Using medaka embryos as a model system to study biological effects of the electromagnetic fields on development and behavior

Ecotoxicology and Environmental Safety 108 (2014) 187–194

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Using medaka embryos as a model system to study biological effects of the electromagnetic fields on development and behavior Wenjau Lee n, Kun-Lin Yang Department of Bioscience Technology, Chang Jung Christian University, No. 1, Changda Rd., Gueiren District, Tainan City, Taiwan

art ic l e i nf o

a b s t r a c t

Article history: Received 10 April 2014 Received in revised form 27 June 2014 Accepted 28 June 2014

The electromagnetic fields (EMFs) of anthropogenic origin are ubiquitous in our environments. The health hazard of extremely low frequency and radiofrequency EMFs has been investigated for decades, but evidence remains inconclusive, and animal studies are urgently needed to resolve the controversies regarding developmental toxicity of EMFs. Furthermore, as undersea cables and technological devices are increasingly used, the lack of information regarding the health risk of EMFs to aquatic organisms needs to be addressed. Medaka embryos (Oryzias latipes) have been a useful tool to study developmental toxicity in vivo due to their optical transparency. Here we explored the feasibility of using medaka embryos as a model system to study biological effects of EMFs on development. We also used a white preference test to investigate behavioral consequences of the EMF developmental toxicity. Newly fertilized embryos were randomly assigned to four groups that were exposed to an EMF with 3.2 kHz at the intensity of 0.12, 15, 25, or 60 mT. The group exposed to the background 0.12 mT served as the control. The embryos were exposed continually until hatch. They were observed daily, and the images were recorded for analysis of several developmental endpoints. Four days after hatching, the hatchlings were tested with the white preference test for their anxiety-like behavior. The results showed that embryos exposed to all three levels of the EMF developed significantly faster. The endpoints affected included the number of somites, eye width and length, eye pigmentation density, midbrain width, head growth, and the day to hatch. In addition, the group exposed to the EMF at 60 mT exhibited significantly higher levels of anxiety-like behavior than the other groups did. In conclusion, the EMF tested in this study accelerated embryonic development and heightened anxiety-like behavior. Our results also demonstrate that the medaka embryo is a sensitive and cost-efficient in vivo model system to study developmental toxicity of EMFs. & 2014 Elsevier Inc. All rights reserved.

Keywords: Electromagnetic field Medaka Development Behavior

1. Introduction The electromagnetic fields (EMFs) are naturally occurring forces, but modern technologies have produced stronger EMFs and made them more ubiquitous in our daily life. Today, EMFs are another class of pollutants that may cause adverse effects on the health of humans and wildlife. The EMFs are divided into several categories based on their frequencies. There are extremely low frequency (ELF) EMFs generated from power lines, domestic appliances and electric transformers, and radiofrequency (RF) EMFs produced by radio and television broadcasts and wireless communication devices. The health effects of these EMFs have been investigated extensively for decades through epidemiological and animal studies, but the results remain inconclusive and the health risk uncertain.

n

Corresponding author. Fax: þ 886 62785010. E-mail address: [email protected] (W. Lee).

http://dx.doi.org/10.1016/j.ecoenv.2014.06.035 0147-6513/& 2014 Elsevier Inc. All rights reserved.

For example, Lagroye et al. (2011) reviewed several EMFs studies and found no biological effect in rodents exposed to ELF EMFs (50–60 Hz) at levels up to 30 mT. There is also no epidemiological association of ELF EMFs with adult cancers (Elliott et al., 2013), cardiovascular disease mortality (He et al., 2013), stillbirth (Auger et al., 2012), pregnancy and birth conditions (Mahram and Ghazavi, 2013), and neurodegenerative diseases (Mattsson and Simkó, 2012). On the other hand, ELF EMFs have also been found to increase the risk of miscarriage (Li et al., 2002), breast cancer (Sun et al., 2013), Alzheimer's disease and dementia in men (Qiu et al., 2004), and acute leukemia in children (Sermage-Faure et al., 2013). There are beneficial effects as well. ELF EMFs at 1 mT can modulate chemokine production and keratinocyte growth, rendering it a potentially therapeutic treatment for skin injury (Vianale et al., 2008). Also, an EMF with 7.5 Hz at 0.4 T would induce antitumor immune response, inhibit tumor growth, and prolong survival time in mice (Nie et al., 2013). The potential health hazards caused by RF EMFs are similarly controversial. As reviewed by Herbert and Sage (2013), the

