Science of the Total Environment 686 (2019) 311–321

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Fish behavior: A promising model for aquatic toxicology research

Xiangsheng Hong a,c, Jinmiao Zha a,b,⁎
a
Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
Beijing Key Laboratory of Industrial Wastewater Treatment and Reuse, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
c
University of Chinese Academy of Sciences, Beijing 100085, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A systematic evaluation of behavioral

traits for zebrafish, medaka, and Chinese
rare minnow.
• Fish exposed to 0.01% v/v carrier sol-
vents have no behavioral perturbations.
• Pesticides disturbed spatial memory of
zebrafish and Chinese rare minnow.
• Zebrafish, medaka, and Chinese rare
minnow are excellent behavioral
models.

a r t i c l e i n f o a b s t r a c t

Article history: Fish behaviors have great potential as models for the study of pharmacology, genetics, and neuroscience.
Received 23 March 2019 Zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Chinese rare minnow (Gobiocypris rarus) are pop-
Received in revised form 2 June 2019 ular freshwater animal models. However, their behavioral use in aquatic toxicology research is generally ham-
Accepted 2 June 2019
pered by oversimplified behavioral tasks and the fact that they are not well-developed animal models for
Available online 04 June 2019
toxicology. Here, this study presented a comparative analysis of multiple behavioral traits (i.e., anxiety-like be-
Editor: Henner Hollert havior, novel object recognition, social preferences, habituation to light-dark stimulus and noise stimulus, and
spatial learning and memory). We found that only medaka (d-rR) presented a weak or no response to repeated
Keywords: light-dark stimulus and noise stimulus. In addition, no significant behavioral changes were observed for the three
Behavior species of juvenile fish models after 7 days of exposure to 0.01% v/v carrier solvents (i.e., ethanol, acetone, and
Habituation DMSO). In contrast to zebrafish and Chinese rare minnow, medaka showed no significant changes in spatial
Spatial learning and memory memory after subacute exposure to 1 mg/L imidacloprid or 2.5 μg/L chlorpyrifos (cpf); instead, a hyperactivity
Neurotoxicity response in the open field test and reduced social time were induced by cpf and imidacloprid, respectively.
Aquatic toxicology
Our results suggest that: (1) behavioral effects are negligible when using b0.01% v/v carrier solvents for behav-
ioral assessment; (2) given the differences in sensitivities of behavioral responses, a single behavior used alone
as an endpoint may be insufficient for estimating the toxic impacts of pesticides or other environmental contam-
inants. In conclusion, these results could have major implications for aquatic toxicology research and water qual-
ity monitoring and ecotoxicological risk assessment.
© 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing
Road, Haidian District, Beijing 100085, China.
E-mail address: jmzha@rcees.ac.cn (J. Zha).

