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Sound production in Japanese medaka (Oryzias latipes) and its alteration by exposure to aldicarb and copper sulfate
Chemosphere 181 (2017) 530e535
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Sound production in Japanese medaka (Oryzias latipes) and its alteration by exposure to aldicarb and copper sulfate Ik Joon Kang a, *, 1, Xuchun Qiu b, 1, Junya Moroishi c, Yuji Oshima b a
International Student Center, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan c SEIKO Electric Co., Ltd., Tenjin 3-20-1, Koga City, Fukuoka, 811-3197, Japan b
h i g h l i g h t s We found that medaka produce sound with constant average interpulse intervals. Sound production is affected by exposure to pesticide and heavy metal. Sound production is an effective endpoint to monitor abnormal water quality.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 January 2017 Received in revised form 19 April 2017 Accepted 20 April 2017 Available online 20 April 2017
This study is the first to report sound production in Japanese medaka (Oryzias latipes). Sound production was affected by exposure to the carbamate insecticide (aldicarb) and heavy-metal compound (copper sulfate). Medaka were exposed at four concentrations (aldicarb: 0, 0.25, 0.5, and 1 mg L1; copper sulfate: 0, 0.5, 1, and 2 mg L1), and sound characteristics were monitored for 5 h after exposure. We observed constant average interpulse intervals (approx 0.2 s) in all test groups before exposure, and in the control groups throughout the experiment. The average interpulse interval became significantly longer during the recording periods after 50 min of exposure to aldicarb, and reached a length of more than 0.3 s during the recording periods after 120 min exposure. Most medaka fish stopped to produce sound after 50 min of exposure to copper sulfate at 1 and 2 mg L1, resulting in significantly declined number of sound pulses and pulse groups. Relative shortened interpulse intervals of sound were occasionally observed in medaka fish exposed to 0.5 mg L1 copper sulfate. These alternations in sound characteristics due to toxicants exposure suggested that they might impair acoustic communication of medaka fish, which may be important for their reproduction and survival. Our results suggested that using acoustic changes of medaka has potential to monitor precipitate water pollutions, such as intentional poisoning or accidental leakage of industrial waste. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: David Volz Keywords: Sound production Japanese medaka Water pollution Sonic fish
1. Introduction All fishes possess inner ears for sound detection (Ladich, 2014), but only some fish species are known to produce context-specific sounds for communication (Fine and Parmentier, 2015). Sound production in fish is generally associated with their reproductive, agonistic, and territorial behaviors (Hawkins and Amorim, 2000; Kasumyan, 2008; Fine and Parmentier, 2015). Acoustic
* Corresponding author. E-mail address: [email protected] (I.J. Kang). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.chemosphere.2017.04.088 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
communication has been suggested as an evolutionary property of fishes that inhabit biotopes where other communication systems (e.g. visual or olfactory sensors) are difficult or ineffective (Ladich, 2000; Kasumyan, 2008). In such habitats, acoustic communication by fish might be essential to their reproduction and survival (Bass and McKibben, 2003; Kasumyan, 2008). Recent years, there is increasing evidence that disturbed acoustic communication due to anthropogenic disturbance can affect the reproductive success and survival of fishes (Amorim and Clara, 2006; Van der Sluijs et al., 2011; Rosenthal and Stuart-Fox, 2012; Tricas and Boyle, 2015). Thus, there are raised concerns about the efficiency of fish acoustic communication in the human-disturbed environments (Slabbekoorn et al., 2010; Radford et al., 2014).
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We found that the Japanese medaka (Oryzias latipes) is a sonic fish and produces stable sounds on a regular basis under normal culture conditions. Since medaka inhabits turbid environments such as paddies, marshes, ponds, slow-moving streams and tide pools (Nakabo, 2002), it is possible that they use sound to increase the reliability of communication for successful social interaction in the natural environment. As a model fish for risk assessment of chemicals in the Organization for Economic Cooperation and Development guidelines (OECD, 1999), the individual and social behaviors of medaka fish have been demonstrated to be disturbed by a wide range of environmental toxicants, generally through disturbances in sensory system and/or neuroendocrine system (Rice et al., 1997; El-Alfy and Schlenk, 2002; Oshima et al., 2003; Kang et al., 2009b; Khalil et al., 2013). Moreover, previous studies have reported that pollutants such as chemicals and metals can affect many aspects of acoustic signal production in birds and frogs, and thereby may impact behaviors essential for their survival and reproduction (Gorissen et al., 2005; Markman et al., 2008; Huang et al., 2015). However, detailed empirical studies of environmental toxicants on acoustic communication in fishes are completely lacking (Slabbekoorn et al., 2010; Van der Sluijs et al., 2011; Rosenthal and Stuart-Fox, 2012). On the other hand, biological early warning systems using vision-based analysis of medaka behaviors have been used to quickly detect water pollution from various toxic chemicals at high concentrations (Kang et al., 2009a; Ren and Wang, 2010). However, on-site application of these visual methods may be limited in water of high turbidity or chromaticity, where it is difficult to record images and observe behavior. If variations in sound production by Japanese medaka were associated with toxic chemical exposure, it would be possible to develop a camera-less and non-invasive biomonitoring method for detecting water pollution due to those chemicals, thereby extending the application of existing biological monitoring systems. In this study, we investigated the alteration of sound production by Japanese medaka exposed to the pesticide aldicarb and the heavy metal copper. The intentional and accidental poisoning in animals and people by carbamates, such as aldicarb, has become a threat to public health and public safety worldwide (MacFarlane rez et al., 2015). The et al., 2011; de Siqueira et al., 2015; Ruiz-Sua heave metal, copper, which is neurotoxic to fishes and can interfere with the normal function of sensory nervous system, is a well known nonpoint source pollutant in aquatic ecosystems worldwide
