Psychotropic in the environment: Risperidone residues affect the behavior of fish larvae

Abstract

The ability to avoid and escape from predators are clearly relevant behaviors from the ecological perspective and directly interfere with the survival of organisms. Detected in the aquatic environment, risperidone can alter the behavior of exposed species. Considering the risk of exposure in the early stages of life, we exposed zebrafish embryos to risperidone during the first 5 days of life. Risperidone caused hyperactivity in exposed larvae, which in an environmental context, the animals may be more vulnerable to predation due to greater visibility or less perception of risk areas.

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Psychotropic in the environment:
risperidone residues aect the
behavior of sh larvae
Fabiana Kalichak1, Renan Idalencio1,2, João Gabriel Santos da Rosa1, Heloísa Helena
de Alcântara Barcellos1,2, Michele Fagundes2,3, Angelo Piato
4 & Leonardo José Gil
Barcellos1,2,3,5
The ability to avoid and escape from predators are clearly relevant behaviors from the ecological
perspective and directly interfere with the survival of organisms. Detected in the aquatic environment,
risperidone can alter the behavior of exposed species. Considering the risk of exposure in the early
stages of life, we exposed zebrash embryos to risperidone during the rst 5 days of life. Risperidone
caused hyperactivity in exposed larvae, which in an environmental context, the animals may be more
vulnerable to predation due to greater visibility or less perception of risk areas.
Emotional states such as fear and anxiety are possible to be observed in zebrash larvae. With a comprehensive
behavioral range, these animals are already responsive to the environment at 24 hpf (hours post-fertilization) and
within a week of life, they already respond to stimuli such as touch, sound, water movement or changes in light1,2.
Unlike adults, zebrash larvae prefer clear areas (dark areas are thought to simulate predator shade), avoid areas
of light oscillation and recognize when placed in open areas2–4. Even during the rst week of life, zebrash larvae
may already are sensitive to the same anxiolytics used for anxiety in humans3,5.
Behavioral changes have the potential to impact directly on the physical condition and the perpetuation of a
species2,4. One of the most known behaviors in nature is the prey-predator relationship. To avoid predators and
consequently potential life risks, the escape behavior is fundamental to the species maintenance. Emotions such
as fear or anxiety can be observed in all vertebrates and are very important to maintainance and survival ofthe
species, and the preservation of these escape patterns is observed in mostsh species. Changes or alterations in
this response may induce a direct risk to the individuals or even in severepopulational consequences1,3.
In this context, aquatic contaminants are involved in many behavioral changes of exposed species. Several
drugs consumed by the population promotes an increase in the amount of drug residues in the aquatic environ-
ment6–9. Even when detected in low amounts, the ability to cause changes in the physiology of non-target organ-
isms has not yet been fully elucidated, but it is known that even at low concentrations (ng/L or μg/L) these drugs
may have eects on exposed species6,8–10.
Risperidone (RISP), an atypical antipsychotic used mainly for the treatment of schizophrenia and bipolar
mood disorder, has already been detected at dierent levels in aquatic environments11,12. RISP levels have already
been recorded up to 0.0014 μg/L in seawater8, 0.0029 μg/L in euent water7, and 0.0034 μg/L in drinking water13.
In Belgium, the highest level of environmental contamination was recorded, presenting 0.364 μg/L in auents
and 0.154 μg/L in Dendre River euent14. A few studies have been carried out to nd out the consequences of
species exposure and this type of contaminant at low concentrations15,16. Despite behavioral changes are com-
mon ndings in mammals exposed to risperidone during embryonic development17, no reports about behavioral
changes in the RISP-exposed sh were found in the current literature. us, in view of the great importance of
1Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000,
Cidade Universitária, Camobi, Santa Maria, RS, 97105-900, Brazil. 2Universidade de Passo Fundo (UPF), BR 285,
São José, Passo Fundo, RS, 99052-900, Brazil. 3Programa de Pós-Graduação em Ciências Ambientais, Instituto de
Ciências Biológicas, Universidade de Passo Fundo (UPF), BR 285, São José, Passo Fundo, RS, 99052-900, Brazil.