https://doi.org/10.1016/j.scitotenv.2019.06.028
0048-9697/© 2019 Elsevier B.V. All rights reserved.
312 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321
1. Introduction
For decades, multiple types of anthropogenic chemicals
(i.e., pesticides, pharmaceuticals and industrial chemicals) have ei-
ther directly or indirectly been continuously released into surface
waters or have entered terrestrial systems (Cai et al., 2016;
Morrissey et al., 2015; Santos et al., 2010). Many of these chemicals
(i.e., chlorpyrifos) have been linked to an increased risk of neuro-
toxicity and may even cause brain injury in humans (Carvalho
et al., 2016; Choi et al., 2012; Grandjean and Landrigan, 2006)
and animal models (Cañadas et al., 2005; Zhou et al., 2015). How-
ever, the current risk assessment strategies mainly depend on the
use of traditional ecological endpoints (such as acute toxicity, re-
production, or survival) in and relatively high concentrations of
the compounds (Boxall et al., 2012). Since no specific endpoints
or proposals for a testing strategy are given for neurotoxicity, as-
sessment of neurotoxic effects on non-target species in the aquatic
environment are still limited (Legradi et al., 2018). Accordingly, in
aquatic toxicology research, a critical question to answer is
whether these chemicals may also constitute a threat to the ner-
vous systems of aquatic organisms.
To become better attuned to the surrounding environment, animals
exhibit a variety of behaviors (Farrell, 2011). Researchers from different
fields show completely different interests for this animal behavior. Typ-
ically, for an ecologist, the richness of natural behavior profiles showing
the adaptation of animals to particular environments is the primary
focus of their research. Neuroscientists, in contrast, may often prefer
to understand how a specific behavior is encoded in the brain and un-
cover the ultimate function of the brain (Orger and de Polavieja,
2017). Given the relatively high demands of time, space and money dur-
ing in vivo high-throughput (HT) behavioral drug screening with tradi-
tional animal models (such as rodents, dogs, and pigs) (Zon and
Peterson, 2005), fish (e.g., zebrafish, Danio rerio) behavioral profiles
have become a widely used tool by researchers in pharmacology
(Bruni et al., 2016; Jordi et al., 2018; Rihel et al., 2010; Sieber et al.,
2019) and toxicology (Kalueff et al., 2016; Noyes et al., 2015; Truong
et al., 2014).
Despite their high sensitivity to various environmental
chemicals during the early developmental stages of fish
(Nishimura et al., 2015, 2016), more complex behaviors are exhib-
ited during the juvenile and adult phases (i.e., social interactions,
learning and memory) (Orger and de Polavieja, 2017) that may
provide more options for aquatic neurotoxicity study (Blaser and
Vira, 2014; Stewart et al., 2014).
Currently, zebrafish have already been proven to be a powerful
aquatic vertebrate model based on their rich behavioral phenotype
(Kalueff et al., 2014; Naderi et al., 2017; Stewart et al., 2014). Medaka
(Oryzias latipes) and Chinese rare minnow (Gobiocypris rarus) (Chinese
native species), two kinds of small teleosts, are widely used as aquatic
model organisms for toxicity testing and chemical safety assessment
in our laboratory (Hong et al., 2018; Sun et al., 2007; Yuan et al.,
2016). Given that the current applications of behavioral profiles based
on these three species of small freshwater fish are mainly focused on
their early stages (i.e., zebrafish), only involve simple tests
(i.e., medaka) or are rarely used (i.e., Chinese rare minnow) (Hong
et al., 2018; Ren et al., 2013; Zhang et al., 2016) in aquatic toxicology re-
search or water quality monitoring and ecotoxicological risk assess-
ment, here we systematically present and compare their behavioral
