Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish

Journal Pre-proofs Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish D. Cerveny, T. Brodin, P. Cisar, ES. McCallum, J. Fick PII: DOI: Reference:

S0048-9697(19)34771-0 https://doi.org/10.1016/j.scitotenv.2019.134780 STOTEN 134780

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Science of the Total Environment

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4 July 2019 20 September 2019 1 October 2019

Please cite this article as: D. Cerveny, T. Brodin, P. Cisar, ES. McCallum, J. Fick, Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134780

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Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish

Cerveny, D.a,b*, Brodin, T.c, Cisar, P.b, McCallum, E. S.c,d, Fick, J.a

a Department b University

of Chemistry, Umeå University, SE-90187, Umeå, Sweden

of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of

Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic c Department

of Wildlife, Fish and Environmental Studies, Swedish University of Agricultural

Sciences, SE-90183, Umeå, Sweden dDepartment

of Ecology and Environmental Science, Umeå University, SE-90187, Umeå,

Sweden

*Corresponding author, [email protected]

Abstract

We studied the adverse effects of four benzodiazepines frequently measured in European surface waters. We evaluated bioaccumulation potential of oxazepam, bromazepam, temazepam, and clobazam in freshwater fish species - perch (Perca fluviatilis) and we conducted a series of behavioral trials to assess their potential to alter boldness, activity, and social behavior. All selected endpoints were studied individually for each target

1

benzodiazepine and as a mixture of all tested compounds to assess possible combinatory effects. We used a three-dimensional automated tracking system to quantify the fish behavior.

The

four

compounds

bioconcentrated

differently

in

fish

muscle

(temazepam>clobazam>oxazepam>bromazepam) at high exposure (9.1, 6.9, 5.7, 8.1 µg L-1, respectively) and low exposure (0.5, 0.5, 0.3, 0.4 µg L-1, respectively) concentrations. A significant amount of oxazepam was also measured in fish exposed to temazepam, most likely because of the metabolic transformation of temazepam within the fish. Bromazepam, temazepam, and clobazam significantly affected fish behavior at high concentration, while no statistically significant changes were registered for oxazepam. The studied benzodiazepines affected behavior in combination, because the mixture treatment significantly changed several important behavioral traits even at low concentration, while no single compound exposure had such an effect at that dose. Based on our results, we conclude that effects of pharmaceuticals on aquatic environments could be underestimated if risk assessments only rely on the evaluation of single compounds. More studies focused on the combinatory effects of environmentally relevant mixtures of pharmaceuticals are necessary to fill the gaps in this knowledge.

1. Introduction

Pharmaceuticals represent an important group of xenobiotics present in the environment. Many of these compounds are an indispensable part of human lives, and their consumption has rapidly increased over the last few decades both for treatment of human diseases and for use in livestock husbandry (Bernhardt et al., 2017). For instance, consumption of 2

antihypertensive drugs almost doubled between 2000 and 2015 in OECD countries and similar trends can be seen for antidiabetics and antidepressants, while consumption of cholesterollowering drugs has nearly quadrupled in the same time period (OECD, 2017). Also, the total number of different pharmaceuticals regularly used in human and veterinary medicine is increasing, as more than 4 000 pharmaceutically active compounds (PhACs) are currently being produced and marketed (Ur Rehman et al., 2015). PhACs enter aquatic environments through several main pathways of which one important pathway is wastewater. The majority of sewage and wastewater in developed countries is centralized and processed in sewage treatment plants (STPs), and PhACs excreted or improperly disposed in sewage enter aquatic environments because they are a common recipient of treated sewage effluents. As most STPs only consist of conventional physical and biological treatment focused on removing nutrients, they are unable to completely remove all PhACs from the sewage water (Zorita et al., 2009; Subedi et al., 2015; Yang et al., 2017). Various classes of PhACs (e.g. antibiotics, antiparasitics, anti-inflammatory drugs, or hormonal growth promotors) that are used in livestock husbandry also enter aquatic environments (Bertram et al., 2018; Charuaud et al., 2019) via STPs, run-off from livestock operations, or run-off from agricultural sites where the manure of treated animals is used as organic fertilizer. Besides the manure, treated sewage sludge from STPs is also frequently used in agriculture and represents yet another source of human PhACs to the environment (Ivanová et al., 2018). Concentrations ranging from sub-ng L-1 to thousands of ng L-1 are reported for a wide range of pharmaceuticals in aquatic environments across the globe, indicating that PhACs are commonly occurring contaminants (Liu et al., 2015; Biel-Maeso et al., 2018; Kallenborn et al., 2018; Fekadu et al., 2019; Charuaud et al., 2019; Kim et al., 2019). Although such 3

