encountered in effluent-influenced waters (1.8 µg L-1) influenced the behavior of Eurasian perch (Perca fluviati-lis) (Brodin et al., 2013). Oxazepam belongs to a family of pharmaceuticals, the benzodiazepines, which share a common chemical structure (a 1,4-benzodiazepine ring) and have similar effects, including anxiolytic (anxiety-reducing), sedative (sleep-inducing), anticonvulsant (anti-seizure) and mus-cle-relaxing properties (Bateson, 2002; Sternbach, 1979). Differences in pharmacokinetic properties between individual benzodiazepines determine their clinical use, with short and intermediate-acting benzodiazepines pre-ferred in the treatment of insomnia while longer-acting benzodiazepines are recommended in the treatment of anxiety (Dikeos et al., 2008). Benzodiaze-pines exert their effects by binding to the receptor of the major inhibitory neurotransmitter γ-aminobutyric acid, the GABAA receptor, at a binding site that is homologous to but separate from to the GABA binding site. In doing so, they facilitate the GABA-induced conformational change of the receptor resulting in the opening of the central ion pore (Masiulis et al., 2018), which in turn leads to the inflow of negatively charged chloride ions into the neu-ron, hyperpolarizing it and driving it away from the threshold for excitation (Olsen and Sieghart, 2008). Occupation of the benzodiazepine binding site in the absence of GABA does not cause activation of the receptor, which is, together with their low toxicity, the main reason why benzodiazepines re-placed the barbiturates as anxiolytics of choice (Bateson, 2002).

Benzodiazepines have been detected in treated WWTP effluent at concen-trations ranging from 0.01 to several µg L-1 (Asimakopoulos and Kannan, 2016; Calisto and Esteves, 2009; Kosjek et al., 2012; Loos et al., 2013; Verlicchi et al., 2012). Fick et al. analyzed surface water samples from 30 European rivers for the occurrence of 13 different benzodiazepines, of which 9 were detected with median concentrations of 5.4 ng L-1 (Fick et al., 2017). Oxazepam, temazepam, clobazam and bromazepam were most commonly detected, their relative occurences covarying with relative local prescription rates. Oxazepam had the highest frequency of occurrence (85% of the sam-ples) and had a maximum concentration of 61 ng L-1, which can be explained by high prescription rates of oxazepam and by the fact that the highly de-scribed benzodiazepine diazepam is metabolized in the human body to oxa-zepam and temazepam (Ono et al., 1996). Björlenius et al. measured the occurrence of 93 selected pharmaceuticals in the Baltic sea and Skagerrak and detected oxazepam as one of the five pharmaceuticals with the highest median concentration (51 ng L-1 in the harbor of the Swedish city of Gävle

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and 36 ng L-1 in the Stockholm argipelago sampling point closest to the out-let of the municipal WWTP) (Björlenius et al., 2018). Close to the outlet of the municipal treatment plant in the city of Uppsala, oxazepam surface water concentrations of 210 ng L-1 were measured, which rapidly decline to 26 ng L-1 8 km downstream in lake Ekoln (Klaminder et al., 2015). In the US, alprozalam is the most commonly prescribed benzodiazepine and as such, was detected with a mean and maximum concentration of 3.3 ng L-1 resp. 25 ng L-1 in urban river sites (Batt et al., 2016).

Analyses of sediment cores from the river Fyris and Lake Ekoln (Up-pland, Sweden) collected in 1995 and 2013 allowed for assessment of oxa-zepam deposition and persistence in situ (Klaminder et al., 2015). Traces of oxazepam were detected in the sediment depth dating back to the early 1970s, which corresponds well with the introduction of this drug on the Swedish market in 1968. A pond experiment comparing oxazepam degrada-tion in a covered and open pond revealed that once adsorbed to the sediment, oxazepam is protected from degradation by solar radiation and the prevailing anaerobic conditions may further prevent microbial degradation. Also, de-sorption from the sediment was observed at two timepoints, indicating bioa-vailable concentrations of oxazepam may vary in response to changes in abiotic factors (Klaminder et al., 2015).

Effect of benzodiazepines on behavior The first discovered benzodiazepine, chlordiazepoxide (Librium), was syn-thesized in 1955, and its pharmacological properties were tested in mice and cats in 1957 (Sternbach, 1979) followed by rats, dogs and monkeys (Randall et al., 1960). Behavioral tests revealed muscle relaxant properties, reductions in avoidance learning as well as ‘taming effects’, including lower irritability of rats with septal lesions and lower aggression towards the experimenter in vicious monkeys and rats. The first experiments applying a benzodiazepine on a fish were done by Figler et al., who found that immersion treatment with 15 or 30 mg L-1 chlordiazepoxide attenuated the attack behavior of male Siamese fighting fish (Beata splendens) (Figler, 1973; Figler et al., 1975). Rehnberg et al. exposed fathead minnows to 1, 10 or 20 mg L-1 chlordiaze-poxide; the highest dosed group lacked the species-typical behavioral re-sponse to alarm pheromone (Rehnberg et al., 1989). Zebrafish became a popular model organism in neuropharmacology at the end of the 2000s (Gerlai, 2003; A. V. Kalueff and Cachat, 2011; Kalueff and Cachat, 2011), and as such anxiolytic effects were confirmed in this species of chlordiaze-poxide (Sackerman et al., 2010) and diazepam (Bencan et al., 2009). These studies provided proof of principle that the function of GABAA receptors and the behavioral effect of benzodiazepines are highly similar in mammals and fish.

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The effect of low concentrations of the benzodiazepine oxazepam (Ox-ascand, Sobril) on the behavior of wild fish was investigated in a series of exposure studies carried out by a team of chemists, geologists and behavioral biologists from the University of Umeå, Sweden. Common freshwater spe-cies including perch (Perca fluviatilis), roach, burbot (Lota lota) and Atlan-tic salmon (Salmo salar) were exposed for approximately one week to oxa-zepam concentrations in the µg L-1 range. All species showed behavioral disruptions indicative of an anxiolytic effect. Perch exposed to ~1 µg or ~1 mg L-1 displayed more locomotory activity in a novel arena (Brodin et al., 2014, 2013; Klaminder et al., 2014), a lower latency to feed (Brodin et al., 2013), less time close to a group of conspecifics (Brodin et al., 2013; Klaminder et al., 2014) and in the high dose, were less hesitant to enter a novel compartment (Brodin et al., 2013; Klaminder et al., 2014). Roach also showed more locomotory activity and in the high dose (280 µg L-1), spent more time in the white side of a light/dark box (Brodin et al., 2017). Burbot (Lota lota) were less affected but spent less time sheltering in response to a low oxazepam dose (1 µg L-1) and showed more movements exceeding 3 body lengths s-1 after receiving the high dose (100 µg L-1) (Josefin Sundin et al., 2019). Hatchery-raised Atlantic salmon increased their migration intensi-ty in response to relatively low doses of oxazepam (1.8 µg L-1) (Hellström et al., 2016) and exposed smolt (200 µg L-1) released along a 21-km natural river-to-sea migratory route had a higher probability of being predated upon (Klaminder et al., 2019).