features from simple (i.e., spontaneous locomotion) to complex tasks
(i.e., learning and memory). In addition, we specifically selected three
commonly used carrier solvents (ethanol, acetone, and dimethyl sulfox-
ide [DMSO]) and two neurotoxic chemicals (imidacloprid and chlorpyr-
ifos [cpf]) to examine whether the fish behavioral responses differed
after exposure to these chemicals and to further elucidate the feasibil-
ities of the use of fish behavioral endpoints, especially for medaka and
Chinese rare minnow.
2. Materials and methods
2.1. Ethics statement
All protocols were reviewed and approved by the Research Center
for Eco-Environmental Sciences, Chinese Academy of Sciences.
2.2. Chemicals
Ethanol (≥99.7% purity) and acetone (≥99.5% purity) were pur-
chased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China),
and both were analytical grade. DMSO (≥99.0% purity) was purchased
from Sigma-Aldrich LLC. (Darmstadt, Germany). Imidacloprid (≥96.0%
purity) and cpf (≥99.5% purity) were purchased from J&K Chemical
Ltd. (Hebei, China). Stock solutions of imidacloprid and cpf were pre-
pared by dilution in acetone and stored at −4 °C. The final vehicle con-
centration of acetone was b0.01%.
2.3. Aquaculture and chemical treatments
Wild-type zebrafish (AB genetic background) were purchased from
the Institute of Hydrobiology, Chinese Academy of Sciences and were
maintained under standard conditions (28.5 °C and 14-h light/10-h
dark) (Westerfield, 2007). Wild-type medaka (d-rR strain) were kindly
provided by the Laboratory of Freshwater Fish (the Bioscience Center of
Nagoya University of Japan) and maintained at 25 ± 1 °C on a 14-h
light/10-h dark cycle (Zha et al., 2006). Chinese rare minnows (a gener-
ous gift from Professor Jianwei Wang, Institute of Hydrobiology, Chinese
Academy of Sciences, Wuhan, China) were raised as described (Hong
et al., 2018). All fish were fed twice daily with laboratory-hatched
brine shrimp (Artemia nauplii, Tianjing, China).
Juvenile fish (~2 months old) (zebrafish, medaka, and Chinese rare
minnow) were acclimated to the laboratory conditions no b2 weeks be-
fore experiments. Fish were exposed to chemicals using a semistatic
aqueous exposure test (daily refreshment of the test solution) at nomi-
nal concentrations of 0 (control), 0.01% ethanol (v/v), 0.01% acetone (v/
v), 0.01% DMSO (v/v), 1 mg/L imidacloprid, and 2.5 μg/L cpf (based on
the reported effective concentration) for 7 days (Ge et al., 2015; Yuan
et al., 2016). There were six replicate control containers and six replicate
containers for each exposure concentration. Each container (20−l)
contained a total of 20 individuals. During acclimatization and the ex-
periments, the room and water temperature were maintained at 25
± 1 °C.
2.4. Behavioral studies
2.4.1. Trajectory tracking and quantification of fish behavior
All fish behavioral tests described in the study were videotaped with
a video camera (SONY Handycam, FDR-AX60, Japan) and analyzed off-
line with animal behavior tracking software, EthoVision XT 14
(v14.0.1322, Noldus, the Netherlands).
2.4.2. Open field test
To explore locomotion and spontaneous activity, we characterized
fish behavior as they freely explored a Plexiglas open-field apparatus
(20 cm diameter; 10 cm height) (Fig. 1a) using a video tracking system.
Fish were transferred individually to the tank for 10 min; trajectories
within the final 8 min were recorded by a top view video camera for
analysis. Time spent (s), total distance traveled (cm) and average veloc-
ity (cm/s) in the center and outer areas were measured during the
session.
2.4.3. Novel tank test
The novel tank testing apparatus comprises a transparent 1.5liter
trapezoidal tank (15.2 × 27.9 × 22.5 × 7.2 cm; height × top × bottom
× width) (Fig. 1c) (Cachat et al., 2010). Fish were placed in this tank
 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321 313