4Programa de Pós-Graduação em Farmacologia e Terapêutica, Instituto de Ciências Básicas da Saúde, Universidade
Federal do Rio Grande do Sul (UFRGS), Av. Sarmento Leite 500/305, Porto Alegre, RS, 90050-170, Brazil. 5Programa
de Pós-Graduação em Bioexperimentação, Faculdade de Agronomia e Medicina Veterinária, Universidade de Passo
Fundo (UPF), BR 285, São José, Passo Fundo, RS, 99052-900, Brazil. Correspondence and requests for materials
should be addressed to L.J.G.B. (email: lbarcellos@upf.br)
Received: 13 June 2017
Accepted: 4 October 2017
Published: xx xx xxxx
OPEN
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preserving all the behavioral repertoire, we sought to identify the eects of RISP exposition on behavioral param-
eters in embryos and larvae zebrash.
Results
Fishexposed to the concentration of 0.03 μg/L showed an increase in mortality when compared to the control
group (Fig.1) (p < 0.0001). No dierences were found in hatching and heart rate.
During the open field test, RISP at a concentration of 0.003 μg/L increased the total distance traveled
(p = 0.0009), the number of entries in the central area (p = 0.0127) and the mean speed in the nal phase of the
test (6–12 min) (p = 0.0009). e concentration of 0.0003 μg/L increased the immobility time in the settling phase
(p = 0.0542) (0–6 min), but this result did not last during the nal test phase (6–12 min) (p = 0.6093) (Fig.2).
Larvae exposed to the concentration of 0.003 μg/L RISP decreased the response to the aversive stimulus and
remained mostly in the stimulus area when compared to the control group (p = 0.0011). e other groups did not
present alterations (Fig.3).
At the concentration of 0.003 μg/L larvae decreased the latency for entry into the dark side of the well
(p = 0.0229), as well as increasing the number of entrances and residence time on this side during the second
phase of the test (6–12 min) (p = 0.1532). In the initial phase of the test, there was no dierence between groups
(Fig.4). ere was no dierence between the groups tested in relation to spontaneous movement.
Discussion
Here we show that the presence of RISP residues in water can alter the exploratory behavior of zebrash embryos
and larvae. In fact, during the rst-time window (0–6 min) in the open eld test, aversive stimuli and light/dark
tests, larvae exposed to 0.0003 µg/L RISP increased the immobility time. e observed eects of extremely low
RISP concentration were surprising since that this concentration was already detected in natural aquatic envi-
ronments13, indicating the potential risk for populations exposed to this type of contaminant10. Moreover, larvae
exposed to higher concentration of RISP displayed an increased mortality (5.68% in relation to the control group)
and no signicant alteration in hatching. ese results are consistent with our previous study, where RISP aects
parameters such as survival, hatching, heart rate and total larval length16.
In the 2nd time window (6–12 min) of the exploration tests, larvae presented increased exploratory activity
and decreased response to the environmental stimuli, both indicating hyperactivity. e dierence between 1st
and 2nd-time window contradicts the actual knowledge that larvae do not present an adaptation period as seen in
adults18. Further studies should be carried out to validate this hypothesis.
RISP is an atypical antipsychotic antagonist of serotonin and dopamine receptors19. Changes in the explor-
atory activity caused by RISP are common ndings in adult rats17 and adult20 as well as larvae21 zebrash. ese
changes in exploratory behavior are expected since that activity of dopaminergic and serotonergic neurons is
related to motor coordination22,23.
e eects observed in our larvae appear to be associated with RISP exposure in early stages of development.
In fact, the exposure to antipsychotics during the embryonic stage is associated with reduced levels of neurotrans-
mitters leading to functional problems in exposed sh24 and mammals17,25,26. In addition, our larvae were exposed
for 5 days and evaluated on the 6th day. is 24h-period without exposure may be related to these eects since the
chronic administration of psychoactive drugs also appears to lead to an increase in the activity of dopaminergic
and serotonergic receptors shortly aer the drug withdrawal25–31.
In our study, the eects were mainly observed in the intermediary RISP concentration, but not in the higher.