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(Hecht et al., 2007; McIntyre et al., 2008; Linbo et al., 2009; Tilton et al., 2011; Wang et al., 2013). The objectives of this study are to describe the sound patterns produced by Japanese medaka and to investigate the responses of sound production to environmental toxicants. We also compared the sensitivity of responses to aldicarb exposure in terms of sound production to that in swimming behavior, in order to evaluate the potential utility of sound parameters in biomonitoring of water quality and pollution. 2. Materials and methods 2.1. Test chemicals Aldicarb (C7H14N2O2S, 100% purity) was obtained from Wako Pure Chemical Industries (Osaka, Japan). Copper sulfate (CuSO4$5H2O) was purchased from Sigma-Aldrich Co., Ltd. (Saint Louis, MO). Aldicarb stock solution (100 mg L1) and copper sulfate stock solution (100 mg L1) were prepared using dechlorinated tap water. 2.2. Test fish Mature Japanese medaka (4e6 months after hatching; body weight, 283 ± 36 mg; total length, 31.5 ± 1.8 mm) were collected from brood stock maintained for several years. The fish were placed under a 14:10-h light:dark photoperiod and fed twice daily with sufficient quantities of Artemia nauplii (<24 h after hatching). The water temperature was maintained at 22 ± 1 C, and the dissolved oxygen concentration was maintained at 7.0 ± 0.2 mg L1. 2.3. Sound production test We performed a preliminary test to determine the conditions suitable for sound production by Japanese medaka. A test chamber (34 cm long 16 cm wide 10 cm high) containing 1.5 L of dechlorinated tap water was placed in a soundproof and vibrationproof box to reduce potential extraneous noise and vibration (Fig. 1E). Dechlorinated tap water was delivered into the test chamber at 100 mL/min using a rotary peristaltic pump (Eyela RP1000; Tokyo Rikakikai Co., Ltd., Tokyo, Japan), and the overflow water drained out from the outlet. A hydrophone (YN02; CTI Science Systems Co., Ltd., Tokyo, Japan) was installed in the test chamber. Ten medaka fish were acclimatized in the test chamber under these continuous flow-through conditions for 12 h. Subsequently, the sounds produced by the Japanese medaka were recorded for 10 h using a digital recorder (PCM-D1; Sony Co., Ltd., Tokyo, Japan). The recorded data were analyzed using Adobe Audition 3 software (Adobe Systems Inc., Tokyo, Japan). The results showed that the Japanese medaka produced sound pulses with a frequency of 0.5e3.5 kHz, and that sounds produced by Japanese medaka under the above conditions can be recorded. 2.4. Exposure tests
Fig. 1. Image (left) and diagram (right) of the exposure test chamber. (A) Lightemitting diode; (B) hydrophone; (C) amplifier; (D) digital recorder; (E) sound- and vibration-proof box.
Test solutions containing aldicarb (0.25, 0.5, and 1 mg L1) and copper sulfate (0.5, 1, and 2 mg L1) were prepared by diluting the stock solution (100 mg L1) with dechlorinated tap water. The control solution (0 mg L1) contained only dechlorinated tap water. Exposure experiments were conducted using the test chamber with continuous flow through as described in section 2.3 (Fig. 1). Each test group consisted of 10 medaka fish placed in the test chamber; each exposure test was repeated four times (40 fish in total for each). The fish were first acclimatized in dechlorinated tap water for 2 h, and then the test solution flowed through the test chamber for 5 h. The flow rate was checked by using a graduated cylinder;
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the test solution in the chamber was exchanged 4 times per hour. Sounds produced by each group of Japanese medaka were recorded 6 times (each recording, 10 min); at 10 min before the initiation of exposure, and then during 50e60, 110e120, 170e180, 230e240, and 290e300 min after exposure. For comparison, we also followed variations in the swimming behavior of Japanese medaka exposed to aldicarb (0, 0.25, 0.5, and 1 mg L1) using the biomonitoring system described by Kang et al. (2009a). Briefly, in each replicate (n ¼ 4), one medaka fish was placed in the test chamber with a continuous flow of dechlorinated tap water. After 30 min acclimation, fish locomotion was recorded for another 30 min to monitor the swimming speed during the preexposure period. Subsequently, the test solution was introduced into the test chamber for 300 min, and the swimming behavior of the medaka was recorded during 30e60, 90e120, 150e180, 210e240, and 270e300 min after the start of exposure. Swimming behavior was recorded in three dimensions (3-D; x, y, and z coordinates) by two digital video cameras (model GE60, Library, Tokyo, Japan) placed in front and on the side of the transparent test chamber. 2.5. Sound and swimming-behavior analysis Successive pulses with an interpulse interval <0.6 s were considered as a pulse group, where the interpulse interval is the silent period between two successive sound pulses (Fig. 2). For each pulse group within a 10-min recording period, we determined the number of pulses it contained (Ni) and its duration (Di, the time from the beginning to the end of the pulse group) (Fig. 2B). The average interpulse interval (IPIave) for all pulse groups within each 10-min recording period was calculated using the following equation:
Pn Di IPIave ¼ Pn i¼1 Ni 1 i¼1
(1)
The 3-D swimming speed (mm/s) of fish was determined using the method described by Kang et al. (2009a).
Fig. 2. Oscillogram (A) and spectrogram (B) of representative sounds produced by Japanese medaka (Oryzias latipes), and the harmonics (C) of a single pulse (indicated by triangles). The downward-pointing arrows indicate each pulse in a pulse group. Di, duration of each pulse group; Ni, number of pulses in each pulse group; IPI, interpulse interval.