Fig. 1. Location preference in the open field tank and novel tank. (a) Schematic of the open field tank (top). The behavioral setup with a single chamber was virtually divided into two
segments by an inner circle. (b) Distance traveled (cm), average velocity (cm/s), and time spent (s) of zebrafish, medaka, and Chinese rare minnows in the “center” and “outer” area of
the open field tank. (c) Schematic of the novel tank (side). (d) The time (%) spent by fish in the different areas in the morning (9:00) and at night (21:00). Data are mean ± SEM, n =
30 fish for each treatment; ⁎Asterisks denote comparisons of zebrafish with medaka and Chinese rare minnow, respectively; ⁎P b 0.05; n.s., not significant.

for 10 min in the morning (9:00) or at night (21:00). Their behavioral
trajectories were recorded for the final 8 min. For the trajectory analysis,
the novel tank was virtually divided into three horizontal segments
(upper, middle, and lower).
2.4.4. Novel object test
Each fish was individually transferred to the transparent test tank
(25 × 20 × 15 cm, length × width × height); each tank contained a
novel object (blue plastic cubic, 3 × 3 × 1 cm, length × width × height)
to determine their responsiveness to novelty (Fig. 3a). Tepid water (25
± 1 °C, pH 7.2–7.6, and hardness 44.0–61.0 mg CaCO3/L) was placed in
the test tank filled to a depth of 10 cm. After an acclimation period of
5 min, the novel object was placed in a corner of the tank and the fish
was allowed to freely explore for 8 min. Their behavioral trajectories
were recorded for the final 6 min. For the analysis, the test tank was vir-
tually divided into two segments (new object zone and no object zone)
(Fig. 3a). We analyzed the total distance traveled (cm) and the time
(s) spent in the two parts of the virtually divided tank.
2.4.5. Social interaction
The test apparatus required to perform social interaction tests in fish
primarily entailed a transparent Plexiglas cross-shaped tank (50 × 50
× 10 cm, length × width × height) (Fig. 3e). Each arm of the tank was
separated by a Plexiglas wall. In the tank, one of the randomly selected
terminal chambers (the other three chambers were empty) and the
center chamber contained 1 individual each from the same treatment.
After the transfer, fish were acclimated for 2 min, their behavior was re-
corded for 8 min. For the data analysis, the time fish spent in the zone
nearest to their companion (social zone) was calculated as the response
to the sight stimulus of conspecifics.
Time in Social Zone ðsÞ
Social preference ð%Þ ¼
Time in Free Swimming Zone ðsÞ
Area of Free Swimming Zone
  100
Area of Social Zone
2.4.6. Habituation to dark or noise stimulus
The open-field tank was used to assess the fish startle response to
dark or noise stimuli. The dark and the noise stimuli experiments are
carried out separately. Briefly, the consecutive dark stimulus
(5 min cycle) was delivered through multiple arrays of automatic on/
off white LEDs placed on the bottom side of the transparent open-field
apparatus. In addition, a series of noise stimuli were produced with a
plastic cube (15 × 9 × 0.5 cm, length × width × height) by raising it to
the height of 100 cm and releasing it by hand onto a hard, flat floor
(Fig. 5a and b). A top-view camera was used to capture the fish
movements.
2.4.7. Spatial learning and memory
Spatial learning and memory is one form of long-term storage of ex-
plicit memory. Like mammals, accumulating evidence suggests that tel-
eost fish also have the properties and neural basis of learning and
memory (Salas et al., 2006). A latent learning apparatus was used to as-
sess and test the spatial learning and memory ability of fish. The appa-
ratus was identical to that described in a previous report (Gómez-
Laplaza and Gerlai, 2010), which is composed of a start chamber, a re-
ward chamber, and two tunnels that connect these two chambers.
314 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321
During the training or testing, the maze was filled with a 10-cm depth of
water at the same temperature as that in the experimental tanks. The
2 weeks of learning training and the final memory assessment were car-
ried out essentially as described elsewhere (Naderi et al., 2016). The
spatial learning training started from 7 days pre-exposure and ended
with another 7 days after exposure. Each training sessions were con-
ducted daily by placing ten individuals per group in the start chamber
and releasing these after 30 s to explore the maze for 30 min (either
the right or left tunnel is left open). The final memory assessment was
10 min long (one individual per group). The total number of fish in
the reward chamber and the time the fish spent in different segments
were calculated in this study.
2.5. Data analysis
All statistical analysis was performed with SPSS software (version
25.0, IBM SPSS Inc., USA) and shown as the mean ± standard error of
the mean (SEM). Normality and homogeneity of variances for the raw
behavioral data were analyzed using Shapiro-Wilk's test and Levene's
test, respectively. Statistical analysis was performed on parametric
data using one way analysis of variance (ANOVA) followed by Dunnett's
post-hoc tests to determine significant differences among treatment
groups. When data are in a state of heteroscedasticity, Welch's test
with the Games−Howell post-hoc test was performed. Significant differ-
ences were considered when the P value b0.05. All of the groups were
compared with the control group.
Pearson's correlation coefficients (r) were calculated and visualized
using ggscatter from the R package of ggplot2-based plotting functions
