Environmental Pollution 206 (2015) 275e281
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Chronic bisphenol A exposure alters behaviors of zebraﬁsh (Danio rerio) Ju Wang a, 1, Xia Wang b, 1, Can Xiong a, Jian Liu a, Bing Hu c, Lei Zheng a, b, * a
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China School of Medical Engineering, Hefei University of Technology, Hefei, 230009, China c School of Life Science, University of Science and Technology of China, Hefei, 230027, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2014 Received in revised form 5 July 2015 Accepted 14 July 2015 Available online xxx
The adult zebraﬁsh (Danio rerio) were exposed to treated-efﬂuent concentration of bisphenol A (BPA) or 17b-estradiol (E2) for 6 months to evaluate their effects on behavioral characteristics: motor behavior, aggression, group preference, novel tank test and light/dark preference. E2 exposure evidently dampened ﬁsh locomotor activity, while BPA exposure had no marked effect. Interestingly, BPA-exposed ﬁsh reduced their aggressive behavior compared with control or E2. Both BPA and E2 exposure induced a signiﬁcant decrease in group preference, as well as a weaker adaptability to new environment, exhibiting lower latency to reach the top, more entries to the top, longer time spent in the top, fewer frequent freezing, and fewer erratic movements. Furthermore, the circadian rhythmicity of light/dark preference was altered by either BPA or E2 exposure. Our results suggest that chronic exposure of treated-efﬂuent concentration BPA or E2 induced various behavioral anomalies in adult ﬁsh and enhanced ecological risk to wildlife. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Zebraﬁsh Bisphenol A Behavior
1. Introduction Behavior is a crucial determinant for survival, growth and reproductive success in animals (Gerlai, 2003; Little et al., 1990; Peichel, 2004), which may affect aquatic community compositions and eco-function in adult life (Brodin et al., 2013). For example, motor behavior, aggression, group preference and novel tank test are regarded as the common and easily measured behavioral responses, which correlate to courting display, foraging, escaping from the risky area (Little et al., 1990). Furthermore, the behaviors of animals are initially presumed to be primitive and instinctive which could promote access to resources such as mates, shelter and foraging positions and antipredator defense (Brodin et al., 2013). In addition, the appropriate preference for light or dark environment is vital to the survival of the diurnal/nocturnal animals through affecting ﬁtness of organisms (Gerlai, 2010). Interestingly, our previous study indicated that the choice of light/ dark area can be affected by circadian rhythm in zebraﬁsh (Wang
* Corresponding author. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China. E-mail addresses: [email protected]
, [email protected]
(L. Zheng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.envpol.2015.07.015 0269-7491/© 2015 Elsevier Ltd. All rights reserved.
et al., 2014). Consequently, the modiﬁcation of a wide range of behaviors in male- and female-typical animals can alter the stability of ecosystem through affecting various ﬁtness functions such as growth, reproduction and body maintenance in the whole aquatic environment (Alvarez et al., 2005; McCarthy and Fuiman, 2008). The endocrine disrupting chemicals (EDCs) are an exogenous agent existed widely in environment, which can be accumulated and stored inside animal body (Clotfelter et al., 2004) and plant (Pan et al., 2013). Bisphenol A (BPA), as one of representative EDCs, is composed of two phenol rings and has structural homology with 17b-estradiol(E2), leading to a strong binding to both estrogen receptors (ERs) and estrogen related receptor gamma (ERRg) (Okada et al., 2008; Washington et al., 2001). BPA persists in wastewater efﬂuent through the incomplete polymerization or gradual breakdown of BPA-containing products (Biedermann et al., 2010; Vandenberg et al., 2010; Welshons et al., 2006; Zhang et al., 2013) and can therefore be found at concentrations ranging from nondetectable to 17200 mg/L in treated efﬂuent (Huang et al., 2012; Suzuki et al., 2004; Yamamoto et al., 2001). The evidence has shown that BPA can alter behaviors by participating in the organization of neural circuits that control wide aspects of neuroendocrine, behavioral, and cognitive functions.