2.6. Statistical analysis Deviation from the normality of the experimental data distribution was examined using a Kolmogorov-Smirnov test, and logarithmic or reciprocal transformation was used when needed. The sound parameters and swimming speed of medaka fish were analyzed using two-way repeated measure analysis of variance (ANOVA) for the exposure period (from pre-exposure), considering exposure concentrations as the between-subjects factors. Subsequently, the significances between responses at the pre-exposure period and those at each 10-min recording period after exposure were checked for each exposure concentration. In the copper sulfate exposure tests, the replicates that did not produce sound within recording period were excluded for further statistical analysis of average interpulse interval. All statistical analyses were performed using SPSS Base 11.0J (SPSS Inc., Chicago, IL, USA). 3. Results Medaka produced sound that can be represented as a sequence of pulses, with peak frequencies ranging from 0.5 to 3.5 kHz (Fig. 2). A single pulse comprised a low fundamental frequency (around 0.5 kHz) and several harmonics (repeating 2e6 times) with multiple frequencies (Fig. 2C). There were variations in the parameters of sound produced by medaka exposed to aldicarb. There were large individual differences in the number of pulses and the number of pulse groups within each test group (reflected in large error bars), and almost no statistically significant differences in the parameters during the pre-exposure period and each recording period after exposure (Fig. 3A and B). On the contrary, we observed a constant average interpulse interval of approximately 0.2 s in all test groups before exposure and in the control group during the 5-h test period (Fig. 3C). Independent of the concentrations of aldicarb, the average interpulse interval of sound produced by medaka became significantly longer (Fig. 4 shows representative sound spectrograms of prolonged interpulse interval) during the recording periods after 50 min of exposure (P < 0.05), and reached a length of more than 0.3 s during the recording periods after 120 min exposure (P < 0.01). After 50 min of exposure, the number of sound pulses and that of pulse groups significantly declined in medaka exposed to copper sulfate at 1 and 2 mg L1 (Fig. 5A and B). Because many medaka fish exposed to copper sulfate at 1 and 2 mg L1 stopped to produce pulse group (i.e. the average interpulse interval in more than 50% replicates cannot be calculated), it was not available to conduct statistical analysis in these concentration treatments. The average interpulse interval in the control group was approximately 0.2 s throughout the experiment (Fig. 5C). Relative shortened interpulse intervals were observed in the treatment of 0.5 mg L1 copper sulfate exposure (Fig. 5C). The significant difference level in interpulse intervals between pre-exposure period and those recorded at 110e120 min (P ¼ 0.057) and at 290e300 min (P ¼ 0.053) after exposure to 0.5 mg L1 copper sulfate were calculated at the P < 0.10 level (Fig. 5C). The Japanese medaka exposed to aldicarb showed variations in swimming speed (Fig. 6). There were significant decreases in swimming speed at 150e180, 90e120, and 30e60 min after exposure to aldicarb at 0.25, 0.5, and 1.0 mg L1, compared with pre-exposure period. 4. Discussion This study is the first to demonstrate that medaka is a sonic fish. It produces sound consisting of a sequence of pulses, with peak
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Fig. 4. Representative sound spectrograms produced by Japanese medaka (Oryzias latipes) before exposure (A) and 5 h after being exposed to aldicarb at 0.25 mg L1 (B). The downward-pointing arrows indicate each single pulse in a pulse group. Fig. 3. Variations in the number of pulses (A), number of pulse groups (B), and average interpulse interval within pulse groups (C) of Japanese medaka (Oryzias latipes) exposed to aldicarb (ald). Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (*P < 0.05; **P < 0.01). Recording periods are in minutes.
frequencies ranging from approximately 0.5 to 3.5 kHz (Fig. 2). These sound characteristics, combined with the harmonic structure of the pulses, suggest that medaka generates a drumming type of sound by rapid contractions of specialized sonic muscles attached to the external wall of the swim bladder (Kasumyan, 2008; Fine and Parmentier, 2015). This inference is supported by a whole-body section of the medaka, which showed conspicuous muscles attached to coupled with the swim bladder (data not shown). Drumming sounds have been reported for various species of fish belonging to the families of Triglidae, Characidae, Batrachoididae, and Sciaenidae, with a high diversity in their sound characteristics (Amorim and Clara, 2006; Tricas and Boyle, 2015). These sonic fishes can provide various sound signals for different purposes by changing the sound characteristics, and the vocal motor system for adjusting those sound characteristics is regulated by peripheral and central auditory neurons (Bass and McKibben, 2003; Amorim and Clara, 2006; Tricas and Boyle, 2015). It is therefore essential to investigate the impacts of stressors such as toxic chemicals and rapid changes in water quality on sound production in fish to be able to fully evaluate the health of fish on the basis of sounds produced. In the present study, we used the numbers of sound pulses and pulse groups, and the average interpulse interval of sounds produced by medaka fish to detect possible impacts of exposure to aldicarb and copper sulfate, because these sound parameters have been demonstrated to be important for signal discrimination and species recognition in various sonic fish (Spanier, 1979; Crawford and Huang, 1999; Bass et al., 2001). Significant decreases in the
number of both pulses and pulse groups were observed in the sound produced by medaka fish exposed to copper sulfate, but not in that of aldicarb exposed fish (Figs. 3 and 5). The number of pulses and pulse rate are the key features for species-specific recognition of sonic fishes, and alterations in those parameters has been reportedly affected by the motivation and social status of the fish, the season, time of a day, and ontogenetic changes (Amorim and Clara, 2006). Thus, we suggest that investigating alterations in these sound parameters induced by various toxicants may be very useful to improve our understanding on the mechanisms involved in their impacts on social behaviors (and organization) of medaka fish. The constancy of the average interpulse interval under normal conditions and its sensitivity to aldicarb (prolonged effect) and copper sulfate (shortened effect) exposure suggests that this sound parameter can possibly serve as a valuable endpoint in studies of medaka ecotoxicology for determining changes of environments. The constancy of interpulse interval has been reported for various sonic fishes (Crawford, 1997; Mann et al., 1997; Hawkins and Amorim, 2000; Kozloski and Crawford, 2000). As one of the most important characteristics for acoustic signal discrimination, changes in interpulse interval of sound produced by some sonic fish have been reported to cause variations in playback behavior and the absence of vocalization response in nearby individuals (Winn, 1972; McKibben and Bass, 1999, 2001). Therefore, the significantly altered interpulse interval of sound due to toxicants exposure might impair the acoustic communication of medaka fish. Such impairment in sonic fishes could further induce deleterious effects on their survival at the population level. Moreover, the different responses in the sound characteristics of fish exposed to aldicarb and copper sulfate suggested that those two chemicals may affect the sound production by different action mechanisms.
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Fig. 5. Variations in the number of pulses (A), number of pulse groups (B), and average interpulse interval within pulse groups (C) of Japanese medaka (Oryzias latipes) exposed to copper sulfate. Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (#P < 0.10; *P < 0.05; **P < 0.01). Recording periods are in minutes. NS, no sound pulse was observed within the record period; NG, no sound pulse group was observed within the record period; NA, not available to calculate the interpulse interval because of none sound pulse.