In fact, our tested concentrations were lower than plasma levels suitable to cause therapeutic eects32 or con-
centrations commonly tested in scientic experiments17,24,30,31 but exerted eects on zebrash larvae, like those
described with higher concentration. us, any comparison between our results and those reported in the liter-
ature is dicult, since no reports were found using RISP concentration as low as we used in the present study.
The last comment is about the possible implications of our results. In fish, dopamine and serotonin are
involved in locomotion, attack/defense, learning/memory and eating behavior33. e ability to capture prey
and escape from predators are clearly relevant behaviors from the ecological perspective, as they directly inter-
fere with the growth and survival of organisms33,34. A prey may be more susceptible to predation as a result of
non-detection of predators, poor escape performance, reduced resistance, inability to learn and greater visibility
Figure 1. e larvae exposed to the highest concentration of risperidone tested showed increased mortality.
Survival curve evaluated by Kaplan-Meier method. *p < 0.05. N = 160.
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due to hyperactivity33,35. Our results highlight that RISP altered larvae activity patterns, which in an environmen-
tal context can directly inuence the ability to avoid or evade predatory behavior which may result in signicant
repercussions on the maintenance of the species as well on the ecosystem.
Figure 2. Open eld test results. (A) e rst phase (0–6 min). e concentration of 0.0003 µg/L RISP increased
the immobility time of the exposed larvae. (B) e second phase (6–12 min). Hyperactivity can be observed by
an increase in the distance, average speed and a number of entries in the central area. Means were compared
by One-way ANOVA followed by Dunnett’s or Kruskal-Wallis test followed by Dunn’s were useddependingon
Figure 3. e concentration of 0.003 µg/L risperidone increased the number of animals that remained in the
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Materials and Methods
Ethical Aspects. is study was approved by the Animal Use Ethics Committee (CEUA) of the University of
Passo Fundo, UPF, Passo Fundo, RS, Brazil (Protocol #9/2015) and complied with the guidelines of the National
Council for Animal Experimentation Control (CONCEA). Approximately 600 larvae were tested during this
experiment.
Study strategy. We exposed embryos and larvae of zebrash (Danio rerio, wild-type) to dierent concen-
trations of RISP already detected in the aquatic environment analyzing eventual changes in the larvae behavioral
repertoire. is analysis was based on the dierent behavioral tests as follows: spontaneous movement, open eld,
light/dark as well as aversive stimulus. We opted for a 5-day chronic exposure to RISP since this period window
correspond to the whole period of zebrash organogenesis (2hpf to 120hpf)36.
Reproduction and maintenance of embryo. For breeding, healthy zebrash wild-type, aged between
3 and 18 months were used. e animals were placed in barred bottom aquaria in ratios of 1:1 (males: females).
Aer 12 hours of dark, during the morning the lights were on and aer 1 hour the embryos were collected36,37. e
methods of reproduction and maintenance of embryos are described in the previous work16.
Aer collection, the embryos were washed and classied as fertilized and unfertilized with the aid of light
microscopy37,38. Embryos were maintained in E3 medium (reverse osmosis water + 60 mg/L Marine Ocean
Instant Ocean) and distributed in 24 well cell culture plates (3 ml/well), 10 embryos per well and incubated in a
water bath at 28 °C39. For the tests, embryos of up to 3hpf were accepted. e embryos were exposed to RISP from
3hpf to 120hpf.
Concentrations tested. e RISP concentrations were based on those already registered in the aquatic
environment: 0.00034 µg/L13, 0.003 µg/L, and 0.03 µg/L. ese concentrations were previously tested and changed
survival, hatching, heart rate and total larval length16. e solutions were prepared and stored in amber glass bot-
tles, where they remained heated in a water bath to be replenished in the wells when necessary.
Survival and hatching analysis. For analysis of survival and hatching, we have monitored all animals
once a day in the morning for 7 days with the aid of a magnifying glass or optical microscopy. Embryos and larvae
that do not show transparency, coagulated or without cell formation, cardiac movement or blood circulation
were considered dead. Animals were considered “hatched” when partially or completely outside of the chorion.