concentrations are not acutely toxic, they may still affect aquatic organisms in different ways, e.g. altering fish metabolism (Burkina et al., 2015; Du et al., 2018), inducing endocrine disruption (Niemuth et al., 2015), affecting early life development (Zhang et al., 2015), or altering natural fish behavior (Brodin et al., 2013; Kellner et al., 2016; McCallum et al., 2017). Furthermore, the processes of bioconcentration, bioaccumulation, and biomagnification within the aquatic food web may result in tissue concentrations that are substantially higher than waterborne concentrations (Grabicova et al., 2014; Liu et al., 2015; Moreno-González et al., 2016; Grabicova et al., 2017; Huerta et al., 2018). Exposure to PhACs in the wild may have important sub-lethal ecological consequences for aquatic organisms (Brodin et al., 2014). Specifically, psychoactive pharmaceuticals are designed to affect human behavior as part of the intended treatment (antidepressants, anxiolytics, antipsychotics) or as a side-effect of certain treatments (analgesics) and may affect aquatic wildlife similarly. This is because many drug-targets/receptors have been widely conserved across the vertebrate taxa, as such similar effects as in humans could be also expected in fish (Gunnarsson et al., 2008; Brown et al., 2014). In ecotoxicology, antidepressants and anxiolytics are two of the most studied classes of PhACs, and several authors have demonstrated that they can alter the behavior of aquatic organisms even at low (environmentally relevant) concentrations (Winder et al., 2012; Kellner et al., 2016; Brodin et al., 2017). Anxiolytics (specifically, oxazepam) are one of the most extensively studied psychoactive compounds, mainly because of their effects on animal behavior (Heynen et al., 2016c; Lagesson et al., 2016; Garcia-Galan et al., 2017; Miller et al., 2017; Saaristo et al., 2019). To date, most behavioral studies have assessed the effect of single PhACs, even though animals are exposed to complex mixtures in the wild that may have potentially combinatory effects (Backhaus, 2016). For instance, exposure to several related PhACs with a similar 4

mechanism of action may result in synergistic effects (Ågerstrand et al., 2015). Only a few studies have addressed the combinatory effects of pharmaceutical mixtures on behavioral endpoints (Schoenfuss et al., 2016; Liu et al., 2017; Melvin, 2017; Porseryd et al., 2017), indicating additive effects of various PhACs. The aim of our study was to describe the combinatory effects of four benzodiazepines (oxazepam, bromazepam, temazepam, and clobazam) on wild juvenile fish behavior. These four benzodiazepines were previously found to be most common in a survey that was done in thirty European rivers and they co-occurred at several sampling sites (Fick et al., 2017).

2. Material and methods 2.1. Experimental fish

Here we used European perch (Perca fluviatilis) as model organism for this study. European perch is a common freshwater fish species across Europe and parts of Asia and often occupy an intermediate position in aquatic food-webs. Using a beach seine, we collected approximately 500 young-of-the-year perch from Lake Bjännsjön (Umeå municipality, Sweden) in June 2017. Fish were transported in aerated tanks to a pond at the Umeå Experimental Ecosystem Facility of Umeå University in Röbäck providing a natural habitat with dense macrophyte vegetation and optimal food resources. At the end of September, fish were recaptured from the pond using umbrella traps and subsequently transferred to aerated, flowthrough tanks at Umeå University. During a six week acclimatization period, fish were fed with frozen chironomids with 10% addition of sinking dry food (Inicio, BioMar). Fish were fed daily

5

and the chironomid/dry food ratio was adjusted every day, reaching a completely dry food diet after two weeks. The flow-through tanks were continuously fed by non-chlorinated tap water of following chemical properties: pH, 8; ammonium [NH4+], <0.004 mg L-1; nitrite [NO2], <0.003 mg L-1; oxygen saturation, >100%. The light/dark regime was set to 12/12 hours. In addition to the perch, four adult pikes (Esox lucius) between 25 and 30 cm of total length were caught by beach seining in the Ume River Delta and kept in a flow-through tank at Umeå University. All experimental animals were handled in accordance with Ethical Committee on Animal Experiments in Umeå (dnr: A18-15), current Swedish law, and institutional guidelines for the protection of human subjects and animal welfare (European parliament and Council, 2010).