As a carbamate, aldicarb binds with acetylcholinesterase (AChE) and allows acetylcholine to accumulate in the synapse, resulting in over-stimulation of the nerves (Coppage, 1977; Baron, 1994). For some fishes producing drumming sounds (e.g. mormyrids), the interpulse interval is controlled by interval-coding neurons in the medulla and midbrain (Crawford, 1997; Kozloski and Crawford, 2000). If the medaka has systems similar to those of the
mormyrid fish, then aldicarb might significantly extend the interpulse interval by inhibiting the signal transference in intervalcoding neurons. Moreover, our results also indicated that the response in interpulse interval of sound was more sensitive than that in swimming speed to aldicarb exposure (compare Figs. 3 and 6). The higher sensitivity of response in sensory nervous systems than that of behavior patterns to the AChE inhibitors (e.g. organophosphate) has been reported by some previous studies. For example, salmon exposed to chlorpyrifos showed that the olfactory neurobehavioral dysfunction can be observed in the absence of pesticide effects on brain AChE (Sandahl et al., 2004, 2005; Wang et al., 2016). Thus, studies about the effects of carbamate and organophosphate on sound productions of sonic fishes may also be helpful to promote our understanding of their adverse effects on aquatic organisms by different mechanisms other than AChE inhibition. On the other hand, the heavy metal copper is well known for producing acute or chronic effects on the sensory nervous systems and related behavior of various aquatic organisms. For example, copper adversely affects olfactory functiondwhich is directly and indirectly connected with behavioral and physiological responses in fishdand impairs behaviors such as reproduction, predator avoidance, food location, and migration (Ji et al., 2006; Wang et al., 2013). Our results from copper sulfate exposure (0.5 mg L1) show shortening of interpulse intervals at a certain extent. Previous studies have showed that exposure to copper, zinc, or their complexes caused an abnormal increase in the coughing rate of rainbow trout Oncorhynchus mykiss (Svecevicius and Kazlauskiene, 2011). As the cough-like behavior was suggested to cause contraction of muscles connected to swim bladder and was associated with shortduration sound pulse generated by some sonic fishes (Allen and Demer, 2003), we therefore infer that copper sulfate exposure may induce an increase in coughing rate in medaka and be linked with the shortening of interpulse intervals observed in the present study. Our results suggest that monitoring of variations in sound parameters would be valuable for detecting chemicals that have the action mechanisms similar to that of aldicarb and copper. Considering the relatively high experimental concentrations of two chemical toxicants used in the present study, further studies testing the effects of various chemical contaminants and physical stress (as well as their mixture effects) on sound parameters are needed, to decipher the sensitivity, accuracy, and reliability of this novel biomarker. Nevertheless, the data provided from the present study can still demonstrate the ability of using acoustic changes of medaka for monitoring precipitate water pollutions from toxicants at high levels, such as intentional poisoning or accidental leakage of industrial waste.
5. Conclusion
Fig. 6. Variations in the swimming speed of Japanese medaka (Oryzias latipes) exposed to aldicarb. Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (*P < 0.05; **P < 0.01). Recording periods are in minutes.
Overall, this is the first study to demonstrate that the Japanese medaka is a sonic fish. The alterations in the sound characteristic of medaka fish exposed to pesticide and heave metal may not only provide important ecotoxicological information for this fish, but also potentially serve as an effective endpoint for biological monitoring of water quality and contamination attributable to those toxic chemicals. Further studies are needed concerning other chemicals at environmentally relevant levels and stressors (as well as their and their mixture effects) on medaka sound production to improve our understanding of the ecological importance of acoustic communication in medaka, and to facilitate the application of sound production in water-quality monitoring and early warning systems.
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deprivation in juvenile coho salmon exposed to dissolved copper under varying water chemistry conditions. Environ. Sci. Technol. 42, 1352e1358. McKibben, J.R., Bass, A.H., 1999. Peripheral encoding of behaviorally relevant acoustic signals in a vocal fish: single tones. J. Comp. Physiol. A 184, 563e576. McKibben, J.R., Bass, A.H., 2001. Peripheral encoding of behaviorally relevant acoustic signals in a vocal fish: harmonic and beat stimuli. J. Comp. Physiol. A 187, 271e285. Nakabo, T., 2002. Fishes of Japan: with Pictorial Keys to the Species. Tokai University Press. OECD, 1999. Final Report from the OECD Expert Consultation Meeting. London, UK, 28e29 October 1998. Report 9906. Environmental Health and Safety Division, Paris, France. Oshima, Y., Kang, I.J., Kobayashi, M., Nakayama, K., Imada, N., Honjo, T., 2003. Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17b-estradiol. Chemosphere 50, 429e436. Radford, A.N., Kerridge, E., Simpson, S.D., 2014. Acoustic communication in a noisy world: can fish compete with anthropogenic noise? Behav. Ecol. aru029. Ren, Z., Wang, Z., 2010. Differences in the behavior characteristics between Daphnia magna and Japanese madaka in an on-line biomonitoring system. J. Environ. Sci. 22, 703e708. Rice, P.J., Drewes, C.D., Klubertanz, T.M., Bradbury, S.P., Coats, J.R., 1997. Acute toxicity and behavioral effects of chlorpyrifos, permethrin, phenol, strychnine, and 2, 4-dinitrophenol to 30-day-old Japanese medaka (Oryzias latipes). Environ. Toxicol. Chem. 16, 696e704. Rosenthal, G., Stuart-Fox, D., 2012. Environmental disturbance and animal communication. In: Candolin, U., Wong, B. (Eds.), Behavioural Responses to a Changing World: Mechanisms and Consequences. Oxford University Press, Oxford, pp. 16e31. Ruiz-Su arez, N., Boada, L.D., Henríquez-Hern andez, L.A., Gonz alez-Moreo, F., rez, A., Camacho, M., Zumbado, M., Almeida-Gonz Su arez-Pe alez, M., del Mar Travieso-Aja, M., Luzardo, O.P., 2015. Continued implication of the banned pesticides carbofuran and aldicarb in the poisoning of domestic and wild animals of the Canary Islands (Spain). Sci. Total Environ. 