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Saili et al. (2012) found that BPA led to hyperactivity of larval ﬁsh and learning deﬁcits in adult zebraﬁsh. Wang et al. (2013) only reported that BPA exposure altered spontaneous movement, decreased touch response and swimming speed in response to light stimulation in larval zebraﬁsh by inducing axial muscle damage. However, numerous behavioral tasks by chronic BPA exposure are not well known. Behavioral change which could affect dominance hierarchies and alter population stability in the whole aquatic ecosystems has been largely overlooked. The behavioral changes of ﬁsh may inﬂuence aquatic community compositions and the functioning of aquatic systems. In the present study, we use adult zebraﬁsh (Danio rerio) to evaluate the ecotoxicology in aquatic environment through behaviors (including motor behavior, aggression, group preference, novel tank test and light/dark preference) by mimicking BPA exposure in waste water efﬂuent. 2. Materials and methods 2.1. Animals Subjects were 150 adult (4-6 months-old) zebraﬁsh from wildtype stock (short-ﬁn phenotype), of mixed gender in a 1:1 male: female ratio, obtained from breeding center at University of Science and Technology of China. Zebraﬁsh was housed in the glass tanks in a dedicated “ﬁsh” room; each tank (35 cm 20 cm 23 cm, length width height) was held 10e15 ﬁsh. The water was dechlorinated water which contained some salts, the PH and conductivity were 7.0e8.0 and 1500e1600 ms/cm, respectively. Water temperature was maintained at about 28 C, with a 14 h light/10 h dark cycle (room ﬂuorescent light, 08:00am-22:00pm). They were fed twice per day, at 09:00am and 14:00pm respectively, with freshly hatched brine shrimps. 2.2. Chemical exposures BPA or E2 was added to exposure tanks from stock solutions that were prepared by dissolving 30 mg BPA (98% purity; Chem Service Inc., China) in 3 mL ethanol or dissolving 10 mg E2 (98% purity; Chem Service Inc., China) in 10 mL ethanol. The ﬁnal concentration of each chemical in experimental tanks was 500 mg/L or 10 mg/L for BPA or E2, respectively. The ﬁnal concentration of ethanol was 0.005% in BPA or 0.001% in E2. All solutions were changed daily during the exposure periods and animals were kept in the same physical conditions of their home tanks. Before drugs exposure, 150 zebraﬁsh were transferred to tanks in groups of 50 animals per tank. The 2 groups of adult ﬁsh were exposed for 6 months to BPA or E2 at concentrations of 500 mg/L or 10 mg/L, respectively. The BPA concentration used in this study was chosen in light of measured concentrations in treated efﬂuent environment (Huang et al., 2012). Despite previous data reported that the lower ethanol concentrations can hardly affect the behavioral parameters of zebraﬁsh (Gerlai, 2003; Wang et al., 2014), the volume of ethanol applied to high exposure tanks was also added to control group to a maximum concentration of 0.005%. 2.3. Behavioral tests The diagram of behavioral devices and the testing parameters of behaviors can refer to Table 1. 2.3.1. Motor behavior To assess motor behavior, the front wall of experimental tank was equally divided into seven segments in vertical direction, and three segments in horizontal direction, so the tank in water area
was consisted of thirty-ﬁve equal grids in total. The video recorded the entire number of times that the ﬁsh moved from one section into another during observation.
2.3.2. Aggressive behavior In aggressive experiment, the bottom of testing tank was divided into four equal segments by three vertical lines. A mirror was placed inclined at 22.5 to the left lateral wall of the tank (Gerlai, 2003). Fish mirror image appeared closer to it when the experimental ﬁsh swam to the left side of the tank. Therefore, ﬁsh spent the amount of time in left-most segment was quantiﬁed as the intensity of aggressive behavior.