2.2. Exposure regime

Experimental fish were exposed to eight single compound treatments: oxazepam, bromazepam, temazepam, clobazam at nominal concentration of 0.5 (low) and 8 (high) µg L-1 and two mixtures of all four benzodiazepines, where each compound was present in the mixture at nominal concentration of 0.5 (mix low) and 8 (mix high) µg L-1. Based on recently published work of Cunha, et. al (2019), low concentration used in our study should be considered as environmentally relevant for oxazepam in surface waters. Each treatment group consists of twenty individuals, and there was no difference in size of fish assigned to different treatments (Table 1). Fish were exposed individually for a period of seven days in 6L static tanks with aeration. . The exposure period was set according to Heynen et. al (2016a) who reported reaching the steady state in perch after 5 days of exposure to oxazepam at 6

concentration of 1.5 µg L-1. Exposure tanks were placed in a temperature-controlled room allowing a stable temperature of 11 °C and a 12/12 hour dark/light regime as in the holding tanks during acclimatization period. Trials were conducted in a staggered design to accommodate the large number of treatments over ten weeks. Therefore, three control treatment groups (no exposure) were included at the start, in the middle, and at the end of experiment. Detailed information about what treatment groups were run at which time is summarized in supplementary material (Figure S1).

2.3. Behavioral trials

All experiments were conducted in water with the same physical-chemical properties as used in the acclimatization and exposure periods. Each experimental fish underwent two different behavioral trials separated by one hour of recovery in their exposure tank. Three ecologically important behavioral traits were quantified across the two trials (boldness, social behavior, and swimming performance). Boldness (risk-taking) was carried out first, followed by a social behavior trial. Because of the total number of individuals used in the experiment and associated time with different trials, assays were only run after the exposure. To measure boldness, fish were placed in a glass aquaria (90x34 cm) containing 25 cm of water from the holding tank where pikes were kept, to saturate the tank with chemical predator-cues. Tanks were divided into two main parts that were separated by a permanent glass wall (Supplementary material, Figure S2a). The smaller compartment housed a pike used as a visual predator cue, while the perch were tested in the larger compartment, called the “testing arena”. The testing arena included a refuge with artificial vegetation made of plastic, 7

and an open “dangerous” area with no cover, both separated by a removable barrier (Supplementary material, Figure S2a). Each perch was tested independently and was introduced into the refuge part of arena before the trial and left to acclimate for 15 minutes. After that, the barrier was removed and fish movement in the testing arena was tracked for 60 minutes. Latency to leave the refuge and enter the potentially dangerous area for the first time, and total time spent out of the refuge were quantified. Individuals that did not leave the refuge during the trial were given the maximum score (3600 sec.). Swimming performance expressed as a total distance swam was also measured during the boldness trial. In the social behavior trial, smaller (60x30 cm) glass aquaria with water depth of 25 cm were used. These tanks were divided into three parts by permanent transparent barriers (Supplementary material, Figure S2b). One of the side compartments housed a shoal of conspecifics (four unexposed perch randomly selected from the holding tank), while the opposite compartment contained only water. The tested fish was then introduced in the center of central compartment (arena), left for 15 minutes to acclimate, and then tracked for 30 minutes. Sociability was quantified based on the spatial use of fish in the central compartment that, for sociability scoring purposes, was divided into 5 zones (software based, not visual for the fish) using a protocol based on (Brodin et al., 2013). Each zone was assigned a sociality factor (3, 1, 0, -1, -3) by which the time the fish spent in that zone was multiplied (e.g., the time fish spent in the zone closest to the shoal of conspecifics was multiplied by 3, the time in the next zone by 1, etc.). The higher the score a fish received the closer to the shoal it stayed (on average) and hence the more social it was.