505, 1093e1099. Sandahl, J.F., Baldwin, D.H., Jenkins, J.J., Scholz, N.L., 2004. Odor-evoked field potentials as indicators of sublethal neurotoxicity in juvenile coho salmon (Oncorhynchus kisutch) exposed to copper, chlorpyrifos, or esfenvalerate. Can. J. Fish. Aquat. Sci. 61, 404e413. Sandahl, J.F., Baldwin, D.H., Jenkins, J.J., Scholz, N.L., 2005. Comparative thresholds for acetylcholinesterase inhibition and behavioral impairment in coho salmon exposed to chlorpyrifos. Environ. Toxicol. Chem. 24, 136e145. Slabbekoorn, H., Bouton, N., van Opzeeland, I., Coers, A., ten Cate, C., Popper, A.N., 2010. A noisy spring: the impact of globally rising underwater sound levels on fish. Trends Ecol. Evol. 25, 419e427. Spanier, E., 1979. Aspects of species recognition by sound in four species of Damselfishes, Genus eupomacentrus (Pisces: Pomacentridae). Z. Tierpsychol. 51, 301e316. Svecevicius, G., Kazlauskiene, N., 2011. Behavioral responses in rainbow trout Oncorhynchus mykiss as indicators of sublethal exposure to heavy metals. In: Environmental Engineering, VIII International Conference, Selected Papers, pp. 19e20. Tilton, F.A., Tilton, S.C., Bammler, T.K., Beyer, R.P., Stapleton, P.L., Scholz, N.L., Gallagher, E.P., 2011. Transcriptional impact of organophosphate and metal mixtures on olfaction: copper dominates the chlorpyrifos-induced response in adult zebrafish. Aquat. Toxicol. 102, 205e215. Tricas, T.C., Boyle, K.S., 2015. Diversity and evolution of sound production in the social behavior of Chaetodon butterflyfishes. J. Exp. Biol. 218, 1572e1584. Van der Sluijs, I., Gray, S.M., Amorim, M.C.P., Barber, I., Candolin, U., Hendry, A.P., Krahe, R., Maan, M.E., Utne-Palm, A.C., Wagner, H.-J., 2011. Communication in troubled waters: responses of fish communication systems to changing environments. Evol. Ecol. 25, 623e640. Wang, L., Bammler, T.K., Beyer, R.P., Gallagher, E.P., 2013. Copper-induced deregulation of microRNA expression in the zebrafish olfactory system. Environ. Sci. Technol. 47, 7466e7474. Wang, L., Espinoza, H.M., MacDonald, J.W., Bammler, T.K., Williams, C.R., Yeh, A., Ke’ale, W.L., Marcinek, D.J., Gallagher, E.P., 2016. Olfactory transcriptional analysis of salmon exposed to mixtures of chlorpyrifos and malathion reveal novel molecular pathways of neurobehavioral injury. Toxicol. Sci. 149, 145e157. Winn, H.E., 1972. Acoustic discrimination by the toadfish with comments on signal systems. In: Winn, H.E., Olla, B.L. (Eds.), Behavior of Marine Animals: Current Perspectives in Research Volume 2: Vertebrates. Springer US, Boston, MA, pp. 361e385.
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Sound production in Japanese medaka (Oryzias latipes) and its alteration by exposure to aldicarb and copper sulfate Ik Joon Kang a, *, 1, Xuchun Qiu b, 1, Junya Moroishi c, Yuji Oshima b a
International Student Center, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan c SEIKO Electric Co., Ltd., Tenjin 3-20-1, Koga City, Fukuoka, 811-3197, Japan b
h i g h l i g h t s We found that medaka produce sound with constant average interpulse intervals. Sound production is affected by exposure to pesticide and heavy metal. Sound production is an effective endpoint to monitor abnormal water quality.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 January 2017 Received in revised form 19 April 2017 Accepted 20 April 2017 Available online 20 April 2017
This study is the first to report sound production in Japanese medaka (Oryzias latipes). Sound production was affected by exposure to the carbamate insecticide (aldicarb) and heavy-metal compound (copper sulfate). Medaka were exposed at four concentrations (aldicarb: 0, 0.25, 0.5, and 1 mg L1; copper sulfate: 0, 0.5, 1, and 2 mg L1), and sound characteristics were monitored for 5 h after exposure. We observed constant average interpulse intervals (approx 0.2 s) in all test groups before exposure, and in the control groups throughout the experiment. The average interpulse interval became significantly longer during the recording periods after 50 min of exposure to aldicarb, and reached a length of more than 0.3 s during the recording periods after 120 min exposure. Most medaka fish stopped to produce sound after 50 min of exposure to copper sulfate at 1 and 2 mg L1, resulting in significantly declined number of sound pulses and pulse groups. Relative shortened interpulse intervals of sound were occasionally observed in medaka fish exposed to 0.5 mg L1 copper sulfate. These alternations in sound characteristics due to toxicants exposure suggested that they might impair acoustic communication of medaka fish, which may be important for their reproduction and survival. Our results suggested that using acoustic changes of medaka has potential to monitor precipitate water pollutions, such as intentional poisoning or accidental leakage of industrial waste. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: David Volz Keywords: Sound production Japanese medaka Water pollution Sonic fish
1. Introduction All fishes possess inner ears for sound detection (Ladich, 2014), but only some fish species are known to produce context-specific sounds for communication (Fine and Parmentier, 2015). Sound production in fish is generally associated with their reproductive, agonistic, and territorial behaviors (Hawkins and Amorim, 2000; Kasumyan, 2008; Fine and Parmentier, 2015). Acoustic
* Corresponding author. E-mail address: [email protected] (I.J. Kang). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.chemosphere.2017.04.088 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
communication has been suggested as an evolutionary property of fishes that inhabit biotopes where other communication systems (e.g. visual or olfactory sensors) are difficult or ineffective (Ladich, 2000; Kasumyan, 2008). In such habitats, acoustic communication by fish might be essential to their reproduction and survival (Bass and McKibben, 2003; Kasumyan, 2008). Recent years, there is increasing evidence that disturbed acoustic communication due to anthropogenic disturbance can affect the reproductive success and survival of fishes (Amorim and Clara, 2006; Van der Sluijs et al., 2011; Rosenthal and Stuart-Fox, 2012; Tricas and Boyle, 2015). Thus, there are raised concerns about the efficiency of fish acoustic communication in the human-disturbed environments (Slabbekoorn et al., 2010; Radford et al., 2014).