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consequences of RF EMF exposures, such as cellular and oxidative stress, mitochondrial dysfunction, and neurodevelopmental disruption, are so similar to the autism spectrum conditions that RF EMFs are suspected to be the cause of the disorder. On the contrary, other review studies have concluded that evidence is either lacking or inconclusive for adverse effects of RF EMFs on the nervous system (van Rongen et al., 2009), cognitive function (Regel and Achermann, 2011), psychophysiological reactions (Augner et al., 2012), and cancer (Health Protection Agency-UK, 2012). Animal studies on developmental toxicity of EMFs have also produced variable results. For example, ELF EMFs exposures (0.5 mT) have been found to slow down development and metamorphosis in tadpoles (63.9–76.4 mT; Severini et al., 2010), but accelerate embryonic development in Daphnia magna (45–500 Hz, 75 mT; Krylov, 2010) and Japanese newt embryos (5–30 mT; Komazaki and Takano, 2007). However, Brent (1999) and Juutilainen (2005), through extensive literature reviews, have concluded that most developmental toxicity studies on ELF and RF EMFs with non-human mammalians result in the absence of adverse effects. Part of the reason for these differences is due to the wide differences in the methodology, exposure designs, and/or cell types. Different environmental conditions may also account for the variability, as EMFs affect water physiochemical properties variably with temperature, illumination, etc. (Naira et al., 2013). In addition, EMF effects can be reverse depending on the length of exposure periods (Bae et al., 2013), which further increases the discrepancies. Though ELF and RF EMFs vary drastically in their frequencies and strengths, they do have common properties. First of all, they are both the type of non-ionizing radiation, as opposed to ionizing radiation, such as X-rays, that have sufficient energy to produce charged particles. Secondly, both EMFs diminish rapidly with distance, either in the air or water. For example, the EMFs produced from underwater cables have been estimated to be 0.46–8.0 mT on the surface and 20–100 mT at 1.0 m distance from the surface, a 23–80 times reduction (Cada et al., 2011). Thirdly, some of their biological effects may not be that different after all. For example, microwave thermal effects are long considered the cause of RF EMFs' health risk, but a safety limit based on nonthermal effects from low intensity RF EMFs has been proposed (Hardell and Sage, 2008). This is because both ELF and RF EMFs are similarly linked to an increase in cancer risk, as DNA may act as a fractal antenna that reacts to EMFs and results in DNA damage (Blank and Goodman, 2011). To add the complexity further, newer studies are exploring the interacting effects of ELF and RF EMFs on living organisms (Murbach et al., 2012). In reality, these two categories of EMFs are commonly mixed in urban environment, and separating the two when assessing EMF health risk may not be environmentally relevant. Because of this uncertainty, both ELF and RF EMFs have been classified as “possible human carcinogens” by the WHO International Agency for Research on Cancer. The WHO has also placed the “effects of early-life and prenatal RF exposure on development and behavior” as high priority for animal studies (World Health Organization, 2010). As to ecological impacts of EMFs, field studies are few. However, ecologically relevant evidence from laboratory studies, using bacteria, plants, insects, amphibians, birds, and the usual mice and rats, indicates that RF EMFs have the potential to cause adverse biological effects on wildlife (Balmori, 2009; Cucurachi et al., 2013). But Bochert and Zettler (2004) exposed several marine benthic animals to a static EMF (3.7 mT) for several weeks and found no significant difference to the control group. A most recent study confirmed that migratory fish use the earth's magnetic field