2.3.3. Group preference In the test of group preference, the testing tank was placed in the middle, and both sides of tank were additional tanks (including empty tank and stimulus tank), the stimulus tank held 15 zebraﬁsh as “stimulus ﬁsh”. The testing tank was divided into two equal sections with a vertical line in the front wall. The amount of time which the tested ﬁsh spent on the side of tank closer to the conspeciﬁcs was regarded as group preference or shoal.
2.3.4. Novel tank test Novel tank test was deﬁned by ourselves which reﬂected the congenital characteristics in swimming behavior of zebraﬁsh. Zebraﬁsh had two behavioral phenomena: erratic movements were deﬁned as sharp changes in direction or velocity and repeated rapid darting behaviors, freezing was deﬁned as a total absence of movement, except for the gills and eyes for 1s or longer. The novel tank was divided into two equal horizontal portions and parameters of these behaviors were recorded: latency to upper half (s), time spent in the upper half (s), number of transitions to the upper half, number of erratic movements, number of freezing bouts and freezing duration (s). Zebraﬁsh were placed individually in the tank. After half a minute habituation period, their behaviors were recorded for 6 min. A video camera with infrared feature was positioned in front of the testing tank to record the behaviors, but the aggressive behavior which was recorded in the above of the tank. The video recordings were later analyzed by recording the behavioral parameters of ﬁsh during 6-min observation. 2.3.5. Light/dark preference Zebraﬁsh is a typical diurnal animal, and the appropriate preference for light or dark environment can certainly help them to regulate responses to the social stimulus. To test the light/dark preference, we recorded the proportion of time of ﬁsh spent in the dark area as the indicator of the light/dark preference. The detailed apparatus and methods for light/dark preference tests have been described in our previous work (Wang et al., 2014). Brieﬂy, the aquarium consisted of a dark chamber and a light chamber, the dark chamber was covered with matter black paper on all side and the top. 10 L fresh ﬁsh water was poured into the experimental tank. Individual zebraﬁsh was placed in the preference tank at 7:30am on day 1 and can swim freely in the entire tank, after a 30 min adaption, the video camera was positioned in the front of the tank and the recording was started from 8:00am on day 1 till 8:00am on day 3 over 48 h. The swimming trajectory of zebraﬁsh were analyzed by ﬁsh tracking software which developed by Prof. John Y. Chiang. The experimental room was closed and kept quiet to minimize the interference from the outside, the experimenter was not visible to the ﬁsh during the recording.
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Table 1 The diagram of devices and the testing behavioral parameters. Behaviors
The number of crossing the 35 segments of the tank
Relative duration of time (%) in left segment 1
Relative duration of time (%) spent by ﬁsh near the stimulus ﬁsh
Novel tank test
Latency to upper half (s), time spent in the upper half (s), number of transitions to the upper half, number of erratic movements, number of freezing bouts and freezing duration (s).
Levin et al., 2007; Stewart et al., 2012
Proportion of time spent in the dark area (%)
Wang et al., 2014
Diagram of behavioral devices
The front view of experimental tank. The vertical view of experimental tank.
3. Statistical analysis
4.2. Chronic exposure to BPA or E2 affects aggression behavior
All experimental data were analyzed by one-way analysis of variance (ANOVA), followed by post hoc comparisons between the experimental groups. Signiﬁcance was set at P < 0.05. Data were presented as mean ± SEM.
Responses to the mirror image of an individual conspeciﬁc were also signiﬁcantly changed as a result of BPA or E2 treatment. From Fig. 2, the aggressive behavior was signiﬁcantly affected by BPA exposure compared with control (F1,18 ¼ 10.67, P < 0.01). Fish exposed to BPA showed less time responding to the mirror stimulus compared to the E2 group (F1,18 ¼ 4.52, P < 0.05). However, no statistically signiﬁcant difference was detected between E2 exposure groups and the control ((F1,18 ¼ 0.63, P > 0.05).