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We found that the Japanese medaka (Oryzias latipes) is a sonic fish and produces stable sounds on a regular basis under normal culture conditions. Since medaka inhabits turbid environments such as paddies, marshes, ponds, slow-moving streams and tide pools (Nakabo, 2002), it is possible that they use sound to increase the reliability of communication for successful social interaction in the natural environment. As a model fish for risk assessment of chemicals in the Organization for Economic Cooperation and Development guidelines (OECD, 1999), the individual and social behaviors of medaka fish have been demonstrated to be disturbed by a wide range of environmental toxicants, generally through disturbances in sensory system and/or neuroendocrine system (Rice et al., 1997; El-Alfy and Schlenk, 2002; Oshima et al., 2003; Kang et al., 2009b; Khalil et al., 2013). Moreover, previous studies have reported that pollutants such as chemicals and metals can affect many aspects of acoustic signal production in birds and frogs, and thereby may impact behaviors essential for their survival and reproduction (Gorissen et al., 2005; Markman et al., 2008; Huang et al., 2015). However, detailed empirical studies of environmental toxicants on acoustic communication in fishes are completely lacking (Slabbekoorn et al., 2010; Van der Sluijs et al., 2011; Rosenthal and Stuart-Fox, 2012). On the other hand, biological early warning systems using vision-based analysis of medaka behaviors have been used to quickly detect water pollution from various toxic chemicals at high concentrations (Kang et al., 2009a; Ren and Wang, 2010). However, on-site application of these visual methods may be limited in water of high turbidity or chromaticity, where it is difficult to record images and observe behavior. If variations in sound production by Japanese medaka were associated with toxic chemical exposure, it would be possible to develop a camera-less and non-invasive biomonitoring method for detecting water pollution due to those chemicals, thereby extending the application of existing biological monitoring systems. In this study, we investigated the alteration of sound production by Japanese medaka exposed to the pesticide aldicarb and the heavy metal copper. The intentional and accidental poisoning in animals and people by carbamates, such as aldicarb, has become a threat to public health and public safety worldwide (MacFarlane rez et al., 2015). The et al., 2011; de Siqueira et al., 2015; Ruiz-Sua heave metal, copper, which is neurotoxic to fishes and can interfere with the normal function of sensory nervous system, is a well known nonpoint source pollutant in aquatic ecosystems worldwide
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(Hecht et al., 2007; McIntyre et al., 2008; Linbo et al., 2009; Tilton et al., 2011; Wang et al., 2013). The objectives of this study are to describe the sound patterns produced by Japanese medaka and to investigate the responses of sound production to environmental toxicants. We also compared the sensitivity of responses to aldicarb exposure in terms of sound production to that in swimming behavior, in order to evaluate the potential utility of sound parameters in biomonitoring of water quality and pollution. 2. Materials and methods 2.1. Test chemicals Aldicarb (C7H14N2O2S, 100% purity) was obtained from Wako Pure Chemical Industries (Osaka, Japan). Copper sulfate (CuSO4$5H2O) was purchased from Sigma-Aldrich Co., Ltd. (Saint Louis, MO). Aldicarb stock solution (100 mg L1) and copper sulfate stock solution (100 mg L1) were prepared using dechlorinated tap water. 2.2. Test fish Mature Japanese medaka (4e6 months after hatching; body weight, 283 ± 36 mg; total length, 31.5 ± 1.8 mm) were collected from brood stock maintained for several years. The fish were placed under a 14:10-h light:dark photoperiod and fed twice daily with sufficient quantities of Artemia nauplii (<24 h after hatching). The water temperature was maintained at 22 ± 1 C, and the dissolved oxygen concentration was maintained at 7.0 ± 0.2 mg L1. 2.3. Sound production test We performed a preliminary test to determine the conditions suitable for sound production by Japanese medaka. A test chamber (34 cm long 16 cm wide 10 cm high) containing 1.5 L of dechlorinated tap water was placed in a soundproof and vibrationproof box to reduce potential extraneous noise and vibration (Fig. 1E). Dechlorinated tap water was delivered into the test chamber at 100 mL/min using a rotary peristaltic pump (Eyela RP1000; Tokyo Rikakikai Co., Ltd., Tokyo, Japan), and the overflow water drained out from the outlet. A hydrophone (YN02; CTI Science Systems Co., Ltd., Tokyo, Japan) was installed in the test chamber. Ten medaka fish were acclimatized in the test chamber under these continuous flow-through conditions for 12 h. Subsequently, the sounds produced by the Japanese medaka were recorded for 10 h using a digital recorder (PCM-D1; Sony Co., Ltd., Tokyo, Japan). The recorded data were analyzed using Adobe Audition 3 software (Adobe Systems Inc., Tokyo, Japan). The results showed that the Japanese medaka produced sound pulses with a frequency of 0.5e3.5 kHz, and that sounds produced by Japanese medaka under the above conditions can be recorded. 2.4. Exposure tests
Fig. 1. Image (left) and diagram (right) of the exposure test chamber. (A) Lightemitting diode; (B) hydrophone; (C) amplifier; (D) digital recorder; (E) sound- and vibration-proof box.
Test solutions containing aldicarb (0.25, 0.5, and 1 mg L1) and copper sulfate (0.5, 1, and 2 mg L1) were prepared by diluting the stock solution (100 mg L1) with dechlorinated tap water. The control solution (0 mg L1) contained only dechlorinated tap water. Exposure experiments were conducted using the test chamber with continuous flow through as described in section 2.3 (Fig. 1). Each test group consisted of 10 medaka fish placed in the test chamber; each exposure test was repeated four times (40 fish in total for each). The fish were first acclimatized in dechlorinated tap water for 2 h, and then the test solution flowed through the test chamber for 5 h. The flow rate was checked by using a graduated cylinder;
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the test solution in the chamber was exchanged 4 times per hour. Sounds produced by each group of Japanese medaka were recorded 6 times (each recording, 10 min); at 10 min before the initiation of exposure, and then during 50e60, 110e120, 170e180, 230e240, and 290e300 min after exposure. For comparison, we also followed variations in the swimming behavior of Japanese medaka exposed to aldicarb (0, 0.25, 0.5, and 1 mg L1) using the biomonitoring system described by Kang et al. (2009a). Briefly, in each replicate (n ¼ 4), one medaka fish was placed in the test chamber with a continuous flow of dechlorinated tap water. After 30 min acclimation, fish locomotion was recorded for another 30 min to monitor the swimming speed during the preexposure period. Subsequently, the test solution was introduced into the test chamber for 300 min, and the swimming behavior of the medaka was recorded during 30e60, 90e120, 150e180, 210e240, and 270e300 min after the start of exposure. Swimming behavior was recorded in three dimensions (3-D; x, y, and z coordinates) by two digital video cameras (model GE60, Library, Tokyo, Japan) placed in front and on the side of the transparent test chamber. 2.5. Sound and swimming-behavior analysis Successive pulses with an interpulse interval <0.6 s were considered as a pulse group, where the interpulse interval is the silent period between two successive sound pulses (Fig. 2). For each pulse group within a 10-min recording period, we determined the number of pulses it contained (Ni) and its duration (Di, the time from the beginning to the end of the pulse group) (Fig. 2B). The average interpulse interval (IPIave) for all pulse groups within each 10-min recording period was calculated using the following equation:
Pn Di IPIave ¼ Pn i¼1 Ni 1 i¼1
(1)
The 3-D swimming speed (mm/s) of fish was determined using the method described by Kang et al. (2009a).