There are also studies reporting that low concentrations of benzodiaze-pines had no effect on fish. Researchers originally involved in the discovery of endocrine disruption in fish reported that a laboratory population of fat-head minnows exposed to oxazepam for 28 days (4.7 µg L-1) showed in-creased diving responses and decreased activity, while a higher dose (30.6 µg L-1) did not affect behavior in the novel tank diving test (Huerta et al., 2016). In zebrafish an increased diving response would be interpreted as an increased anxiety-like state and/or a sedative effect (Cachat et al., 2010), but fathead minnows may not respond in the same way. Reproductive behavior of fathead minnows also appears unaffected by a 21-day exposure to diaze-pam (0.1, 1.04 or 13.4 µg L-1) (Lorenzi et al., 2014). Thus, the current evi-dence indicates that short-term exposure to low concentrations of oxazepam has anxiolytic effects in wild fish, while laboratory populations do not al-ways show the same effects.

Development of tolerance After the chemical and pharmacological discovery of chlordiazepoxide (trade name Librium), the compound was quickly introduced to the market in 1960 (Sternbach, 1979). There was great enthusiasm about the relatively simple chemical manufacture method, strong potency and low toxicity, an the enthusiasm became even more fervent when the more potent benzodiaz-

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epine diazepam (Valium) was discovered (Sternbach, 1979). By the begin-ning of the 70s however, evidence began to accumulate that benzodiazepines are not equally effective in long-term administration (Wise et al., 1972). Sandra File from the department of pharmacology at the University of Lon-don devoted a number of studies on the subject, investigating effects on be-havior and monoamine neurotransmitter turnover (e.g. Vellucci and File, 1979, de Angelis and File, 1979; File, 1982, 1981, 1983; see final paragraph of this introduction). Interestingly, tolerance to the sedative effects of benzo-diazepines seemed to appear faster than tolerance to the anxiolytic effects (Bateson, 2002).

Effect of benzodiazepines on physiology Effect of benzodiazepines on the HPI axis Situations in which environmental demands exceed the natural regulatory capacity of an organism, in particular those with low predictability and low controllability, exert stress on animals (Koolhaas et al., 2011). Acute stress-ors like capture, excessive exercise, hypoxia, osmotic or temperature shocks and social defeat trigger the activation of the physiological integrated stress response, an evolutionarily highly conserved mechanism important for phys-iological adaptation to a wide range of stressors (Schreck and Tort, 2016; Selye, 1976, 1936; Wendelaar Bonga, 2002). The two main players of the physiological stress response are the sympathetic adrenomedullary (SAM) system and the hypothalamic pituitary adrenocortical (HPA) axis in mam-mals (Koolhaas et al., 2011); the homologous systems in fish are the hypo-thalamic-sympathetic-chromaffin cell axis resp. hypothalamic pituitary inter-renal (HPI) axis (Wendelaar Bonga, 2002). These ‘stress axes’ function to-gether to stimulate oxygen uptake and transfer, mobilize energy substrates and reallocate energy away from growth, reproduction and immune func-tions.

In fish, perception of an acute stressor leads to a massive release of cate-cholamines (adrenalin and noradrenalin) from the chromaffin cells of the head kidneys within seconds to minutes, which increases ventilation, bran-chial blood flow, gas exchange and blood glucose levels (Wendelaar Bonga, 2002). Simultaneously, pertubation of the external and internal environment is relayed to corticotropin releasing hormone (CRH) producing cells located in the parvocellular and magnocellular nuclei of the preoptic area (POA) in the hypothalamus. Pulses of CRH induce the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland, which stimulates the secretion of the main end product, the hormone cortisol, from the interrenal cells of the head kidney tissue. Cortisol release is slower than that of cate-cholamines and typically occurs a few minutes after the stressor was first perceived, increasing by 10- to 100-fold (Wendelaar Bonga, 2002). Return to

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basal levels takes one or more hours, which is thought to result from a nega-tive feedback mechanism, although in chronic stress cortisol levels may re-main elevated (albeit well below peak levels) (Wendelaar Bonga, 2002). Cortisol functions to restore the hydromineral disturbances caused by circu-lating catecholamines, to promote long-term energy substrate mobilization and to inhibit growth, reproduction and immune functions.

It is important to emphasize that activation of these ‘stress axes’ of simi-lar magnitude can also be measured in appetitive and rewarding situations such as feeding, sexual behavior or social victory (Koolhaas et al., 2011), or during the final stages of gonadal maturation and during spawning and smol-tification (Wendelaar Bonga, 2002). Hence elevated cortisol levels are not synonymous with stress. Koolhaas et al. argue that stressful situations are characterized by low predictability and low controllability (Koolhaas et al., 2011). Physiologically, an unpredictable situation can be distinguished by the absence of an anticipatory rise in for example cortisol, blood pressure and heart rate. Uncontrollability (e.g. when an expected food reward is not received or when losing a fight), plasma adrenalin rises and the recovery of the HPA axis is slower (Koolhaas et al., 2011).

In rats, injections with chlordiazepoxide or diazepam can reduce the cor-ticosterone (the major glucocorticoid in rats) response to stress (Krulik and Cerny, 1971; Lahti and Barsuhn, 1974). The effect was significant for intra-peritoneal (IP) diazepam injections of 5 or 10 mg kg-1 while 1.25 or 2.5 mg kg-1 did not affect peak corticosterone plasma concentrations (Lahti and Barsuhn, 1974). Chronic administration of chlordiazepoxide (IP injections with 5 or 50 mg kg-1 day-1 for 10 days) failed to reduce the stress-induced corticosterone response (File, 1982).