2.4. Automated tracking system 8

Behavioral trials were recorded with a monitoring system based on the 3D – camera (Xbox One Kinect Sensor V2), which was placed above the aquaria for each trial. The system automatically records the 3D position of a moving fish to a log file with the sampling frequency of 30 locations per second and spatial resolution of 2 mm (Saberioon and Cisar, 2016). The analysis of the 3D fish track was done by in-house implemented software to calculate the time spent in defined regions, swimming distance and crossing of defined regions. Functionality of the tracking system was validated prior to the experiment. For that purpose, we used the same aquaria as used in the boldness trial, which was divided lengthwise into four sectors, one representing refuge with artificial vegetation. A perch of the same size as in exposure groups was introduced in the refuge sector and recorded for 40 minutes by both Kinect Sensor and a regular RGB camera (Sony HDR PJ50). Data obtained by manual inspection of recorded videos and those obtained from automated tracking system were evaluated statistically. Both datasets were strongly positively correlated with mean r2=0.974 (N=22 for both datasets). Detailed results of the validation experiment are presented in Supplementary materials (Figure S3).

2.5. Preparation of samples for chemical analyses

All experimental fish were sacrificed immediately after their last behavioral trial and kept frozen at -20°C until analysis. Seven of 20 individuals were sampled for chemical analyses from

9

each exposure treatment group. In case of control fish, four of 20 individuals were sampled from each of three control treatments, resulting in 12 individuals in total. When preparing the samples for analysis, fish were defrosted at room temperature, measured, weighed, and a muscle fillet from the left side of body was taken. Samples of muscle tissue (0.1 g) with added internal standard (50 ng per sample) and 1.5 mL of extraction solvent (acetonitrile) were extracted precisely following the same protocol as described in our previous study (McCallum et al., 2019a). Samples of water were taken from all individual tanks on both the first and the last day of exposure and immediately frozen in 10 mL plastic tubes. Before analysis, water samples were defrosted in a room temperature, 5 mL was filtered through a 0.45 µm syringe filter Filtropur S (Sarstedt, Nümbrecht, Germany) into the 10 mL autosampler glass vials, and 5 ng of internal standard was added.

2.6. Chemicals and reagents

LC/MS grade of acetonitrile and methanol (LiChrosolv—hypergrade) were purchased from Merck (Darmstadt, Germany). Formic acid (Sigma-Aldrich, Steinheim, Germany) was used to prepare the 0.1% mobile phases. Working mixtures of native compounds and surrogate standard were prepared in methanol at a concentration of 1 µg mL-1 and stored at -18 °C. All native compounds were of analytical grade (>98%). Bromazepam (CAS 1812-30-2) and temazepam (CAS 846-50-4) were purchased at LGC Standards (Teddington, UK), clobazam (CAS 22316-47-8) was purchased at British Pharmacopoeia (UK), oxazepam (CAS 604-75-1)

10

and mass labeled 2H5-oxazepam (CAS 65854-78-6), used as internal standard (IS) were purchased at Sigma Aldrich (Steinheim, Germany).

2.7. Instrumental analysis

A triple-stage quadrupole mass spectrometer Quantum Ultra EMR (Thermo Fisher Scientific, San Jose, CA) coupled to an Accela LC pump (Thermo Fisher Scientific, San Jose, CA) and a PAL HTC autosampler (CTC Analytics AG, Zwingen, Switzerland) was used for instrumental analyses of both water and tissue extracts samples. A C18 phase Hypersil gold column (50 mm x 2.1 mm ID x 3 µm particles, Thermo Fisher Scientific, San Jose, CA, USA) was used for the separation of target analytes. Water analysis followed the protocol of Fick et al. (2017), based on an online solid phase extraction system coupled with liquid chromatography-tandem mass spectrometry (SPE LCMS/MS), which has been also described in previous work (Khan et al., 2012). Concerning the tissue extracts analysis, method of LC-MS/MS with exactly the same analytical instrumentation was used. More details about the basic set-up of the electrospray ionization interface and the gradient and flow of the mobile phase are presented in supplementary material (Tables S1-S3). Chromatograms of target benzodiazepines in calibration curve and in muscle tissue samples are also presented in Supplementary material (Figures S4, S5) Quality assurance and quality control (QA/QC) of the analytical method for fish muscle samples was evaluated regarding its linearity, repeatability, limit of quantification (LOQ), and recovery. Instrumental LOQ was derived from the six-point calibration curve (0.5 to 50 ng g-1). 11

Peak area corresponding to this LOQ was then used for calculation of LOQs in individual samples. Corresponding values reflect differences among IS recovery, weight, and final volumes of the extract in each sample. The mean LOQs ranged from 0.3 to 0.5 ng g-1 depending on target compound. Quantification of target compounds in fish samples was done using internal standard approach. More information about repeated measures, recoveries, and LOQs is given in Supplementary material (Table S4). Several blanks were measured with each series of samples, target compounds were not found above the LOQ in any of them.