Fig. 2. Oscillogram (A) and spectrogram (B) of representative sounds produced by Japanese medaka (Oryzias latipes), and the harmonics (C) of a single pulse (indicated by triangles). The downward-pointing arrows indicate each pulse in a pulse group. Di, duration of each pulse group; Ni, number of pulses in each pulse group; IPI, interpulse interval.
2.6. Statistical analysis Deviation from the normality of the experimental data distribution was examined using a Kolmogorov-Smirnov test, and logarithmic or reciprocal transformation was used when needed. The sound parameters and swimming speed of medaka fish were analyzed using two-way repeated measure analysis of variance (ANOVA) for the exposure period (from pre-exposure), considering exposure concentrations as the between-subjects factors. Subsequently, the significances between responses at the pre-exposure period and those at each 10-min recording period after exposure were checked for each exposure concentration. In the copper sulfate exposure tests, the replicates that did not produce sound within recording period were excluded for further statistical analysis of average interpulse interval. All statistical analyses were performed using SPSS Base 11.0J (SPSS Inc., Chicago, IL, USA). 3. Results Medaka produced sound that can be represented as a sequence of pulses, with peak frequencies ranging from 0.5 to 3.5 kHz (Fig. 2). A single pulse comprised a low fundamental frequency (around 0.5 kHz) and several harmonics (repeating 2e6 times) with multiple frequencies (Fig. 2C). There were variations in the parameters of sound produced by medaka exposed to aldicarb. There were large individual differences in the number of pulses and the number of pulse groups within each test group (reflected in large error bars), and almost no statistically significant differences in the parameters during the pre-exposure period and each recording period after exposure (Fig. 3A and B). On the contrary, we observed a constant average interpulse interval of approximately 0.2 s in all test groups before exposure and in the control group during the 5-h test period (Fig. 3C). Independent of the concentrations of aldicarb, the average interpulse interval of sound produced by medaka became significantly longer (Fig. 4 shows representative sound spectrograms of prolonged interpulse interval) during the recording periods after 50 min of exposure (P < 0.05), and reached a length of more than 0.3 s during the recording periods after 120 min exposure (P < 0.01). After 50 min of exposure, the number of sound pulses and that of pulse groups significantly declined in medaka exposed to copper sulfate at 1 and 2 mg L1 (Fig. 5A and B). Because many medaka fish exposed to copper sulfate at 1 and 2 mg L1 stopped to produce pulse group (i.e. the average interpulse interval in more than 50% replicates cannot be calculated), it was not available to conduct statistical analysis in these concentration treatments. The average interpulse interval in the control group was approximately 0.2 s throughout the experiment (Fig. 5C). Relative shortened interpulse intervals were observed in the treatment of 0.5 mg L1 copper sulfate exposure (Fig. 5C). The significant difference level in interpulse intervals between pre-exposure period and those recorded at 110e120 min (P ¼ 0.057) and at 290e300 min (P ¼ 0.053) after exposure to 0.5 mg L1 copper sulfate were calculated at the P < 0.10 level (Fig. 5C). The Japanese medaka exposed to aldicarb showed variations in swimming speed (Fig. 6). There were significant decreases in swimming speed at 150e180, 90e120, and 30e60 min after exposure to aldicarb at 0.25, 0.5, and 1.0 mg L1, compared with pre-exposure period. 4. Discussion This study is the first to demonstrate that medaka is a sonic fish. It produces sound consisting of a sequence of pulses, with peak
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Fig. 4. Representative sound spectrograms produced by Japanese medaka (Oryzias latipes) before exposure (A) and 5 h after being exposed to aldicarb at 0.25 mg L1 (B). The downward-pointing arrows indicate each single pulse in a pulse group. Fig. 3. Variations in the number of pulses (A), number of pulse groups (B), and average interpulse interval within pulse groups (C) of Japanese medaka (Oryzias latipes) exposed to aldicarb (ald). Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (*P < 0.05; **P < 0.01). Recording periods are in minutes.