Effect of benzodiazepines on monoamine neurotransmitters Monoamine neurotransmitters are modulatory neurotransmitters with simi-larities in chemical structure (an amino group connected to an aromatic ring by a two-carbon chain), receptors (most are G-protein coupled receptors), biosynthesis (all are synthesized from aromatic amino acids), intracellular transporters (vesicular monoamine transporters, VMAT) and metabolism (monoamine oxidases, MOA). The most well-studied monoamines include the catecholamines (dopamine, noradrenaline, adrenaline), the tryptamines (serotonin and melatonin) and histamine (Maximino and Herculano, 2010; Panula et al., 2010). Monoamine transmission has been implicated in a varie-ty of behaviors including agonistic behavior, arousal, stress, reward, appe-tite, sleep-wakefullness, motor control and cognition (Maximino and Hercu-lano, 2010; Winberg and Thörnqvist, 2016 and references therein). Mono-amine systems are highly conserved across the vertebrates, with only few distinct differences between mammals and zebrafish, including the duplica-tion of the tyrosine hydroxylase gene in zebrafish, one (zebrafish) instead of two (mammals) monoamine oxidase genes, as well as distinct dopamine and

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serotonin neuronal populations in addition to highly conserved populations (Lillesaar, 2011; Lillesaar et al., 2009; Panula et al., 2010).

Roughly a decade after the discovery of the first benzodiazepine (chlordi-azepoxide, in 1957, Sternbach, 1979), benzodiazepines were found to de-crease the turnover of monoamine neurotransmitters norepinephrine, dopa-mine (Corrodi et al., 1971; Taylor and Laverty, 1969) and serotonin (Chase et al., 1970). Wise et al. (1972), using a variety of different noradrenergic and serotonergic agonists and antagonists, concluded that decreased sero-tonergic turnover under benzodiazepine treatment (especially in the mid-brain), rather than decreased norepinephrine turnover, mediates the anxiolyt-ic effects of benzodiazepines (Wise et al., 1972). This view has been gradu-ally refined and it is now generally accepted that benzodiazepines exert their anxiolytic actions at a diversity of loci involving both monoaminergic and non-monoaminergic mechanisms (Pellow and File, 1984; Millan, 2003). Rodent studies where benzodiazepines were administered to specific brain areas and behavior was evaluated in the Vogel conflict test for anxiolytic activity, have indicated multiple supraspinal loci (i.e. the hippocampus, mammillary bodies, amygdala, lateral septum and dorsal periaqueductal gray), while these studies also further strengthened the evidence that benzo-diazepines inhibit the largest serotonergic cell population in the dorsal raphe nuclei (DRN), thereby suppressing serotonin release (Millan, 2003). Using fear-conditioning paradigms, the amygdala has recently received considera-ble attention as an important component of the neuronal systems involved in acquisition, storage and expression of fear (LeDoux, 2000). Engin et al. (2018) review the current literature regarding the question where in the brain the GABAA receptors involved in specific drug effects and behaviors are located, taking a subtype and circuit-specific approach, and building on re-sults from cell- and subunit-selective genetic knock-down mice models (Engin et al., 2018). This reveals a complex and intricate organization of GABAergic inhibition, involving microcircuits in the amygdala and hippo-campus, in addition to areas such as the medial prefrontal cortex, septum, thalamus, hypothalamus, periaqueductal grey, raphe nuclei and locus co-eruleus, as well as having profound influence over the ventral tegmental area dopamine neurons in the mesocorticolimbic dopamine system. It is a chal-lenging task to find the homologous brain areas in fish, but homologues of these mammalian brain areas in fish have been proposed (reviewed in Mueller, 2012, Soares et al., 2018; see also Cocco, 2018).

The inhibitory effect of short-term benzodiazepine administration on serotonin turnover has been shown to be reduced or absent in chronic treat-ment with anxiolytic doses. Rats treated for 5 days with chlordiazepoxide (5 mg kg-1 d-1, IP) had increased serotonin (5-HT) concentrations in hypothala-mus, midbrain and cerebral cortex, while levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) were reduced in hypothalamus after 5 and 15 days, and in cerebral cortex after 15 days, indicating reduced seroto-

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nin turnover. However, after 25 days there were no longer any significant differences from controls (Vellucci and File, 1979). Tolerance to the inhibi-tory effects on noradrenalin did not coincide with serotonin recovery (File and Hyde, 1978; Vellucci and File, 1979).

Mitigation Pharmaceutical pollution is an environmental problem for which a general engineering solution might be feasible, and trials with activated carbon and ozonation (among other methods) are currently being executed (Prasse et al., 2015). Although it is necessary to evaluate the effects of these removal tech-niques on fish and other aquatic vertebrates (Pohl et al., 2018, 2019), their development offers a truly optimistic view.

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Aims of the thesis

The overarching aim of this PhD thesis work was to evaluate the effect of near-future levels of CO2 and low concentrations of the anxiolytic drug oxa-zepam on the behavior and physiology of fish. Specifically, I address the following questions:

• Are zebrafish (Danio rerio) affected by elevated pCO2? In paper I, we exposed zebrafish of the AB strain for ~1600 µatm CO2 and evaluated behavior in the open field and detour tests.

• Are three-spined stickleback (Gasterosteus aculeatus) affected by elevated pCO2? In paper II and III, we exposed marine stickle-back to ~1000 µatm pCO2. In paper II we evaluated reproductive behaviors including male courtship behaviors, mating probability, nest building and egg fanning as well as sexual ornamentation. In paper III, we evaluated female behavior in the open field, light/ dark and detour tests, and male-male aggressive behavior. We al-so quantified neurotransmitter turnover rates of serotonin, dopa-mine and norephinephrine in winner and loser males at control or elevated pCO2.

• Can the GABAA receptor antagonist gabazine restore behavioral disruptions caused by elevated pCO2? In paper I and III, we treated control and elevated pCO2 exposed fish with gabazine. In paper III, we also include a dose-response curve for gabazine as well as GABAA receptor agonist muscimol, evaluating the dose most commonly used in fish.

• Are laboratory and wild-caught zebrafish differentially affected by low concentrations of oxazepam? In paper IV, we compare the diving responses and cortisol levels of oxazepam exposures in the µg L-1 range between AB strain zebrafish and wild-caught zebrafish from India.