2.8. Statistical analyses

Analysis of variance – one-way ANOVA was used to test for differences between the treatments where data were normally distributed (length and weight of fish), whereas nonparametric Kruskal-Wallis tests were used to evaluate the effect of exposure on fish behavior because these data deviated from a normal distribution. Shapiro-Wilk and KolmogorovSmirnov tests were used to test for normality. All statistical analyses were performed using Statistica 12 software (StatSoft Inc., USA). To assess the effects of treatments on behavior, each benzodiazepine exposure-type (i.e. single or mix) was evaluated individually by comparing results obtained at both concentration levels with the control group. When significant, post-hoc multiple comparisons of mean ranks (two-sided significance levels with a Bonferroni adjustment) were done. The rationale behind choosing this statistical approach was in part due to a statistical significance in boldness was found between control groups, most likely as a result of time dependent habituation of fish to lab conditions (i.e. the different time that fish spent in holding tanks). Because of this, each 12

compound/mixture was compared to its most relevant control group (based on the date, when behavior trials were run) to avoid false positive results and misinterpretation of data. Results of statistical analyses are presented in Supplementary material (Tables S5—S8).

3.

Results and discussion

3.1. Chemical analyses

The highest bioconcentration potential in the single exposure scenarios was from temazepam, followed by clobazam, oxazepam, and bromazepam, both when measured as muscle tissue concentrations or bioconcentration factor (BCF) (Table 2). Relatively high concentrations of oxazepam were also measured in fish exposed to temazepam (Figure 1). As no oxazepam was detected in water from temazepam treatments, its presence in fish muscle is likely the result of metabolic transformation of temazepam within the fish body, which was previously described in humans and animals (specifically, mouse, rat, and dog) (Schwarz, 1979). It should be noted that oxazepam is not a metabolite of bromazepam or clobazam. This finding emphasizes the need to include biologically active metabolites of pharmaceuticals in ecotoxicology studies. Concentrations of benzodiazepines measured in muscle of fish were lower when present together in the water with the exception of oxazepam. Higher oxazepam concentrations were found in fish exposed to mixture treatments than in those from single exposures at both concentrations (Table 2, Figure 1). This was especially evident in the high mixture concentration, where oxazepam expressed the highest concentration from all four benzodiazepines tested. We suggest again that this finding is mostly likely a result of a

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combination of direct exposure to oxazepam in the water and from metabolic transformation of temazepam into oxazepam. To the best of our knowledge, only the uptake of oxazepam and temazepam has been previously measured in fish (Brodin et al., 2013; Huerta et al., 2016; McCallum et al., 2019b; Saaristo et al., 2019). Most of these studies have focused on oxazepam and reported slightly different tissue concentrations and bioconcentration factors (BCFs), most likely as a result of species-specific differences in uptake/elimination and the concentration of oxazepam in the water during exposure. Besides the species-specific differences, other factors may also affect uptake and bioconcentration. It was reported by Heynen et al. (2016b) for oxazepam, that its uptake in perch was higher when fish were exposed individually compared to those exposed in groups, most likely as a result of elevated stress associated with being housed in isolation. This same study also showed a negative correlation between BCF and fish weight. Unlike oxazepam, only one previous study has measured the uptake of temazepam. Temazepam concentration in sea trout (Salmo trutta) was found to be 5.4 ng g-1 in muscle tissue after a 6-day exposure to 0.8 µg L-1 (BCF=6.4) (McCallum et al., 2019b), which is comparable with our findings in perch. Based on data presented in this paper and findings of other authors, benzodiazepines typically have a low bioconcentration potential. In our study and other previous works, a BCF<10 was always reported. As such, benzodiazepine BCF values are far below the regulatory thresholds. Despite their low bioconcentration potential, benzodiazepines and some other psychoactive pharmaceuticals have been shown to have ecological consequences for wild aquatic animals, even at low concentrations and low BCF values. For such reasons, the importance of BCFs as the criterion in risk assessment of pharmaceutically active substances should be discussed. We also assume that a broad range of both biotic and abiotic factors are 14

playing role in the mechanisms of uptake and possible bioconcentration of pharmaceuticals. Some of those factors, e.g. stress or fish size can be addressed when planning experiments, while others e.g. temperature and metabolites should be studied in more detail. Temperature driven effects are an especially big gap in current knowledge, even though this represents one of the most important abiotic factors for aquatic organisms that live with seasonal variability.