as a cue, but the fish only temporarily altered their swimming direction when crossing the EMF-producing submarine cables (Gill and Bartlett, 2012). As fish can be electrically and magnetically sensitive, the lack of information regarding the health risk of EMFs to fish needs to be addressed, especially when undersea cables and marine technological devices are increasingly being laid as now (Boehlert and Gill, 2010; Tricas and Gill, 2011). Therefore, in the present study, we aimed to test the feasibility of using the medaka fish (Oryzias latipes) as a model system to study biological effects of EMFs on development and behavior. The fish is an oviparous freshwater teleost. It has become a popular animal model in recent years because of its small size (adult 2–4 cm in length), hardiness (tougher than zebrafish), fecundity (spawn 10–20 eggs daily), and short generational time (2–3 months). Most of all, as medaka embryos are transparent, their development can be easily observed in whole living embryos. These qualities have rendered the fish ideal for the studies of EMF developmental toxicity. Here we investigated the effects of EMF exposures on (1) development by directly observing the morphological development of embryos, and on (2) behavior by conducting the white preference test on four-day-old hatchlings of EMF-exposed embryos. This behavioral test is commonly used as a measure for anxiety-like behavior in fish (Echevarria et al., 2011).

2. Materials and methods 2.1. Experimental animals A colony of medaka had been established in the current facility for over five years. The fish was maintained at 28 1C under a constant 14 h light:10 h dark photoperiod in glass tanks filled with flow-through filtered water (pH 7.5–7.8). Embryos were incubated in embryo medium (0.1 percent NaCl, 0.003 percent KCl, 0.004 percent CaCl2  2H2O, and 0.016 percent MgSO4  7H2O, pH 7.5–7.8). All salts in the medium were supplied by J.T.Baker (Phillipsburg, NJ, USA). Newly hatching larvae were transferred to the tanks and fed with ground fish flake for one week, then fed three times daily with brine shrimp (within 24 h after hatching). All procedures were carried out in accordance to the “Guidelines for Animal Experimentation” of Chang Jung Christian University, Taiwan.

2.2. Embryo exposures The procedures were based on the OECD guideline for fish embryo toxicity test (OECD, 2006). Briefly, medaka embryos from breeding pairs were collected within 5 h postfertilization, and randomly placed in 24-well plates, one embryo per well. Each well contained 1 ml embryo medium with a depth about 1 cm, short enough for the penetration of EMFs. The embryo medium was completely replaced every 24 h. The plates were randomly assigned to different exposure groups [0.12 (n¼ 27), 15 (n¼ 17), 25 (n¼ 16), and 60 mT (n ¼16)]. To keep each observation session within a 2-h period, each batch of embryos was limited to n¼ 2–4 per group, and the experiment was repeated seven times. The wells in the plates were then positioned at the distance of 70, 2, 1, and 0.5 cm from a magnetic field generator (described below). The plates and the generator were placed on a bench in an air-conditioned room, with temperature ranging from 25 to 28 1C. The room was also subjected to natural sunlight and artificial lighting during regular working hours. The group exposed to 0.12 mT served as the control, which was the general background reading around the room. The intensity of the EMF at each position was monitored daily with a Gaussmeter (EMDEX II, Enertech Consultants), which varied within 9.42% of the designated values. The embryos were exposed until hatch, and the hatchlings were photographed and then transferred to glass dishes for the behavioral test. The magnetic field generator consisted of an AC/DC power converter, an adjustment knob, a function generator chip, and a copper wire coiling around four magnetic poles with an inner diameter of 31 mm. An alternating current of 60 Hz, 110 V, was applied to the generator and electrified the coil. The frequency of 3.2 kHz used in this study was chosen randomly and later verified with a digital oscilloscope (Tektronix dpo3014).