• Do zebrafish develop tolerance to the anxiolytic effect of oxaze-pam in chronic exposure? In paper V, we exposed wild-caught zebrafish to ~7 µg L-1 oxazepam for 7 to 28 days and evaluated behavior in the novel tank diving test and Pavlovian avoidance learning test, gene expression of GABAA receptor subunits and CRH, cortisol levels and neurotransmitter turnover rates of sero-tonin, dopamine and norepinephrine.

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Materials and methods

Study species Zebrafish The zebrafish (Danio rerio) is a small cyprinid fish species native to the Bengal region of India and Bangladesh, and neighboring regions in Pakistan, Myanmar and Nepal (Engeszer et al., 2007). Over the past decades, zebrafish have grown in popularity as a biological model organism, being used in bi-omedical, ecotoxicological, and neuropharmacological research, among oth-er research areas. Why just zebrafish became so popular compared to other small fish species is likely due to its popularity as a pet animal and therefore its availability in pet stores the world over, in addition to the species’ short generation time and transparency of larvae. Currently, the zebrafish is amongst the most well described vertebrate species with regards to devel-opmental, genomic and translational information available, its behavior is well described and standardized behavioral tests have been developed (Kalueff and Cachat, 2011; Kalueff and Cachat, 2011; Kalueff et al., 2013; Kawakami et al., 2016) and effects of (neuro)pharmacological treatments have been documented. The increasing number of genomic and translational tools available for zebrafish enhances its popularity further, and in some research areas zebrafish have already taken the place of rodents as the most common laboratory animal.

Yet despite its popularity as a laboratory organism, relatively little is known about the species ecology. Spence et al. (2006), Engeszer et al. (2007) and Sundin et al. (2019) describe the geographical range, habitat and life history of zebrafish, as compiled from both their own and other docu-mented field studies (Spence et al., 2006; Engeszer et al., 2007; J Sundin et al., 2019). Typical habitats of adults include rivers, small streams, stagnant or slow moving pools near streams, as well as ‘rice paddies’, often with silt-covered bottoms and submerged or overhanging vegetation, and adults are thought to feed primarily on insects. At the sites visited by Engeszer et al., zebrafish were found at temperatures from 24.6 to 38.6°C, pH 5.9 to 8.1, and conductivities of 10 to 271 µS cm-1. With the onset of the rainy season (ap-proximately between April and August), adults move into ‘nalas’ (seasonal streams that feed into larger rivers) and rice paddies, where they spawn in groups amid flooded vegetation. Larvae and juveniles remain in these patch-

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es, and young zebrafish likely move back into the streams as the seasonal waters recede. A wide variety of small cyprinid species co-occur with zebrafish (Engeszer et al., 2007). Natural predators likely include snake-heads (of the genus Canna), needlefish (Xenenton cancila), the catfish (Mys-tus bleekeri) and the knifefish (Notopterus notopterus).

Based on genetic variation in single nucleotide polymorphisms (SNPs), three major clades of the species zebrafish can be distinguished: 1) northern India and western and eastern Nepal; 2) Bangladesh/ southern India; 3) cen-tral Nepal (Whiteley et al., 2011). The three laboratory strains examined in Whiteley et al. (2011) (AB, SJA and TM1) were likely collected from the Ganges/ Brahamaputra region near the major city of Calcutta, and had clear-ly reduced SNP diversity compared to wild populations. In paper I and IV, we used AB strain laboratory zebrafish, while in paper IV and V we ana-lyzed wild-caught zebrafish from different populations from West Bengal, India (for precise catching locations, see Morgan et al., 2019).

Three-spined stickleback The three-spined stickleback is a small fish species with both marine and anadromous ecotypes (Ahnelt, 2018) that occurs in coastal seas, estuaries and fresh waters in temperate regions (above 30° latitude), including tide pools and brackish marshes (Östlund-Nilsson et al., 2007). In these habitats CO2 concentrations fluctuate heavily in relation to the tide and time of day (Truchot and Duhamel-Jouve, 1980; Waldbusser and Salisbury, 2014). The behavior of three-spined stickleback has been intensely studied by ethol-ogists since the 1930s, notably by the one of the winners of the 1973 Nobel Prize in physiology and medicine, Niko Tinbergen (Russell and Russell, 2010). Especially the species reproductive behavior is well documented in the ecological and evolutionary literature, while also genetic techniques have been adapted for this species, the combination of which makes it an excellent model for a variety of laboratory as well as field studies (von Hippel, 2010).

In paper II, we evaluated the effect of elevated pCO2 on this species’ re-productive behaviors. An excellent overview of three-spined stickleback reproductive behavior is given by Sara Östlund-Nilsson (Östlund-Nilsson et al., 2007 and references therein), which I briefly summarize here. During the winter, stickleback form larger shoals consisting of both males and females, that reside in deeper waters. From late April to July, males leave the shoal to establish a territory, and develop sexual coloration including conspicuous orange-red breeding coloration combined with UV reflectance in the cheek region (Hiermes et al., 2016). After construction of a nest (using filamentous algae glued together with a proteinaceous substance called ‘spiggin’ that is secreted from the kidney), males attract females with characteristic courtship dances including the zigzag dance. After successful courtship, a female lays eggs inside the males nest, upon which a male will start to perform paternal

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care for the offspring, consisting of egg fanning behavior. The male posi-tions himself head-down in front of the nest and uses his pectoral fins to produce a jet-stream of oxygenated water over the eggs. The duration that the male displays this behavior per minute increases as the eggs mature, which usually takes 7-9 days (Mayer and Páll, 2007). During egg incubation, males also defend the nest from egg predation by conspecific and heterospe-cific fish – male aggressive behavior is quantified in paper III.