3.2. Behavioral analyses

Except for oxazepam, exposure to other three benzodiazepines resulted in a change of important behavioral traits in perch. Statistically significant effects were measured for boldness and activity, while no changes in sociality were induced by any of treatments. The results also indicate that benzodiazepines had a combinatory effect on fish behaviour because exposure to even the low concentration mixture of compounds increased boldness and activity in perch. In contrast, the single-compound exposures only had significant effects at the high concentration and they only affected boldness behaviours.. However, we are not able to confirm at this point whether the increased effect in our mixture treatments was caused by compound additivity or synergism. More research would be necessary to clarify the physiological basis of observed combinatory effect on fish behavior. Three endpoints were quantified in the boldness trial (latency to leave the refuge, time spent out of refuge, and swimming performance). Latency to leave the refuge was most sensitive to benzodiazepine exposure, as single exposures to bromazepam (p=0.001), temazepam (p<0.001), and clobazam (p=0.039) at high (8 µg L-1) concentration decreased this latency (i.e., fish left the refuge faster). 15

For the time that fish spent out of the refuge in the boldness trial, there was no clear effect following any of the individual benzodiazepine exposure scenarios. In general, most individuals spent most of their time in the refuge. Only 32% of fish across all the individual and mixture treatments spent more than 3 minutes (5% of trial duration) in the potentially dangerous zone. Similar results were obtained in case of swimming performance with no significant changes observed in fish exposed to the single compounds. Contrary to single exposure scenarios, fish exposed to both low and high concentration mixture treatments were bolder and swam longer during the trial (Figure 2). Moreover, these effects did not increase with exposure concentration in mixture treatments, which was different from single exposure scenarios. This might indicate that binding at the benzodiazepine site on the GABAA receptor was saturated or at a level to induce therapeutic effects even in the perch exposed to low concentration mixture of studied compounds. These results are the first to show that benzodiazepines can have combinatory effects on the behavior of aquatic organisms. Oxazepam is the most studied benzodiazepine to date, and the anxiolytic effects of oxazepam on behavior in different fish species has been reported by various authors (Brodin et al., 2013; Hellstrom et al., 2016; Brodin et al., 2017; Saaristo et al., 2019; Sundin et al., 2019). In our study, oxazepam was the only benzodiazepine that did not affect any behavioral endpoints. Several reasons might explain these findings. One is the design of the boldness trial in our experiment that combined both the chemical and visual predator cue to produce a very stressful and dangerous environment in the testing arena. In previous studies, where an effect of oxazepam on boldness was reported, the scototaxis assay (light/dark preference, (Maximino et al., 2010)) was used (Brodin et al., 2017; Saaristo et al., 2019). This explanation is supported by the fact that Saaristo et al. (2019) showed that chemical predator cues alone 16

(no oxazepam exposure) reduced boldness in perch, i.e. affected boldness in the opposite direction as oxazepam exposure. Based on these findings, we hypothesize that behavioral effects of oxazepam might be counteracted by strong predator cues. Oxazepam concentrations, both in water and internally, is another likely factor playing an important role in explaining the differing effects of oxazepam on boldness or swimming performance, as previous works reported effects following exposure to concentrations over 100 µg L-1 (Brodin et al., 2013; Brodin et al., 2017; Sundin et al., 2019). There is very limited literature on the possible effects on aquatic organisms of the other benzodiazepines (excluding oxazepam) in our study. Temazepam was recently found to alter fish behavior, as sea trout (Salmo trutta) exposed to 0.05 µg L-1 migrated faster in a field based experiment (McCallum et al., 2019b). Also bromazepam has been shown to affect various behavioral traits in zebrafish (Danio rerio), but the concentration used in that study (1.5 mg L-1) is much higher than what could be considered environmentally relevant (Gebauer et al., 2011). No statistically significant effects on fish social behavior were found in any of single exposure or mixture treatments in our study (Figure 3). Again, few studies have evaluated the effect of benzodiazepines on fish social behavior (but see Brodin et al., 2013; Brodin et al., 2017), and only the effect of oxazepam on perch and roach. In the case of roach, no effect on sociality was found even at the high concentration (280 µg L-1), while a concentration of 1.8 µg L-1 resulted in decreased sociality in juvenile perch. In the present study, the same behavioral sociality protocol was used as in the two previously mentioned, but our experiment was done at a lower temperature. It was previously reported that temperature itself has a potential to affect behavior in different fish species (Biro et al., 2010; Forsatkar et al., 2016). In the work of Maulvault et al. (2018), increased temperature was found to magnify the effect 17