For this hatching and survival measurements, we analyzed 160 embryos by concentration (control and the three
concentrations tested), totalizing 640 embryos
Figure 4. During the LDT, larvae exposed to the intermediate concentration of risperidone increased the time
on the darkside and the number of crosses of the center line. Means were compared by One-way ANOVA
followed by Dunnett’s or Kruskal-Wallis test followed by Dunn’s were useddependingon data normality.
*p < 0.05. N = 35.
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Spontaneous movement. In 24 hpf the embryos present spontaneous movements of the tail still inside
the chorion. ey are thus considered because they are induced by the development of the motoneurons without
any control by the central nervous system37,40,41. ese movements were recorded in 1 minute40 in 64 embryos by
group (total of 256 embryos).
Heart rate. Heart rate was assessed at 48hpf in all groups during the morning. e heart rate was manually
counted by light microscopy for 1 minute42 in 48 embryos by group (total of 192 embryos).
Open eld test. To perform the open eld test, the larvae at 6 dpf were placed in 10 mL wells containing only
E3 medium and lmed (Canon EOS Rebel T5 Macro Lens EF 100mm) for 12 minutes. Similar to the behavior
seen in mammals, zebrash larvae also present thigmotaxis and recognize when placed in a new environment3,4,43.
For thigmotaxic behavior analysis, we lmed 30 larvae by group (total of 120 larvae). In the videos, the well
was virtually divided into a central and peripheral area (Fig.5A)18 and the period were divided into two phases:
adaptation period (0–6 min) and the exploratory period (6–12 min). ANY-maze soware was used to analyze
the following parameters: total distance travelled, time in the central area, distance travelled in the central area,
entries in the central area, immobility time and mean speed.
Light/dark test (LDT). For this test, a 6-well cell culture plate was used with one well (5 ml) divided into a
dark (black) area and a clear (white) area. Unlike adults, zebrash larvae prefer to stay in the white area of the well
(Fig.5B). It is believed that the dark area represents the shadow of a possible predator43,44.
irty-ve larvae by group (total of 140 larvae) were placed in the test area and lmed for 6 minutes. e
latency for entry into the dark side, the number of entries and dwell time on the dark side were evaluated using
the ANY-maze soware. As in the open eld test, the LDT was divided between the initial phase (0–6 min) and
nal phase (6–12 min).
Aversive stimulus test (AST). Aversive stimulus aims to test the cognitive ability of the larva to identify
areas of danger. Tests with colorations have been used for their ecological relevance, since dierent species of sh,
like zebrash, use colors to dierentiate possible foods, recognize specics as well as avoid predators18.
For this test, the larvae were placed in 6-well cell culture plates (5 larvae per well, n = 55 by group totalizing
220 larvae) above an LCD monitor. Aer the adaptation period (2 min), using PowerPoint soware (Microso
Oce Professional Plus 2013), we started the exposure to a visual stimulus area with a red sphere of 1.35 cm in
diameter with a trajectory that traveled only half the well (Fig.5C). e animals were stimulated for 5 min and
at the end of the test were recorded the number of animals that remained in the stimulus area and those that
remained in the non-stimulated area45.
Statistical analysis. For statistical analysis and graphing we used GraphPad Prism soware version 6.01 for
Windows. Survival and hatchability data were evaluated by Kaplan-Meier method. For the analysis of the heart
rate data, spontaneous movement, open eld test, LDT and AST, One-way ANOVA followed by Dunnet’s (a group
of parametric data) or Kruskal-Wallis test followed by Dunn’s were used (a group of non-parametricdata). All
groups were compared to the control group. Statistical signicance was accepted when p ≤ 0.05.
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SCIentIfIC REPORts | 7: 14121 | DOI:10.1038/s41598-017-14575-7
Acknowledgements
is study was funded by the Universidade de Passo Fundo and CNPq. L.J.G.B. holds CNPq research fellowship
(301992/2014–2).
Author Contributions
F.K. and L.J.G.B. conceptualize the experiments, wrote the manuscript and prepare the gures. F.K., R.I., J.G.S.R.,
H.H.A.B. and M.F. conducted experimental procedures. A.L.P. analyzed the results. All authors have read and
approved the manuscript for publication.
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