Behavioral tests Open field test (OFT) The open field test (OFT) is a widely used behavioral test for quantifying anxiety-like behavior in non-human vertebrates, including rodents, cows, pigs, sheep, rabbits and primates (Prut and Belzung, 2003). It was devised by Hall (1934) who exposed rats to a brightly lid circular arena of about 1.2 m in diameter closed off by a 0.45 m high wall (Hall, 1934; Prut and Belzung, 2003). A rat was placed alone in this arena and an experimenter observed horizontal locomotion, frequency of rearing, grooming and movement along the walls, the latter behavior being termed ‘thigmotaxis’ (attraction to the wall). Increased time spent along the walls, decreased movement in the cen-ter and increased latency to enter the central part of the arena are usually interpreted as anxiety-like behavior. Compounds that elicit thigmotaxis are considered anxiogenic, while compounds that reduce thigmotaxis are con-sidered anxiolytic. Acute administration of classical benzodiazepines (such as diazepam and oxazepam) reduce thigmotaxis and are therefore considered anxiolytic compounds, while such effects are not always observed for the triazolobenzodiazepines (such as alprazolam) and in chronic administration with SSRIs (such as fluoxetine) (Prut and Belzung, 2003). In zebrafish, the width of the wall zone is often taken as 2 times the length of the fish (~5 cm) and the total arena measures 30 by 30 centimeters with a water depth of 4 cm (Godwin et al., 2012). In paper I, we evaluated the effect of exposure to elevated pCO2 and the GABAA receptor selective antagonist gabazine using the open field test for zebrafish. In paper III, we tested three-spined stickle-back in the OFT to evaluate whether CO2 exposure had an anxiogenic effect.

Light/ dark test (LDT) Like the OFT, the light/ dark test makes use of the tendency of many verte-brate species to seek shelter when placed individually in a novel environ-ment, but in contrast to the OFT, the light/ dark test measures ‘scototaxis’ (attraction to a dark or covered part of an open arena) instead of thigmotaxis (Stewart et al., 2011). Half of the area of a rectangular arena is covered with

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a plastic sheet and the time spent in the darkened and light side of the arena is quantified, either using manual scoring or using automated video tracking software. We have made use of this test in paper III.

Novel tank diving test (NTDT) The novel tank diving test (NTDT) provides yet another measure of anxiety-related behavior, this time making use of the natural tendency of zebrafish (and many other small tropical fish) to dive to the bottom of a novel arena, a behavior also termed the ‘diving response’ (Cachat et al., 2010). This behav-ior may be more specific to zebrafish since three-spined stickleback do not show such a distinct movement to the bottom in this situation (personal ob-servation). The NTDT has been pharmacologically validated with acute treatment with diazepam, which reduces the time spent in the bottom and increases time in the top half of the arena, while having no effect on swim-ming speed (Bencan et al., 2009).

In paper IV and V, we adapted the standard NTDT by adding conspecif-ic skin extract to the tank water 12 minutes after introducing the fish to the diving tank. Zebrafish and other fish of the superorder Ostariophysi contain alarm pheromones in specialized epidermal cells, which are released upon injury to the skin, such as during a predator attack. These alarm pheromones were first described by in minnows by Karl von Frisch who named these compounds “schreckstoff” (von Frisch, 1942; Pfeiffer, 1977). The method of preparing conspecific alarm cue is described in detail in the corresponding papers.

Detour test The detour test of behavioral lateralization is a behavioral test devised to measure cerebral lateralization in fish. In this test, an individual fish is placed in the middle of a runway, after which the fish moves through the runway until it reaches a T-shaped crossroad and moves into one of the arms. The choice of left or right arm is noted down by the experimenter (al-ternatively, the animal is filmed), and the animal moves back into the run-way to make the next choice for the lower left or right arm. The test is com-pleted when the animal has made ten (paper III) or twenty (paper I) left or right choices. Three-spined stickleback in paper III were encouraged with plastic rods to move around the arena while the zebrafish in paper I moved readily back and forth through the runway without encouragement.

The direction taken by a fish is assumed to be dependent on its eye pref-erence, and therefore the detour test is considered to reflect cerebral laterali-zation important for schooling (Bisazza and Dadda, 2005). The test has been used extensively in the OA literature (Domenici et al., 2014, 2012; Jutfelt et al., 2013; Welch et al., 2014; Lopes et al., 2016, 2016; Sundin and Jutfelt,

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2016; Jarrold et al., 2017). Disruptions in detour behavior (although often in different directions) in CO2 exposed groups have been argued to result from a CO2-induced disruption of cerebral lateralization that might negatively influence fish survival.

Marking individuals with elastomer tags In paper V, we marked individual fish with two elastomer tags at the base of the dorsal fin, in order to compare individual behavior before and after expo-sure to oxazepam. We followed the procedure described by (Hohn and Petrie-Hanson, 2013).

Microdissection of fish brain tissue In paper III and V, we dissected the brains of stickleback and zebrafish, resp. into fore- and hindbrain according to the procedure described in (Dahlbom et al., 2011). Forebrains were incubated with RNA-later, stored at -70°C and later used in gene expression analyses, while hindbrains were snap-frozen in liquid nitrogen and stored at -70°C, for analysis of monoam-ine neurotransmitters using HPLC-EC.

Physiological measurements RT-qPCR Quantitative real-time polymerase chain reaction (RT-qPCR) is a method to measure gene expression of an a priori decided gene of interest. In paper V, we quantified relative transcript levels of 20 genes encoding for GABAA receptor α, β and γ subunits as well as two genes encoding CRH in zebrafish exposed to control tapwater or oxazepam.

RNA was extracted from forebrain samples of experiment 2 using the GeneElute Mammalian Total RNA Miniprep Kit (RTN70-1KT, Sigma-Aldrich, Sweden) followed by DNAse treatment using the Invitrogen Turbo DNA-free Kit (AM1907, ThermoFisher Scientific, Sweden). RNA concen-tration and purity was evaluated using a Nanodrop ND-1000 spectrophotom-eter (ThermoFisher, Sweden). cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (K1641, ThermoFisher Sci-entific, Sweden). The resulting cDNA reaction volume of 20 µL was diluted 40 times and 4 µL of diluted cDNA was used in each qPCR reaction. Diluted cDNA samples were plated out in triplicates on a single 384-well plate (Corning Inc., USA) per gene using a Pipetman M 12-channel electronic

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pipette (Gilson Inc., USA). The RT-qPCR reaction was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) with a reaction volume of 10 µL, of which 5 µL of Maxima SYBR Green/ROX qPCR Master Mix (K0223, ThermoFisher Scientific, Sweden), 0.5 µL of both forward and reverse primer (5uM) and 4 µL of template. The thermal profile was 10 min at 95 °C, followed by 45 cycles at 95 °C (30 sec), 60 °C (30 sec) and 72 °C (30 sec). A melting curve was included consisting of 95 °C, 60 °C, 95 °C, each temperature lasting for 15 sec.

