Journal Pre-proofs

Bioconcentration and behavioral effects of four benzodiazepines and their en-

vironmentally relevant mixture in wild fish

D. Cerveny, T. Brodin, P. Cisar, ES. McCallum, J. Fick

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

To appear in: Science of the Total Environment

Received Date: 4 July 2019

Revised Date: 20 September 2019
Accepted Date: 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 of Chemistry, Umeå University, SE-90187, Umeå, Sweden

b University 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, cerveny@frov.jcu.cz

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 cholesterol-

lowering 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, flow-

through 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 LC-

MS/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 non-

parametric 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 Kolmogorov-

Smirnov 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

13
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
80 oxazepam
70 bromazepam
temazepam
60
clobazam
50
ng g-1

40
30
20
10
0
w

gh

gh

gh

gh

ow

gh
lo

lo

lo

lo
hi

hi

hi

hi

hi
l
am

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am

ix
am

am

am

ix
m
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m
za
ep

ep

ep
ep

ep

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ba
az

az

az
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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
oxazepam bromazepam clobazepam temazepam mix
Treatment control
low high low high low high low high low high
n1 12 7 7 7 7 7 7 7 7 7 7

total length 70.5 69.0 67.6 67.3 68.4 71.4 69.3 68.7 68.0 70.4 72.0
(mm ± SD) ± 5.3 ± 3.0 ± 6.1 ± 6.1 ± 7.5 ± 6.5 ± 4.2 ± 5.7 ± 4.8 ± 9.0 ± 7.5

weight 3.1 2.9 2.8 2.6 3.2 3.4 3.0 2.8 2.9 3.4 3.4
(g ± SD) ± 1.0 ± 0.9 ± 0.9 ± 0.5 ± 1.2 ± 1.1 ± 0.6 ± 1.0 ± 0.6 ± 1.9 ± 1.5

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

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.

water muscle tissue

target compound/matrix n µg L-1 ng g-1 BCF

mean ± SD (min — max)

0.4 ± 0.02 1.6 ± 0.4

low 7 4
(0.3 — 0.4) (0.8 — 2.1)
oxazepam
5.7 ± 0.3 16.5 ± 6.4
high 7 2.9
(5.3 — 6.3) (4.2 — 25.8)

0.4 ± 0.5 0.6 ± 0.2

low 7 1.5
(0.4 — 0.5) (0.4 — 0.9)
bromazepam
8.1 ± 1.2 10.5 ± 2.6
high 7 1.3
(6.7 — 10.1) (7.5 — 14.5)

0.5 ± 0.04 1.4 ± 0.1

low 7 2.8
clobazam (0.4 — 0.5) (1.2 — 1.6)

high 7 6.9 ± 0.6 26.3 ± 4.3 3.8

27
 (6.4 — 8.2) (21 — 34.2)

0.5 ± 0.03 5.0 ± 3.8

low 7 10
(0.5 — 0.6) (2.1 — 13.8)
temazepam
9.1 ± 0.9 51.6 ± 19.1
high 7 5.7
(7.8 — 10.3) (29 — 79.4)

0.4 ± 0.03 2.3 ± 1

oxazepam 7 5.7
(0.3 — 0.4) (1.2 — 4.2)

0.7 ± 0.1 0.6 ± 0.2

bromazepam 7 0.9
(0.6 — 1) (0.3 — 1)
mix low
0.5 ± 0.1 3.9 ± 2.4
temazepam 7 7.8
(0.5 — 0.7) (1.3 — 9.2)

0.5 ± 0.1 1.9 ± 1

clobazam 7 3.8
(0.4 — 0.6) (1.3 — 4.3)

6.3 ± 0.7 54.1 ± 27.1

oxazepam 7 8.6
(5.5 — 7.2) (25.8 — 106.1)

14 ± 2.1 6.4 ± 2.2

bromazepam 7 0.5
(11.4 — 16.6) (4.5 — 9.9)
mix high
9.4 ± 0.6 38.7 ± 11.6
temazepam 7 4.1
(8.9 — 10.5) (22.6 — 59.4)

9.5 ± 0.5 20.2 ± 3.5

clobazam 7 2.1
(9 — 10.3) (15.4 — 26.1)

28
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