4. Results 4.1. Chronic exposure to BPA or E2 affects motor behavior Motor behavior of zebraﬁsh, as measured by the number of grid lines when the ﬁsh crossed during a 6 min period, was affected by BPA or E2 exposure. As seen from Fig. 1, ﬁsh exposed to BPA reduced their locomotor activity slightly compared with the control (F1,18 ¼ 1.18, P ¼ 0.2917). E2 showed serious effects on zebraﬁsh activity (F1,18 ¼ 24.74, P < 0.01), besides, there was no statistically signiﬁcant difference between BPA and E2 exposure groups (F1,18 ¼ 14.01, P > 0.05).
4.3. Chronic exposure to BPA or E2 affects group preference BPA or E2 exposure also altered group preference of both male and female zebraﬁsh as measured by the total time when ﬁsh closed to the shoal per 6 min (F1,20 ¼ 4.75, P < 0.05; F1,20 ¼ 4.44, P < 0.05, respectively) (Fig. 3). Additionally, there was no clear difference between the BPA or E2 exposure groups.
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Fig. 1. The effects of chronic BPA or E2 exposure on locomotion activity behavior in individual adult zebraﬁsh are tested. Statistically signiﬁcant differences between control and E2 are indicated (*P < 0.05 or **P < 0.01). The sample sizes are as following: control, n ¼ 10; BPA, n ¼ 10; E2, n ¼ 10.
4.4. Chronic exposure to BPA or E2 affects novel tank test Both BPA and E2 can affect the novel tank test of zebraﬁsh (Fig. 4). As seen from Fig. 4A, BPA or E2 exposure resulted in a signiﬁcantly lower latency to exposure the upper half of the tank compared with control (F1,18 ¼ 9.78, P < 0.01; F1,18 ¼ 35.22, P < 0.01, respectively). Treatment with BPA or E2 increased the number of transitions to the upper portion compared with the control group (F1,18 ¼ 6.04, P < 0.05; F1,18 ¼ 5.88, P < 0.05, respectively) in Fig. 4B. Additionally, in Fig. 4C, chronic administration of BPA or E2 made zebraﬁsh spend more time in the top half of the tank compared with the control group (F1,18 ¼ 26.63, P < 0.01; F1,18 ¼ 16.80, P < 0.01, respectively). Besides, compared with the control group, the BPA or
Fig. 3. The effects of chronic BPA or E2 exposure on group preference in individual adult zebraﬁsh are tested. Statistically signiﬁcant differences between control and BPA or E2 are indicated (*P < 0.05 or **P < 0.01). n ¼ 11 for each group.
E2 exposure decreased the amount of erratic movement performed by zebraﬁsh (F1,18 ¼ 7.67, P < 0.05; F1,18 ¼ 5.44, P < 0.01, respectively) (Fig. 4D). From Fig. 4E and F, the amount and duration time of freezing were also decreased in response to the E2 exposure group (F1,18 ¼ 40.09, P < 0.01; F1,18 ¼ 13.78, P < 0.01, respectively), but no statistically signiﬁcant difference was detected between BPA exposure group and the control group (P > 0.05). 4.5. Chronic exposure to BPA or E2 affects light/dark preference As seen from Fig. 5, the control group displayed the circadianlike trend of light/dark preference in 2 days with the dark preference increased initially from morning (29.62%, 8:00am) to midnight (74.06%, 2:00am), and subsequently decreased till next morning (43.53%, 8:00am). Compared with the control, zebraﬁsh from BPA or E2 exposure altered the circadian trend of light/dark preference. Although the proportion of dark preference in BPA group was slightly higher than control at 8:00am on day 1 (29.62% vs 39.81%, P > 0.05), there was a signiﬁcant difference between the control and BPA group at 2:00am on day 2 (74.06% vs 55.56%, P < 0.05). The mean proportion of time spent in the dark area for the E2 exposure group was higher than the control, for example, the mean proportion of dark preference was a signiﬁcant difference between the control and E2 group at 8:00am on day 1 (29.62% vs 50%, P < 0.01), but there was no difference at 2:00am on day 2 (74.06% vs 81.48%, P > 0.05). 5. Discussion
Fig. 2. The effects of chronic BPA or E2 exposure on aggressive behavior in individual adult zebraﬁsh are tested. Statistically signiﬁcant differences between control and BPA or E2 are indicated (*P < 0.05 or **P < 0.01). The sample sizes are as following: control, n ¼ 10; BPA, n ¼ 10; E2, n ¼ 10.