HPLC with electrochemical detection In paper III and V, we determined the concentration of serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and norepinephrine (NE) in hindbrain samples using high perfor-mance liquid chromatography with electrochemical detection (HPLC-EC) (Øverli et al., 1999). In short, the frozen hind-brains were homogenized in 4% (w/v) ice-cold perchloric acid containing 10 ng mL-1 3,4-dihydroxybenzylamine (DHBA, as internal standard) using a Sonifier cell disruptor B-30 (Branson Ultrasonics, Danbury, CT, USA) and centrifuged at 21 000 g for 10 min at 4 °C. The supernatant was used for HPLC-EC and the pellet was saved for protein quantitation using a Qubit 2.0 Fluorometer and the Qubit Protein assay kit (Invitrogen, Sweden). The HPLC-EC system consisted of a solvent delivery system model 582 (ESA, Bedford, MA, USA), an autoinjector Midas type 830 (Spark Holland, Emmen, the Nether-lands), a reverse phase column (Reprosil-Pur C18-AQ 3 µm, 100 mm × 4 mm column, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) kept at 40 °C and an ESA 5200 Coulochem II EC detector (ESA, Bedford, MA, USA) with two electrodes at reducing and oxidizing potentials of −40 mV and +320 mV, respectively. A guarding electrode with a potential of +450 mV was employed before the analytical electrodes to oxidize any contaminants. The mobile phase consisted of 75 mM sodium phosphate, 1.4 mM sodium octyl sulphate and 10 µM EDTA in deionised water contain-ing 7% acetonitrile brought to pH 3.1 with phosphoric acid. Samples were quantified by comparison with standard solutions of known concentrations using HPLC software Clarity™ (DataApex Ltd, Prague, Czech Republic). For normalization of monoamine levels the brain protein content was used (Qbit 2.0 Fluorometer, Invitrogen, UK).

Radio-immunoassay (RIA) In paper IV and V, we analyzed cortisol concentrations in whole-body tis-sue samples of zebrafish collected after a 30-minute confinement stress. Zebrafish bodies (brain removed) were thawed, individually weighed and

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homogenized in phosphate-buffered saline solution (PBS) in a 15 mL centri-fuge tube with 1 mL PBS buffer, using a VDI12 dispenser with S12N-7S rotor (VWR international, Sweden). Ethyl acetate was added and the sample was thoroughly vortexed and centrifuged, after which the cortisol containing ethyl acetate phase was transferred into a borosilicate glass vial. Ethyl ace-tate was evaporated with a steady flow of nitrogen gas over the surface area and simultaneous heating to 40° C (Bibby Scientific Stuart sample concen-trator and block heater, Cole Parmer, USA). The sample was reconstituted in ethyl acetate, and 3 replicates of the sample were transferred into three 1.5 mL microcentrifuge tubes.

Samples were compared to a standard curve containing known concentra-tions of cortisol (Sigma-Aldrich, Sweden) ranging from 0.125 to 80 ng mL-1. [1, 2, 6, 7-3H(N)]-Hydrocortisone (Perkin-Elmer, USA) was added to each sample and the remaining ethyl acetate was evaporated using a SpeedVac (Savant, Thermo Fisher Scientific, Sweden). The samples were incubated overnight at 4° C with rabbit polyclonal antibodies to cortisol (Cambio, UK) in a bovine serum albumin buffer (Sigma-Aldrich, Sweden). The next day, unbound antibodies were washed away using dextran-coated charcoal (Sig-ma, Sweden) incubated at 4°C for 5 minutes and centrifuged. The superna-tant was added to a scintillation tube with EcoLite(+) scintillation cocktail (VWR, Sweden) and analyzed with the Tri-Carb 2910TR liquid scintillation analyzer (Perkin-Elmer, USA). Three controls were added containing i) no antibodies and no DCC; ii) no antibodies and DCC; iii) antibodies and DCC. Each assay contained an internal standard consisting of a mixture with equal volume of each extracted sample. The detection limit was 0.25 ng mL-1. Cor-tisol concentrations were corrected for dilution factor and weight of the fish.

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Results

Paper I Adult zebrafish were exposed to control (~400 µatm) or elevated pCO2 (~1600 µatm) for 26 to 48 days and behavior was measured in the open field test and detour test. Half of the fish from each pCO2 group (i.e. control-CO2 females, control-CO2 males, elevated-CO2 females and elevated-CO2 males) furthermore underwent immersion treatment with the GABAA receptor an-tagonist gabazine (4 mg L-1 for 30 minutes). CO2 exposure did not affect locomotory activity or thigmotaxis in the open field test in either sex. Gaba-zine treatment increased thigmotaxis in both sexes. Males treated with gaba-zine swam faster compared to drug controls, irrespective of CO2 treatment. CO2 exposure increased the proportion of fish taking more right turns, whereas gabazine had no effect on behavior in the detour test.

Paper II Breeding pairs of three-spined stickleback were exposed to control (~400 µatm) or elevated pCO2 (~1000 µatm) for 14 to 34 days. No differences be-tween CO2 exposed and control groups were detected in the development of male sexual coloration, male nest building, male courtship behavior (time spent performing zigzag dance, displacement fanning, gluing on nest and crawling through nest), male mating behavior, probability of receiving eggs from a female, number of offspring hatched and time spent nest-fanning.

Paper III Adult three-spined stickleback were exposed to control (~450 µatm) or ele-vated pCO2 (~1000 µatm) for 24 to 31 days. At day 12 of CO2 exposure, females were assessed in the detour test of behavioral lateralization, but no differences between exposure groups were detected, in contrast to earlier findings. After CO2 exposure was completed, female swimming activity, thigmotaxis and scototaxis were assessed and these also proved resilient to elevated pCO2. Gabazine treatment (immersion in 4 mg L-1 for 30 minutes) increased thigmotaxis but did not alter swimming speed of females regard-

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less of CO2 exposure. Male territorial aggression (aggression of breeding males to a flask containing a conspecific male) as well as pairwise aggres-sion (aggressive interaction between two males after breeding was complet-ed) were not affected by pCO2. After 24 hours of pairwise interaction, sub-ordinate males had higher brain serotonin turnover (5-HIAA/ 5-HT ratio) than dominant males, but there was no effect of CO2 level.

A dose-response experiment was carried out where control CO2 females were treated with GABAA receptor antagonist gabazine (immersion in 0.8, 4 or 40 mg L-1 for 30 minutes). Females became hyperactive in the highest dose (40 mg L-1) while the most commonly used dose in OA studies (4 mg L-1) and the lowest dose (0.8 mg L-1) did not affect behavioral activity. Mus-cimol did not affect behavioral activity for any of the doses used in this ex-periment (immersion in 0.5, 1 or 10 mg L-1 for 30 minutes).