of antidepressant venlafaxine on behavior of a marine fish species (Argyrosomus regius). It was also reported that temperature affects the behavior of perch, but no interaction effects with oxazepam were found (Saaristo et al., 2019). It is without the doubt that temperature represents an important environmental factor to consider when assessing the effects of pharmaceuticals on wild fish. Despite this, there is a huge lack of knowledge regarding the interactions between temperature and both the bioconcentration potential of pharmaceuticals and their effects on fish behavior. The need of experiments addressing these interactions is essential for understanding the bioconcentration and effects of pharmaceuticals across seasons and also to predict their effects under different scenarios of climate change. Besides the studied effects of exposure to benzodiazepines, we noted that the time that fish spent in laboratory conditions (holding tanks) was another factor affecting some of the studied behavioral traits. All endpoints measured in the boldness trial differed between the control groups tested at the start, middle, and end of the experiment, indicating that the more time the fish spent in laboratory, the more bold they acted. Therefore, time in the laboratory needs to be considered when planning and analyzing data from behavioral ecotoxicology experiments with wild fish.

4. Conclusions

We conducted a mixed-toxicology experiment focused on bioconcentration and important ecological effects of four benzodiazepines (oxazepam, bromazepam, temazepam, and clobazam). When fish were exposed to individual compounds, temazepam had the highest 18

potential to bioconcentrate. Interestingly, a significant amount of oxazepam was also measured in fish exposed to temazepam, likely as a result of its metabolic transformation. This emphasizes the need to identify and study the biologically active metabolites of pharmaceuticals. Except for oxazepam, the other three benzodiazepines showed lower bioconcentration when fish were exposed to the benzodiazepine mixture when compared to the single exposure scenarios. The contrasting pattern of bioconcentration of oxazepam, we suggest, is due to combined accumulation of oxazepam from water and from metabolic transformation of temazepam in the fish body. Concerning fish behavior, temazepam and clobazam increased boldness in perch at 8 µg L-1. We confirmed combinatory effects of the four benzodiazepines on fish behavior, as both boldness and activity of perch were significantly altered by mixture treatments at both exposure concentrations. The benzodiazepines chosen for this study are frequently found in aquatic environments, and our findings suggest that wild fish might show changes to their behaviour if exposed to these psychoactive compounds in the wild. Nevertheless, further studies, especially field based experiments, are necessary to validate conclusions regarding ecological effects obtained in laboratory experiments before we can reliably predict effects in the natural environment.

Acknowledgements The study was financially supported by Swedish Research Council Formas (2013-4431) to Tomas Brodin. The Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA“ (LM2018099),“CENAKVA Center Development“ (No. CZ.1.05/2.1.00/19.0380). Daniel Cerveny was supported by the Kempe foundation.

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23

90

oxazepam bromazepam temazepam clobazam

80 70 60

ng g-1

50 40 30 20 10

gh hi ix m

ix l m

hi m

ba

za

ow

gh

w lo clo

ba

za

m

hi clo

am

te

m az

ep

ep m az

te

gh

w lo am

hi am

om

az

ep

ep br

az om br

gh

w lo am

hi am ep

ox az

ox az

ep

am

lo

gh

w

0

Figure 1. Concentrations of target benzodiazepines in muscle tissue of perch after 7 days of exposure to nominal concentration of 0.5 and 8 µg L-1 in single and mixture exposure scenarios. Mean with standard deviation of 7 analyzed individuals per each group is presented.

24

25

Figure 2. Boldness trial results (mean ± 1 SE) of perch exposed to four different benzodiazepines and their mixture at nominal concentration of 0.5 and 8 µg L-1. Letters indicate significant differences (p<0.05) between groups. a) Latency to leave the refuge shows the time that fish first enter the potentially danger zone, lower value indicate bolder fish; percentage shows how many fish from the group left the refuge during the trial. b) Time spent out of refuge shows cumulative time that fish spent in potentially danger zone, higher value indicate bolder fish. c) Swimming performance is quantified as a total distance swam during the boldness trial.