BPA is a primary ingredient to manufacture polycarbonate plastic and epoxy resin, and widely used as an intermediate in the production of numerous consumer products including polycarbonate bottle, resin-lined cans and some dental sealants. However, the incomplete polymerization or gradual breakdown of BPAcontaining products results in potential leaching of BPA into food or water (Biedermann et al., 2010; Liao and Kannan, 2013; Welshons et al., 2006). BPA can be found at concentrations ranging from 0.04 to 370,000 ng/L in water (Huang et al., 2012), and even can attain hundreds of thousands of ng/L in some industrial waste waters from the industrial park (Fukazawa et al., 2001). The
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Fig. 4. Novel tank test effects of chronic BPA and E2 exposure (500 mg/L or 10 mg/L for 6 month, respectively) in individual adult zebraﬁsh (5 male and 5 female) tested in the novel tank test during 6 min *P < 0.05 or **P < 0.01. n ¼ 10 for each group.
purpose of this work is to assess whether and how low dose of BPA based on the concentration of treated efﬂuent affects key behaviors of aquatic organisms with chronic exposure in ecotoxicological research. The individuals' behavioral traits of motor behavior, aggression, group preference and light/dark preference were tested by adult zebraﬁsh with 6 months BPA or E2 exposure. Motor behavior is regarded as one of the most common and easily measured behavioral responses that correlate to the physiological
capacity of ﬁsh because it can generate and coordinate the locomotive energy required for basic functions such as migration or escaping predators (Little et al., 1990). One example of the importance of motor behavior for rainbow trout is maintaining position against ﬂowing water while feeding (Little et al., 1990). Fish exposed to the BPA reduce their locomotor activity slightly, while E2 can seriously decrease the activity compared with the control (Fig. 1). BPA exposure cannot cause serious motor deﬁcits like E2 does, the reason may be that the concentration of BPA is
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Fig. 5. Plot of the proportion of time spent in the dark area at each time of day in zebraﬁsh in different drugs exposure (control (closed diamonds), BPA (closed squares), E2 (closed triangle)). n ¼ 9 for each group.