Paper IV Adult zebrafish of the laboratory strain AB and wild-caught zebrafish from West-Bengal, India, were exposed to three different concentrations of the benzodiazepine oxazepam (0.6, 5.6 or 60 µg L-1), or exposed to control tap-water. After 7 days of exposure, behavior was assessed in the NTDT. Upon addition of conspecific skin extract (an alarm pheromone), control exposed wild-caught zebrafish showed a clear diving response, while oxazepam ex-posure resulted in reduced diving responses at all concentrations. Wild-caught males showed a reduced diving response only for the highest dose (60 µg L-1). Control exposed laboratory strain AB zebrafish showed no div-ing response after addition of the alarm cue, suggesting that this strain has lost its ability to respond to conspecific skin extract. No consistent effect of oxazepam exposure was detected in AB strain zebrafish. Post-confinement cortisol concentrations were not affected by oxazepam exposure in any of the experimental groups.

Paper V Wild-caught zebrafish from West-Bengal, India, were exposed to ~7 µg L-1 oxazepam or control tapwater, for 7 or 28 days. After 7 days of exposure, exposed females responded less to addition of an alarm cue (conspecific skin extract) compared to controls, but this effect had disappeared by 28 days, indicating that females had developed tolerance to the anxiolytic effect of oxazepam. Males were not affected by ~7 µg L-1 oxazepam at either time points. Serotonin turnover (ratio 5-HIAA/ 5-HT) at 28 days was reduced in exposed animals of both sexes, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of

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corticotropin releasing hormone (CRH) were not affected by oxazepam. Gene expression of GABAA receptor subunits was not altered by the 28-day oxazepam exposure, suggesting that some other still unknown mechanism caused the developed tolerance in females.

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Discussion

According to the GABA hypothesis (Nilsson et al., 2012), increases in ocean pCO2 induce acid-base regulatory changes in fish resulting in reduced extra-cellular chloride concentrations and increased bicarbonate concentrations. In turn, the altered chloride (and bicarbonate) gradient over the neuronal mem-brane would lead to the outflow of chloride ions out of the neuron upon binding of GABA to the GABAA receptor, increasing the membrane poten-tial and bringing it closer to the threshold for excitation. In other words, CO2 would render the GABAA receptor excitatory rather than inhibitory. While there is substantial support for acid-base regulatory changes in response to near-future pCO2 (Esbaugh, 2018), the methodological difficulties with measuring chloride transmembrane gradients mean that this part of the GABA hypothesis still remains speculative. Instead, researchers have point-ed at behavioral measures such as the ‘reversal of olfactory preferences’ (Dixson et al., 2010; Munday et al., 2010; Nilsson et al., 2012; Welch et al., 2014), increases in distance moved from a shelter and associated decreased survival probability (Munday et al., 2010) and changes in behavioral lateral-ization (Domenici et al., 2012, 2014) which could be restored with gabazine treatment (Nilsson et al., 2012; Chivers et al., 2014; Chung et al., 2014; Lai et al., 2015 but see Tresguerres and Hamilton, 2017 for a discussion of the studies using gabazine). Leaving methodological concerns with these studies aside (regarding the manipulation and measurement of CO2 (Cornwall and Hurd, 2016; Moran, 2014), the use of ‘Atema choice flumes’ (Jutfelt et al., 2017) and the repeatability of the detour test of lateralization (Roche et al., 2019)), an important question that has been left unattended is whether any CO2-induced reduction in GABAergic inhibition would really result in a reduction in anxiety-related behavior. Generally speaking, pharmacological compounds that inhibit the action of GABA (such as gabazine and bicucul-line) have anxiogenic and convulsant effects, while compounds that enhance the action of GABA (such as muscimol, benzodiazepines, barbiturates and alcohol) have anxiolytic, anticonvulsant and sedative effects. So why would CO2, by reducing the inhibitory tone in the brain of fish, have an anxiolytic effect, i.e. making fish attracted to predator odor? And why would CO2 have an anxiolytic effect only at these relatively low concentrations, while the very high CO2 concentrations used for CO2 stunning have anxiogenic effects (Marx et al., 1997)?

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We found no evidence to suggest that zebrafish (paper I) or three-spined stickleback (paper II and III) were affected by an approximately month long exposure to elevated CO2 concentrations. This is not surprising since both species are likely to encounter high CO2 concentrations in their natural environment, and as a result, may have evolved mechanisms to cope with the relatively small increase in pCO2 resulting from anthropogenic emissions. Hence these study species may not be the best choice for investigating this biological question (Krogh, 1929). Considering the methodological concerns with the early studies on the effects of elevated pCO2 on coral reef fish, there is nevertheless a need for replication of these studies. Three of such studies have now been published. Long-term exposure to ~1000 µatm of juvenile spiny chromis damselfish (Acanthochromis polyacanthus) did not affect behavioral activity or the behavioral response to predator odor (Sundin et al., 2017). Adult blue-spotted rockcod (Cephalopholis cyanostigma) exposed to ~1000 µatm for 8-9 days showed no significant differences from controls in post-release swimming behavior (Raby et al., 2018). Maximal thermal limits of coral reef damselfishes were resilient to near-future pCO2 (Clark et al., 2017).

In contrast to the lack of an effect of exposure to elevated pCO2, we did find evidence to suggest that zebrafish were affected by µg L-1 concentrations of the anxiolytic drug oxazepam (paper IV and V). The effects were depend-ent on the sex and strain of the fish (paper IV). Female zebrafish showed anxiolytic effects at concentrations approximately one hundred fold lower than males. Laboratory zebrafish of the AB strain did not show anxiolytic effects, at least not when using the diving response to conspecific skin ex-tract as a read-out. It is possible that other (behavioral or physiological) read-outs can be used to quantify anxiolytic effects also in AB zebrafish, which would facilitate high-throughput screening for effects of a broad range of pharmaceuticals detected in the environment.

The anxiolytic effect of oxazepam was no longer significant after chronic (28 days) exposure to ~7 µg L-1 (paper V). This finding emphasizes the im-portance of using pharmacological knowledge to inform ecotoxicological risk assessment. It also highlights the current lack of knowledge regarding the spatiotemporal patterns present in this particular type of environmental pollution. If oxazepam concentrations vary considerably over the season, then tolerance builds up when the concentrations peak, and is lost when con-centrations are low. Spatiotemporal variation may result from temperature-dependent biological wastewater treatment processes, seasonal changes in river flow rates, but can also result from seasonal increases in pharmaceuti-cal use, such as a peak in antihistamines in spring.