ggpubr (v0.1.4, http://www.sthda.com/english/rpkgs/ggpubr).
3. Results
3.1. Open field test
In the open field test, zebrafish, Chinese rare minnows, and medaka
spent more time (s) and swam a greater distance (cm) in the outer area
than other areas (P b 0.05) (Fig. 1a and b). No significant differences be-
tween the center and outer average velocity (cm/s) were detected
among the three fish species (P N 0.05) (Fig. 1b).
After 7 days of exposure, the locomotor parameters (distance trav-
eled and speed) of the three fish species showed no significant changes
in response to any of the solvents employed in the current study when
compared with the control group (P N 0.05) (Fig. 2a). The average speed
(cm/s) of medaka was significantly increased at 2.5 μg/L cpf treatments
(P b 0.05) (Fig. 2a). However, no significant changes were found among
all treatment groups for time spent and swimming distance in the cen-
ter area (data not shown).
3.2. Novel tank test
In the novel tank test, the three fish species exhibited a “position
preference” profile both in the morning (9:00) and at night (21:00)
(Fig. S1). Compared to the night (21:00), the time spent (s) in the
lower row by zebrafish and Chinese rare minnows significantly higher
in the day (9:00) (P b 0.05) (Fig. S1). However, medaka presented no
differences between the morning and night in behavioral endpoints
such as distance traveled, speed, or time spent in different rows
(Fig. S1b).
In the novel tank test, compared to the zebrafish (9:00), Chinese rare
minnows spent more time (s) in the lower row and less time (s) in the
middle row (P b 0.05) (Fig. 1d); in contrast, less time spent in the lower
row and more the time spent (s) in the upper and middle row when
compared with the zebrafish (21:00) (P b 0.05) (Fig. 1d).
3.3. Novel object test
Results from the novel object test presented a high exploratory be-
havior towards novelty (Fig. 3b). For example, zebrafish (% of the total
distance in the new object zone: 74.90%, P b 0.001) and Chinese rare
minnows (% of the total distance in the new object zone: 68.49%, P =
0.023) displayed more distance traveled in the new object zone than
medaka (P N 0.05) (Fig. 3c). Furthermore, zebrafish (% of time in the
new object zone: 71.32%, P b 0.001) and medaka (% of time in the new
object zone: 66.46%, P = 0.013) spent more time in the new object
zone (Fig. 3d). No significant differences were found for the Chinese
rare minnow for time spent between the new object zone and the no
object zone (P N 0.05) (Fig. 3d). Compared to the zebrafish, both medaka
and Chinese rare minnow showed no significant differences for % of dis-
tance traveled and time spent in the new object zone (P N 0.05) (Fig. 3c
and d).
3.4. Social interaction
All three species of fish presenting a preference for social interaction
and spent more time and swam frequently in the area close to their con-
specifics (Fig. 3f). Compared with the Chinese rare minnow (% of total
time in the social zone: 51.53%; P b 0.05, compared to the non-social
zone) and medaka (% of total time in the social zone: 50.36%; P b 0.05,
compared to the non-social zone), zebrafish showed a stronger social
preference (% of total time in the social zone: 66.73%; P b 0.01, compared
to the two other different fish species) (Fig. 3f).
We next examined whether 7 days of exposure to carrier solvents
(ethanol, acetone, and DMSO) and neurotoxic chemicals (imidacloprid
and cpf) would reduce their social responses. In these experiments, no
significant changes for both zebrafish and Chinese rare minnows were
found for social time (s) (Fig. 2b). However, a significant reduction of so-
cial time (s) for medaka was observed in response to the 1 mg/L
imidacloprid treatments (P b 0.05) when compared with the control
groups (Fig. 2b).
3.5. Habituation to dark stimulus
To determine the fish habituation to inconsequential stimulus, we
first tested a three times consecutive dark stimulus to see if it affected
the speed of these three species of fish. During the 5 min dark period,
the average speed for zebrafish was clearly increased during Stim 1
(4.7485 ± 0.7696; P b 0.01, compared to the 5 min base line period
prior to light off; Games−Howell post-hoc test) and was subsequently
reduced in response to the Stim 2 (3.1453 ± 0.1541; P = 0.071, com-
pared to the average speed of Stim 1; Games−Howell post-hoc test)
and Stim 3 (2.7843 ± 0.2020; P b 0.05, compared to the average
speed of Stim 2; Games−Howell post-hoc test) (Fig. 4a). No significant
changes were observed for the average speed for all poststimulation
speeds of medaka (Fig. 4b). In comparison, the poststimulation speed
of Chinese rare minnows was also significantly increased for the Stim
1 (2.3592 ± 0.5011, P b 0.05); we found no marked changes for the
Stim 2 (2.9560 ± 0.5339), but there was a statistically significant reduc-
tion for the subsequent Stim 3 (1.4803 ± 0.5182, P b 0.05) (Fig. 4c).
To better understand the relationship between fish's habituation to
dark stimulus and the number of dark stimuli, we extracted the first
one min of the postdark-stimulation speed from the video data
(Fig. 4a–c). As illustrated in Fig. 4d–f, a significant negative correlation
was observed only in zebrafish (R = −0.66, P = 0.00046, Fig. 4d).
3.6. Habituation to noise stimulus
Next, we designed a consecutive noise stimulus with different time
intervals (5, 10, 20, 30 min) to further examine the fish habituation to
inconsequential stimulus. Compared with the prestimulation speeds
of Stim 1, the poststimulation speeds (the first 10 s) were significantly
 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321 315