not high enough to disrupt the axonal growth of primary and secondary motor neurons in zebraﬁsh (Wang et al., 2013), and E2 is associated with the defect of neurobehavioral development which may seriously affect the activity (Hamad et al., 2007). The decreased locomotor performances can increase lethargy in the shoal of ﬁsh. Aggression has extended the whole animal kingdom, and most animal species use aggression to establish dominance hierarchies with dominant individuals chasing (Basquill and Grant, 1998) and biting subordinate individuals in order to compete for food or mates. Levels of aggressiveness are related to territory ownership (Whoriskey and FitzGerald, 1994), territory size (van den Assem, 1967) and reproductive success. Zebraﬁsh exposed to BPA shows less aggressive to the mirror stimulus compared to the control. Similar results have been observed after exposure to the other EDCs, such as nonylphenol (NP) (Xia et al., 2010) and ethinyloestradiol exposure (Bell, 2001). The mechanisms of the decreased aggression by BPA exposure associate with the regulation of testosterone and 11-ketotestosterone (Villars, 1983), inhibiting the binding of native androgens to androgen receptor or downregulation of the androgen production via BPA acts as an androgen receptor antagonist in male zebraﬁsh (Bell, 2001; Bonefeld-Jorgensen et al., 2007; Lee et al., 2003; Sohoni and Sumpter, 1998; Xu et al., 2010). The effects of BPA or E2 on the behavior of individual animals inﬂuence the dynamics of ﬁsh populations in aquatic environments. Group preference is essential for all shoaling teleosts, and shoaling behavior can associate with foraging, spawning security, predator recognition (Pitcher, 1986). Zebraﬁsh is a shoal species and exhibits group preference (Spence et al., 2008), shoaling behavior appears to be innate and commences soon after hatching (Engeszer et al., 2007), and ﬁsh rear in isolation quickly form shoals when they are placed together (Kerr, 1962). Shoaling behavior has many advantages for zebraﬁsh. For example, shoal can provide a defense against predators, enhance the ability of the ﬁsh to ﬁnd their own prey or mate and increase foraging success. In our results, group preference was signiﬁcantly affected by BPA or E2 exposure for both male and female zebraﬁsh compared with the control group. Similar results are found in rainbow trout (Ward et al., 2006) and killiﬁsh (Ward et al., 2008) after exposure to nonyphenol. The mechanism of group preference associate with dopaminergic system by the change of the level of dopamine, DOPAC, serotonin and 5HIAA, respectively (Scerbina et al., 2012). BPA or E2 exposure decreased the group preference which could affect dopaminergic system. Zebraﬁsh may suffer from increased predation, decreased food acquisition and decreased the chance of reproduction if the
properties of a shoal were broken down as a result of exposure to BPA or E2. To assess more directly ecological effects of BPA and E2 exposure, we measured individual novel tank test, which are initially presumed to be primitive and instinctive (de Perera, 2004; Laland et al., 2003). Individuals spend the majority of time at the bottom when introduced into a novel condition and then expand their position of swimming to the higher portions of the tested tank (Levin et al., 2007). However, chronic BPA or E2 exposure can alter these behaviors of individuals, with lower latency to reach the top, more entries to the top, longer time spent in the top, lower and fewer frequent freezing, as well as reduced erratic movements. These ﬁndings suggest that zebraﬁsh with BPA or E2 exposure is probably weaker adaptability to the novel environment, which may increase predation risk. Naturally, ﬁsh had the preference in light or dark area, and the appropriate preference was vital to the survival of ﬁsh, moreover, our previous research indicated that circadian clock could affect the light/dark preference of zebraﬁsh (Wang et al., 2014). In our present results, the circadian trend of light/dark preference was signiﬁcantly affected by BPA or E2 exposure compared with the control group (Fig. 5). The less obvious preference may associate with down-regulated of clock genes expression via BPA or E2 exposure in the pituitary, brain, muscle, and skin in ﬁsh (Rhee et al., 2014). BPA or E2 exposure may affect circulating pineal 5-hydroxytryptaming (5-HT) levels (Ho et al., 1985; Weber et al., 2015). As seen from Fig. 5, ﬁsh lack of sensitive to the variation from day to night by the modiﬁcation of light/dark preference through BPA or E2 exposure, because circadian rhythm can alter intensity of social behavior and direct locomotor activity (Weber and Spieler, 1994; Pankseep et al., 2008). The locomotor activity may inﬂuence the proportion of dark preference in BPA and E2 group. This environmentally relevant concentration of BPA affects zebraﬁsh behaviors is alarming, considering the extensive BPA products that are found in waters worldwide. It should also be emphasized that BPA has direct effect on zebraﬁsh behavior which allocate to various ﬁtness functions such as growth, reproduction and body maintenance. Our results highlight ecologically important effects, previously underappreciated effects of BPA that enters aquatic ecosystems, and calls for new attention to examine the full environmental impact of BPA residues. 6. Conclusions The research demonstrated that chronic exposure of treatedefﬂuent concentration BPA or E2 could be able to induce various
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