frequencies ranging from approximately 0.5 to 3.5 kHz (Fig. 2). These sound characteristics, combined with the harmonic structure of the pulses, suggest that medaka generates a drumming type of sound by rapid contractions of specialized sonic muscles attached to the external wall of the swim bladder (Kasumyan, 2008; Fine and Parmentier, 2015). This inference is supported by a whole-body section of the medaka, which showed conspicuous muscles attached to coupled with the swim bladder (data not shown). Drumming sounds have been reported for various species of fish belonging to the families of Triglidae, Characidae, Batrachoididae, and Sciaenidae, with a high diversity in their sound characteristics (Amorim and Clara, 2006; Tricas and Boyle, 2015). These sonic fishes can provide various sound signals for different purposes by changing the sound characteristics, and the vocal motor system for adjusting those sound characteristics is regulated by peripheral and central auditory neurons (Bass and McKibben, 2003; Amorim and Clara, 2006; Tricas and Boyle, 2015). It is therefore essential to investigate the impacts of stressors such as toxic chemicals and rapid changes in water quality on sound production in fish to be able to fully evaluate the health of fish on the basis of sounds produced. In the present study, we used the numbers of sound pulses and pulse groups, and the average interpulse interval of sounds produced by medaka fish to detect possible impacts of exposure to aldicarb and copper sulfate, because these sound parameters have been demonstrated to be important for signal discrimination and species recognition in various sonic fish (Spanier, 1979; Crawford and Huang, 1999; Bass et al., 2001). Significant decreases in the
number of both pulses and pulse groups were observed in the sound produced by medaka fish exposed to copper sulfate, but not in that of aldicarb exposed fish (Figs. 3 and 5). The number of pulses and pulse rate are the key features for species-specific recognition of sonic fishes, and alterations in those parameters has been reportedly affected by the motivation and social status of the fish, the season, time of a day, and ontogenetic changes (Amorim and Clara, 2006). Thus, we suggest that investigating alterations in these sound parameters induced by various toxicants may be very useful to improve our understanding on the mechanisms involved in their impacts on social behaviors (and organization) of medaka fish. The constancy of the average interpulse interval under normal conditions and its sensitivity to aldicarb (prolonged effect) and copper sulfate (shortened effect) exposure suggests that this sound parameter can possibly serve as a valuable endpoint in studies of medaka ecotoxicology for determining changes of environments. The constancy of interpulse interval has been reported for various sonic fishes (Crawford, 1997; Mann et al., 1997; Hawkins and Amorim, 2000; Kozloski and Crawford, 2000). As one of the most important characteristics for acoustic signal discrimination, changes in interpulse interval of sound produced by some sonic fish have been reported to cause variations in playback behavior and the absence of vocalization response in nearby individuals (Winn, 1972; McKibben and Bass, 1999, 2001). Therefore, the significantly altered interpulse interval of sound due to toxicants exposure might impair the acoustic communication of medaka fish. Such impairment in sonic fishes could further induce deleterious effects on their survival at the population level. Moreover, the different responses in the sound characteristics of fish exposed to aldicarb and copper sulfate suggested that those two chemicals may affect the sound production by different action mechanisms.
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Fig. 5. Variations in the number of pulses (A), number of pulse groups (B), and average interpulse interval within pulse groups (C) of Japanese medaka (Oryzias latipes) exposed to copper sulfate. Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (#P < 0.10; *P < 0.05; **P < 0.01). Recording periods are in minutes. NS, no sound pulse was observed within the record period; NG, no sound pulse group was observed within the record period; NA, not available to calculate the interpulse interval because of none sound pulse.
As a carbamate, aldicarb binds with acetylcholinesterase (AChE) and allows acetylcholine to accumulate in the synapse, resulting in over-stimulation of the nerves (Coppage, 1977; Baron, 1994). For some fishes producing drumming sounds (e.g. mormyrids), the interpulse interval is controlled by interval-coding neurons in the medulla and midbrain (Crawford, 1997; Kozloski and Crawford, 2000). If the medaka has systems similar to those of the
mormyrid fish, then aldicarb might significantly extend the interpulse interval by inhibiting the signal transference in intervalcoding neurons. Moreover, our results also indicated that the response in interpulse interval of sound was more sensitive than that in swimming speed to aldicarb exposure (compare Figs. 3 and 6). The higher sensitivity of response in sensory nervous systems than that of behavior patterns to the AChE inhibitors (e.g. organophosphate) has been reported by some previous studies. For example, salmon exposed to chlorpyrifos showed that the olfactory neurobehavioral dysfunction can be observed in the absence of pesticide effects on brain AChE (Sandahl et al., 2004, 2005; Wang et al., 2016). Thus, studies about the effects of carbamate and organophosphate on sound productions of sonic fishes may also be helpful to promote our understanding of their adverse effects on aquatic organisms by different mechanisms other than AChE inhibition. On the other hand, the heavy metal copper is well known for producing acute or chronic effects on the sensory nervous systems and related behavior of various aquatic organisms. For example, copper adversely affects olfactory functiondwhich is directly and indirectly connected with behavioral and physiological responses in fishdand impairs behaviors such as reproduction, predator avoidance, food location, and migration (Ji et al., 2006; Wang et al., 2013). Our results from copper sulfate exposure (0.5 mg L1) show shortening of interpulse intervals at a certain extent. Previous studies have showed that exposure to copper, zinc, or their complexes caused an abnormal increase in the coughing rate of rainbow trout Oncorhynchus mykiss (Svecevicius and Kazlauskiene, 2011). As the cough-like behavior was suggested to cause contraction of muscles connected to swim bladder and was associated with shortduration sound pulse generated by some sonic fishes (Allen and Demer, 2003), we therefore infer that copper sulfate exposure may induce an increase in coughing rate in medaka and be linked with the shortening of interpulse intervals observed in the present study. Our results suggest that monitoring of variations in sound parameters would be valuable for detecting chemicals that have the action mechanisms similar to that of aldicarb and copper. Considering the relatively high experimental concentrations of two chemical toxicants used in the present study, further studies testing the effects of various chemical contaminants and physical stress (as well as their mixture effects) on sound parameters are needed, to decipher the sensitivity, accuracy, and reliability of this novel biomarker. Nevertheless, the data provided from the present study can still demonstrate the ability of using acoustic changes of medaka for monitoring precipitate water pollutions from toxicants at high levels, such as intentional poisoning or accidental leakage of industrial waste.
5. Conclusion
Fig. 6. Variations in the swimming speed of Japanese medaka (Oryzias latipes) exposed to aldicarb. Data are shown as mean ± SD (n ¼ 4). Statistical significance is in comparison with pre-exposure period (*P < 0.05; **P < 0.01). Recording periods are in minutes.
Overall, this is the first study to demonstrate that the Japanese medaka is a sonic fish. The alterations in the sound characteristic of medaka fish exposed to pesticide and heave metal may not only provide important ecotoxicological information for this fish, but also potentially serve as an effective endpoint for biological monitoring of water quality and contamination attributable to those toxic chemicals. Further studies are needed concerning other chemicals at environmentally relevant levels and stressors (as well as their and their mixture effects) on medaka sound production to improve our understanding of the ecological importance of acoustic communication in medaka, and to facilitate the application of sound production in water-quality monitoring and early warning systems.
I.J. Kang et al. / Chemosphere 181 (2017) 530e535
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