Figure 3. Sociality trial results (mean ± 1 SE) of perch exposed to four different benzodiazepines and their mixture at nominal concentration of 0.5 and 8 µg L-1. Sociality index represents cumulative time (sec) multiplied by a specific sociality factor (-3, -1, 0, 1, 3) according to the distance from a group of conspecifics. Means and standard deviations are presented. Lower value indicates less social behavior.

26

Table 1. Characteristics of experimental fish n1

12

oxazepam bromazepam clobazepam low high low high low high 7 7 7 7 7 7

total length (mm ± SD)

70.5 ± 5.3

69.0 67.6 ± 3.0 ± 6.1

67.3 68.4 ± 6.1 ± 7.5

71.4 69.3 ± 6.5 ± 4.2

68.7 68.0 ± 5.7 ± 4.8

70.4 72.0 ± 9.0 ± 7.5

weight (g ± SD)

3.1 ± 1.0

2.9 2.8 ± 0.9 ± 0.9

2.6 3.2 ± 0.5 ± 1.2

3.4 3.0 ± 1.1 ± 0.6

2.8 2.9 ± 1.0 ± 0.6

3.4 3.4 ± 1.9 ± 1.5

Treatment

control

temazepam low high 7 7

mix low high 7 7

n is lower because measurements were only done on individuals sampled for chemical analyses 1

Table 2. Concentrations of oxazepam, bromazepam, clobazam, and temazepam in perch muscle tissue and water from exposure tanks in both individual exposure scenarios and mixture treatments. Bioconcentration factors were calculated from water and muscle tissue concentrations of target compounds.

target compound/matrix

n

water

muscle tissue

µg L-1

ng g-1

BCF

mean ± SD (min — max) 0.4 ± 0.02 low

1.6 ± 0.4

7

4 (0.3 — 0.4)

(0.8 — 2.1)

5.7 ± 0.3

16.5 ± 6.4

(5.3 — 6.3)

(4.2 — 25.8)

0.4 ± 0.5

0.6 ± 0.2

(0.4 — 0.5)

(0.4 — 0.9)

8.1 ± 1.2

10.5 ± 2.6

oxazepam high

low

7

2.9

7

1.5

bromazepam high

low

7 (6.7 — 10.1)

(7.5 — 14.5)

0.5 ± 0.04

1.4 ± 0.1

(0.4 — 0.5)

(1.2 — 1.6)

6.9 ± 0.6

26.3 ± 4.3

7

clobazam high

1.3

7

27

2.8

3.8

low

(6.4 — 8.2)

(21 — 34.2)

0.5 ± 0.03

5.0 ± 3.8

7

10 (0.5 — 0.6)

(2.1 — 13.8)

9.1 ± 0.9

51.6 ± 19.1

(7.8 — 10.3)

(29 — 79.4)

0.4 ± 0.03

2.3 ± 1

(0.3 — 0.4)

(1.2 — 4.2)

0.7 ± 0.1

0.6 ± 0.2

(0.6 — 1)

(0.3 — 1)

0.5 ± 0.1

3.9 ± 2.4

temazepam high

oxazepam

bromazepam

7

5.7

7

5.7

7

0.9

mix low temazepam

clobazam

oxazepam

bromazepam

7

7.8 (0.5 — 0.7)

(1.3 — 9.2)

0.5 ± 0.1

1.9 ± 1

7

3.8 (0.4 — 0.6)

(1.3 — 4.3)

6.3 ± 0.7

54.1 ± 27.1

7

8.6 (5.5 — 7.2)

(25.8 — 106.1)

14 ± 2.1

6.4 ± 2.2

7

0.5 (11.4 — 16.6)

(4.5 — 9.9)

9.4 ± 0.6

38.7 ± 11.6

(8.9 — 10.5)

(22.6 — 59.4)

9.5 ± 0.5

20.2 ± 3.5

(9 — 10.3)

(15.4 — 26.1)

mix high temazepam

clobazam

7

4.1

7

28

2.1

Highlights Bioconcentration and behavioral effects of four benzodiazepines in fish were examined Temazepam showed the highest bioconcentration potential Oxazepam is produced by metabolic transformation of temazepam in fish All studied compounds expressed potential to alter important fish behavior Additive effect of benzodiazepine mixture on behavior of perch was confirmed

29

Graphical abstract

30