While oxazepam no longer had an anxiolytic effect on behavior in chronic exposure, we detected a reduction in serotonin turnover in zebrafish exposed to oxazepam for 28 days. This was likely due to a direct inhibitory effect of

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oxazepam on the release of serotonin from serotonergic cells in the dorsal raphe nuclei. Reduced brain serotonin and/ or 5-HIAA have also been re-ported in fish exposure studies using low concentrations of antidepressants fluoxetine (Clotfelter et al., 2007; Gaworecki and Klaine, 2008; Winder et al., 2009) and venlafaxine (Bisesi Jr et al., 2014). Hence this finding under-lines the importance of studying interaction effects between different phar-maceutical residues found in environmental matrices.

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Acknowledgements

There are many people who I would like to thank for their continuous sup-port throughout my PhD. Starting with my supervisors, Svante. I still don’t understand why you took me on (I had a MSc in evolutionary/ behavioral ecology with very little physiology) but I am very grateful that I got to do my PhD in your lab, with you. You are one of the most kind, generous, loyal and knowledgeable people I know. You never stressed me out and gave me access to all the things I needed to pursue my own interests and research questions. Though at times I have been frustrated that you didn’t tell me what to do, or exactly how to do it, I guess that was just the point. You let me think alongside with you, giving me important insights without taking the decisions. Now to academic dad no. 2, P-O. It took me a while to learn when your Swedish “hm..”, “kanske”, “mja..” really meant: “yes”, “I don’t know” and “no” (Swedish and Dutch culture could not be more dissimilar when it comes to being direct). But now that I got the hang of it (or did I?) I have grown so fond of your respectful and reliable work attitude that I’m afraid I will deeply miss you at my next working place. Thank you for all your extra efforts and for your tolerance with my not so Swedish tempera-ment. Fredrik, my co-supervisor, during our field work at Kristineberg in 2014 you set a great example as an inventor/ engineer type of behavioral physiologist who is basically just having fun in the lab. You have also been a great help at the writing stage, giving positive but constructive feedback. But most importantly you taught me throughout my doctoral education to rely on my own data and to publish all data that is technically reliable (even if it refuted your earlier findings). This may seem like trivial advice but it has been the most helpful when the literature was confusing. Josefin, you were never officially my co-supervisor but you have had a tremendous influence on me. From the way you made literature overviews to your hands-on and systematic way of setting up an experiment, to taking me along to confer-ences. I greatly admire your courage and matter of fact attitude, not letting academic politics get in the way of the main message. Behavioral ecotoxi-cology needs scientists like you, who take the effort to publish (many!) nega-tive findings, also if this compromises one’s career. Tomas, also you have been like a supervisor to me. Without hesitation you and Jerker took the nearly 600 km long trip to Uppsala to discuss my oxazepam project (we hadn’t even met before!). Your and Jerker’s strong and complimentary ex-pertise have been extremely useful, and I really hope that there will be an

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opportunity to work more with ‘the Umeå team’ in the future! Bryndis and Zhe, your doors were always open for my silly GABA or qPCR related questions, thank you for sharing your knowledge.

My fellow PhD students, Arianna Cocco, Arshi Mustafa, Johanna Ax-ling, and Oly Sen Sarma have all contributed in their own way. Arianna through her thorough gene expression study of the GABA system in zebrafish, which paved the way for my oxazepam tolerance article. Arshi set an example with her work ethic. So did Johanna by producing an amazing N equals 2500 dataset on salmon aggression (not to mention the selection experiment, keep an eye out for these studies)! Oly, you were ‘my’ dedicat-ed and creative MSc student (I am very proud!), and I am confident that you will flourish even more as a PhD student.

Daniel Červený made sure the water samples got analyzed for oxazepam in no time, thanks-an-… inverted microgram! Johannes Pohl helped out with zebrafish liver sampling, and we also had fishy discussions about sew-age and ozone. Bertil Borg and Mirjam Amcoff took me on a trip to catch three-spined stickleback in Skåne one cold February weekend. Sjannie Lefevre, Göran Nilsson, Floriana Lai and Öyvind Överli hosted a very nice Värmland retreat. Colleagues at the department made for great interdis-ciplinary reading clubs, including Dan Larhammar, David Lagman, Erika Roman, Åsa Konradsson-Geuken, Martina Blunder, Kate Shebanits, Helen Haines, Olga Dyakova, Nikita Tjernström, Stina Lundqvist and many more. Corridor mates Olga, Atieh and Hayma always shared their knäckebröd with me when I was stuck in the lab. Helgi Schiöth provided guidance as a ‘studierektor’. I had great BSc and MSc students over the years that all did their very best, in chronological order Johan Rudin, Sara Karakechili, Carolina Rönnberg, Emma Åkerman Fulford, Ben Petters-son, Marcus Österkrans, Tom Abbu, Elona Metse, Airon Liu and Clara Jeong. Sulayman Mourabit and later Imdad Ullah and Humaira Rehman were all visiting our lab for some months and made sure the coffee breaks were never boring.

Finally, a big warm thank you to my family and friends. Rikard, my ‘jä-mställd’ husband, who always insists doing the dishes more often than me. I love our discussions about ethology, ethnology and politics. Above all, you are the best dad I could have imagined to our daughters Kerstin and ‘Lasse’ (working name). My parents Trees and Peter raised three environmentally conscious kids and encouraged me to be active in the Dutch youth organiza-tion for nature studies (NJN). My sister Cathelijne and family saved me time researching cloth diapers and baby-wearing (Marien & Debbie, watch out). My aunt Marian financed my violin lessons as a teenager, thanks to which I could join the wonderful Uppsala University orchestra Akademiska Kapellet! My Dutch friends, Ymke, Pauline & Floris, Maud & Jaap, Marieke & Egon, Naomi & Boudewijn, Jelmer, and friends (met) in Swe-den, Froukje & Josefin, Una & Thorgeir, Frida E, Phoebe, Fia (who

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made the fabulous cover art!) & Aaron, Lena & Oskar, Hanna & Jonas, Kasper & Vala, and in the States, David H… thank you all for being you!

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