Fig. 2. Behavioral effect of carrier solvents (ethanol, acetone, and DMSO) and neurotoxic chemicals (imidacloprid and cpf) treatment on zebrafish, medaka, and Chinese rare minnow in the
open field tank and social tank test after 7 days of exposure. Data are mean ± SEM, n N 20 fish for each treatment; ⁎Asterisk indicates a significant difference of fish speed, distance traveled
or time spent in social zone in comparison to the control;⁎P b 0.05; n.s., not significant.

increased in zebrafish (P b 0.05) (Fig. 5c) and Chinese rare minnows (P b
0.05) (Fig. 5e). Subsequently, the following Stim 2, Stim 3, and Stim 4 of
zebrafish and Chinese rare minnows showed a speed tendency of rever-
sion back to what was seen in nonstimulated conditions (Fig. 5c and e).
However, there were no significant differences between the pre- and
poststimulation speed throughout the entire four times of the stimulus
procedure (Stim 1, Stim 2, Stim 3, and Stim 4) for medaka (Fig. 5d).
3.7. Spatial learning and memory
During the 7 days training of the pre-exposure, all three species of
fish showed strong spatial learning abilities in the learning apparatus.
For example, with an increase in training times, the time spent showed
a trend of decreasing, and approximately 70% of fish individuals could
reach the target chamber in 30 min (Fig. 6). In addition, on the 14th
day of training, approximately 80% of the individual fish in the control
groups could reach the target chamber in 10 min (Fig. 6).
To test for possible effects of the 7 days exposure to chemicals on the
fishes' spatial memory, we next analyzed the time they spent in five dif-
ferent areas of the learning apparatus (start chamber, holding chamber,
incorrect tunnel, correct tunnel, and target chamber). In this test, all
three carrier solvents (0.01% v/v) showed no obvious changes on the
spatial memory of zebrafish and medaka (Fig. 7). The 0.01% (v/v) etha-
nol treatment (7 days) significantly decreased the time the Chinese rare
minnows spent in the correct tunnel (P = 0.038), but the fish were un-
affected by acetone (0.01% v/v) and DMSO (0.01% v/v) (Fig. 7). For
zebrafish, both imidacloprid (1 mg/L) (P = 0.021) and cpf (2.5 μg/L)
(P = 0.045) significantly decreased the time spent in the target cham-
ber (Fig. 7). A similar result occurred for imidacloprid and cpf exposed
Chinese rare minnows (all P b 0.05) (Fig. 7), but 1 mg/L imidacloprid
and 2.5 μg/L cpf exposure for 7 days showed no disruption of the spatial
memory of medaka (Fig. 7).
4. Discussion
Although medaka and Chinese rare minnows are two commonly
used small freshwater models for aquatic toxicology research, they
have received relatively little attention in behavioral studies compared
to zebrafish, especially regarding their learning capabilities. Here, we
designed a battery of behavioral assays in juvenile fish to determine
whether the medaka and Chinese rare minnow can be used in behav-
ioral studies in a way similar to how zebrafish are used and whether
316 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321

Fig. 3. Novel object recognition and social interaction. (a) Schematic of the novel object recognition test tank (top). The behavioral setup is virtually divided into two segments along the
diagonal, including the “new object zone” and “no object zone” area. (b) Distance distributions of fish during 6 min of exploration that began when a new object was put into one corner of
the open field tank. Distance traveled (cm) (c) and time spent (s) (d) in the “new object zone” and the “no object zone”. A statistically significant difference of distance traveled or time
spent between “new object zone” and “no object zone” (one-way ANOVA and Dunnett post-hoc test; ⁎P b 0.05). (e) Schematic of the social preference test tank (top). (f) Time spent (s) in
the “social zone” area. Heat maps represent the spatial preferences of fish in the cross-shaped tank after 8 min of social interaction. A statistically significant difference between “time in
social zone” and “time in free swimming zone ” (one-way ANOVA and Dunnett post-hoc test; ⁎P b 0.05). Data are mean ± SEM, n = 30 fish for each treatment.

their behavioral profiles can be useful for screening the effects of phar-
maceutical and toxicological chemicals.
Numerous behavioral paradigms from rodents have been adapted
and applied to zebrafish (Maximino et al., 2010; Stewart et al., 2012).
Similar to the rodent open field test (Prut and Belzung, 2003), open
field tests and novel tank tests are two very commonly used paradigms
for assessing anxiety-like behavior in fish models (Ansai et al., 2016;
Cachat et al., 2010; Kalueff et al., 2014; Ziv et al., 2013). Here, we
found that all of the tested fish models, zebrafish, medaka, and Chinese
rare minnow, showed anxiety-like behavior driven by a new/unfamiliar
 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321 317

Fig. 4. Habituation responses of three species of fish to dark stimulus. (a–c) Distribution of speed (cm/s) in a consecutive-dark-stimulation interval for the population of zebrafish, medaka,
and the Chinese rare minnow in the open field tank (mean ± SEM, n = 30 fish for each treatment). The shaded area is the error range. Stim: stimulation. (d–f) Scatter points represent the
average speed of the first one min after the dark stimulus of Stim 1, Stim 2, and Stim 3. Pearson correction coefficients (r) were used to determine the correlations between the number of
dark stimulations and the habituation responses of the fish. P-values were calculated using Pearson correlation analyses. Areas shaded with light gray are the 95% confidence interval.