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2.3. Observations and image analysis The procedures were as described in the previous study (Lee et al., 2012). Briefly, the heart rate of embryos was counted twice, each for 30 s, under a

yideo camera

189

dissection microscope, then the embryo was anaesthetized with 0.06 percent MS222 (pH 7.25, Sigma-Aldrich), and their images recorded under a microscope (IX2-SLP, Olympus, Tokyo, Japan). The length, width, and the pigmentation density (eye density) of the eye, the distance between the eyes (eye distance; representing head growth), and the midbrain width were analyzed from the images with ImageJ image processing and analysis software (http://rsbweb.nih.gov/ij/). All images were obtained and analyzed under identical conditions without any alteration.

3.6X magnifier 2.4. White preference test

LED light water filled to 10 mm in height height 15 mm

width 40 mm length 60 mm

squares 25 mm2

Fig. 1. Schematic drawing of the white preference test apparatus. The apparatus was a rectangular box made of clear plastic (L 60 mm  W 40 mm  H 15 mm). The box was placed on a piece of cardboard, whose surface was divided into two equal areas, one black and one white. A square pattern of 5  5 mm2 was drawn on the surface. By counting the number of squares the hatchlings entered, we could easily estimate the distance of the larval movement. The four sides of the plastic box were also covered with black or white cardboard accordingly. The box was filled with filtered water to 10 mm in height. An LED lamp fitted with a magnifier was positioned directly above the box, and a video camera was placed above the lamp.

1 dpf

The white preference test was based on the “light versus dark preference test” (Gerlai et al., 2000) with modifications. The test apparatus consisted of a video camera (HTC Incredible S), an LED lamp fitted with a magnifier, and a black/white rectangular box (Fig. 1). The test was conducted in a dark room, so the LED lamp was the only light source. Individual hatchling (four-day old) was first held with a plastic pipet for 30 s to raise its anxiety level, and then released at the center of the black area. The hatchling was allowed to explore freely for the duration of 2 min without any disturbance. Each hatchling was tested only once. The video recording of the hatchling movement was analyzed and the following endpoints were determined: time spent in black or white area, number of squares entered, latent time of first crossing to the white area, and the total time of swimming along the sides or around the center of the box.

2.5. Statistical analysis The statistics software SPSS 17 (SPSS Inc., Chicago, USA) was used for all data analysis. One-way ANOVA was conducted, followed by LSD tests to compare

3 dpf

control

60 µT

Fig. 2. Representative images of medaka embryos at 1 dpf (A and C) and 3 dpf (B and D). Medaka embryos were exposed to the background EMF (controls; A and B) or 60 mT EMF (C and D) until hatched. A and B were the same individual, and C and D were another individual. The images were cropped, but their relative proportion was maintained, and no other alteration was made. Bar ¼100 mm.

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variables of each endpoint among groups. Values were considered as significantly different when p o 0.05.

3. Results 3.1. Effects of the EMF on embryonic development The medaka embryos exposed to the EMF at 3.2 Hz developed significantly more advanced at early stages of development. As shown in Fig. 2, at 1 dpf, the embryo exposed to the EMF at 60 mT already had lenses in the eyes, and its midbrain (the swelling posterior to the eyes) was much larger than that of the control. At 3 dpf, the darker eyes of the embryos from the 60 mT group were clearly discernable. The measurements of various developmental endpoints are listed in Table 1 and graphed as Fig. 3. The changes of these endpoints are summarized in Table 2. Overall, development of the EMF-exposed medaka embryos was significantly faster than that of the control in many of the endpoints examined. At 1 dpf, all of the EMF-exposed embryos

had in average 2.0–2.7 more somites than the control did. This accelerated development was also evident in the eye length and width, and the midbrain width. Development of the head (eye distance) also increased at 2 and 3 dpf. The advanced development of the eye in the EMF-exposed embryos was prominent at 3 dpf, as the eye pigmentation density was 13–17 percent higher than that of the control. There was no difference in the heart rate of the EMF-exposed groups, except that of the 25 mT group at 2 dpf. The embryos exposed to the EMF at 60 mT hatched in average 4.5 days faster than the control did. Their hatching rate was also the highest among the groups, though not statistically significant. There was no difference in the hatchling body length among all of the groups. In addition, no malformation or teratogenic effect was observed, and there was also no difference in hatchling mortality rate.