environment (Fig. 1). Specially, they showed a robust place preference
including thigmotaxis (staying close to the walls in the open field
test), a diving response (novel tank test) and a reduction of exploration.
In humans and other mammals, anxiety has been linked to difficulties in
decision making, which is associated with increased engagement in
threat-avoidance behaviors (Bishop and Gagne, 2018; Prut and
Belzung, 2003). Our data further indicated that teleosts and other verte-
brates, including humans, may potentially share evolutionarily con-
served neural circuits in regards to anxiety-like behaviors (Lee et al.,
2010; Stewart et al., 2012).
To date, depending on visual attention stimuli towards a novel ob-
ject or conspecifics, researchers have demonstrated that mammalian
318 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321

Fig. 5. Habituation responses of three species of fish to noise stimulus. (a) Schematic drawing of the noise production (side). (b) Scheme of the noise stimulus. Stim: stimulation. Line
graphs of zebrafish (c), medaka (d), and Chinese rare minnows (e) represent the distribution of speed (cm/s) in consecutive-noise-stimulation intervals in the open field tank. The
shaded area is the error range. Data are mean ± SEM, n = 30 fish for each treatment; ⁎P b 0.05; n.s., not significant.

and nonmammalian models show similar exploratory behavior (novel
object recognition) (Blaser et al., 2015; May et al., 2016) and social pref-
erences (Polavieja and Orger, 2018; Stewart et al., 2012). Since even dis-
tantly related species share similar behavioral traits, it is common to
expect that medaka, Chinese rare minnows (Cyprinidae), and zebrafish
may also exhibit similar behavioral traits (Blaser et al., 2015). Following
this logic, two behavioral assays, a novel object recognition test and a
social preference test, were designed. In the novel object recognition
test, fish in a familiar context (5 min acclimation) but with a novel ob-
ject within that context, showed a tendency to move more frequently
(spent more time or traveled a greater distance) into the quadrant of
a novel object (Fig. 3c and d). Notably, although this exploration bias
was first noted for rats (Ennaceur and Delacour, 1988) and then gradu-
ally adapted to other animals (i.e., mice and monkeys), our data and
previous studies in zebrafish suggested that this novel object preference
but not familiar object preference could be driven by an object with a
moderate size for Chinese rare minnows and medaka (May et al., 2016).
Like humans, social behaviors are also common in many nonmam-
malian vertebrates, such as zebrafish, which show a diversity of social
traits (i.e., aggression or mating) (Dreosti et al., 2015; Orger and de
Polavieja, 2017). In the present study, our data from two classic param-
eters of social time and social preference % for social behavior strongly
suggested that Chinese rare minnows exhibited excellent social traits
(Fig. 3f). In addition, the rest of the data about the social preferences
of medaka and zebrafish are consistent with previous studies (Fig. 3f)
(Ansai et al., 2016; Kirsten et al., 2018). Given no food requirement,
no aversive motivations, and no need for time-consuming training in
behavioral tasks of novel object recognition and social preference,
these advantages could greatly facilitate their application in aquatic tox-
icology research, particularly for Chinese rare minnows.
Habituation, a conserved form of neuroplasticity, is classified into
two forms of unconscious implicit learning (non-associative learning,
sensitization is another form). Commonly, when animals experience re-
peated exposure to a sudden inconsequential stimulus (neither
 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321 319

Fig. 6. Spatial learning of zebrafish, medaka, and Chinese rare minnow during 7 days of consecutive training. Data are mean ± SEM, n = 40 fish for each treatment; ⁎Asterisk indicates a
significant difference of fish number in target region in comparison to the day 1; ⁎P b 0.05.

beneficial nor harmful), their response tends to weaken and they grad-
ually begin to ignore this repeated stimulus, which typically can be mea-
sured through their locomotory activity changes over time (Best et al.,
2008; Blaser and Vira, 2014). We demonstrate here that both zebrafish
and Chinese rare minnow exhibited non-associative learning habitua-
tion after stimulus of light or noise (Fig. 4a and c; Fig. 5a and c). In
contrast, although the dark stimulus did induce a weaker startle re-
sponse during the first several seconds of poststimulus, we did not ob-
serve any obvious attenuation of the response to repeated stimulus
presentation in medaka (Fig. 4b; Fig. 5b). Similarly, in comparison to
the startle behavior among four inbred strains of medaka fish (HNI-I,
HNI-II, HO5, and Hd-rR-II1), Tsuboko et al. (2014) also revealed that

Fig. 7. Spatial memory test of zebrafish, medaka, and Chinese rare minnow after 7 days of exposure. Data are mean ± SEM, n N 30 fish for each treatment; ⁎Asterisk indicates a significant
difference of fish number in specific regions in comparison to the control; ⁎P b 0.05.
320 X. Hong, J. Zha / Science of the Total Environment 686 (2019) 311–321
the Hd-rR-II1 (an inbred strain derived from d-rR stock) (Yamamoto,
1975) exhibited a low response rate, low sensitivity, and low response
stability to light-dark stimulus.
Spatial recognition, one of conscious implicit learning, has been
widely used to reveal the capabilities of spatial learning and memory
in many species, including teleosts (Brown et al., 2008). The latent
learning apparatus for the spatial memory test can be used for high
throughput screenings due to its feature to operate under minimal
monitoring in parallel with other similar apparatus (Gómez-Laplaza
and Gerlai, 2010). Here, similar to previous research (Gómez-Laplaza
and Gerlai, 2010; Zhu, 2007), our data showed that zebrafish and Chi-
nese rare minnow had quick spatial learning capabilities, i.e., were
able to explore a new environment, acquire spatial information, and
transform the spatial information to implicit memory (Fig. 6). Addition-
ally, to our knowledge, our results are the first to indicate that the d-rR
strain medaka might have sufficient spatial learning capability (Fig. 6).
There is no doubt that ethanol, acetone and DMSO are universally
used as carrier solvents in aquatic toxicity tests. It is usually assumed
that low concentrations of these carrier solvents (0.01% v/v) would
have no influence on commonly used toxicity endpoints but this is actu-
ally unknown for fish behavior. Although a number of previous studies
have reported that carrier solvents can alter fish behavioral traits, many
of them were focused on relatively high concentrations (N0.1%)
(Fernandes and Gerlai, 2009; Huang et al., 2018; Li et al., 2015;
Sterling et al., 2015). Here, our behavioral data (open field test, social
preference, and spatial memory) indicated that carrier solvents at con-
centrations of 0.01% v/v had no effects on all test fish (juveniles), except
for Chinese rare minnow (Figs. 2; 7). Inconsistent with our data, Chen
et al. (2011) reported that 0.01–1% DMSO or ethanol can induce a hy-
peractivity response in 6-day-old zebrafish. This dissimilarity may be
explained by interspecies and age differences.
The behavioral response sensitivity could vary between model or-
ganisms and among the chemicals (Ansai et al., 2016; Steele et al.,
2018). Here, in contrast to the other fish species studied, medaka
showed a different behavioral response sensitivity, including hyperac-
tivity responses (open field tank) and a decline in social preference
after 7 days of exposure to cpf (2.5 μg/L) and imidacloprid (1 mg/L), re-
spectively (Fig. 2). The hyperactivity responses were consistent with
previous research (Khalil et al., 2013), which may be attributed to the
action of low levels of AChE impairment by anti-cholinesterase agents
(Beauvais et al., 2000). However, different from the medaka and
zebrafish, the Chinese rare minnow showed a decline in performance
in the spatial memory assay (Fig. 7). Our results show that single behav-
ior endpoints may be insufficient for obtaining full toxicity information.
Taken together, we suggest that further investigation into complex be-
havioral responses and a combination of multiple behavioral endpoints
will provide more comprehensive and valuable information for discern-
ing the impacts of potential aquatic environmental contaminants. Be-
sides, with the development of 3D video tracking and 3D swim
trajectories reconstruction technologies, its advantages of high-
resolution and high-precision object positioning make it be increasingly
used in neuropharmacology studies utilizing adult zebrafish (Stewart
et al., 2015). We expect this novel 3D method of behavioral analysis to
be applied in aquatic toxicology research.
5. Conclusions
This paper systematically analyzed the differences in behavioral
traits among three commonly used aquatic toxicological fish models, in-
cluding zebrafish, medaka, and Chinese rare minnow. Our results
showed that the commonly used 0.01% v/v carrier solvents (ethanol,
acetone, and DMSO) in aquatic toxicity tests had no influence on the be-
havioral traits of juvenile stage fish models. Due to their different sensi-
tivity and behavioral responses to environmental contaminants,
medaka and Chinese rare minnow may provide useful model systems
for aquatic toxicology research that would complement the widely
used zebrafish. Specially, as Chinese native species, the Chinese rare
minnow would be more useful in early warning for water quality mon-
itoring and chemical risk assessment in the future.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2019.06.028.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the Major International Joint Research
Project of the National Natural Science Foundation of China
(51420105012) and the National Natural Science Foundation of China
(21677165).
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