3.2. Effects of the EMF on hatchling behavior The EMF exposure during embryonic development appeared to significantly affect the anxiety-like behavior of the hatchlings

Table 1 Measurements of endpoints from medaka embryos exposed to various intensties of the electromagnetic field. Data were expressed as mean7 standard error; dpf, day post fertilization; –, not applied; nsignificantly different from the control, p o 0.05; nnp o 0.01. Endpoint

Group

Mean 7 standard error 1 dpf

2 dpf

3 dpf

Number of somites

Control 15 mT 25 mT 60 mT

6.6 7 0.5 8.7 7 0.7nn 8.6 7 0.7n 9.3 7 0.6nn

– – – –

– – – –

Heart rate (beat/min)

Control 15 mT 25 mT 60 mT

– – – –

131.3 71.4 134.8 7 2.4 137.9 7 3.0n 135.87 2.4

152.4 7 2.0 152.0 7 4.2 151.2 7 2.9 153.2 7 1.8

Eye width (mm)

Control 15 mT 25 mT 60 mT

75.4 7 2.6 89.6 7 3.6nn 88.2 7 3.8n 90.6 7 6.2nn

150.8 7 2.4 166.37 3.0nn 166.77 4.4nn 173.3 7 3.9nn

205.2 7 7.7 221.5 7 8.3 205.8 7 4.3 219.7 7 10.7

Eye length (mm)

Control 15 mT 25 mT 60 mT

137.0 7 4.2 146.7 7 5.3 151.4 77.0n 156.2 7 4.7nn

222.17 4.1 238.7 7 6.5nn 243.87 6.2nn 254.7 7 6.4nn

270.17 7.7 291.5 77.3n 299.6 7 5.5nn 283.6 7 8.6

Eye density (pixels)

Control 15 mT 25 mT 60 mT

– – – –

– – – –

3173707 11272 3685717 11005nn 3597397 10970nn 371299 7 7914nn

Eye distance (mm)

Control 15 mT 25 mT 60 mT

84.4 7 2.5 90.0 7 2.6 89.0 7 2.6 91.3 72.7

123.6 72.6 134.2 7 4.3n 131.2 74.9 138.6 7 4.6nn

171.7 7 3.5 190.9 7 2.6nn 183.17 5.8n 193.4 7 4.2nn

Midbrain width (mm)

Control 15 mT 25 mT 60 mT

132.4 7 5.6 158.7 7 9.2n 149.17 6.9 179.0 7 16.7nn

391.5 7 6.8 412.2 7 5.8n 414.6 7 7.8n 430.3 7 9.5nn

472.5 7 4.1 495.47 4.9nn 490.17 7.9n 508.9 7 8.2nn

Hatching rate (percent)

Control 15 mT 25 mT 60 mT

79.2 7 8.7 68.87 12.4 78.6 7 11.8 87.5 7 8.8

Day to hatch (day)

Control 15 mT 25 mT 60 mT

12.5 7 0.8 11.5 7 1.9 10.17 0.8 8.0 7 0.4nn

Hatchling body length (mm)

Control 15 mT 25 mT 60 mT

1855 720.9 1879 732.4 1888 725.5 18187 22.4

W. Lee, K.-L. Yang / Ecotoxicology and Environmental Safety 108 (2014) 187–194

Number of Somites

Eye Length 150

%Control

175

**

150

*

** 125

*

**

125

Control

* **

15 µT 25 µT

75

75

50

50

60 µT

Eye Density

%Control

** ** **

100

100

Eye Distance

150

150

**

125

**

**

125

100

100

75

75

50

50

*

Day to Hatch %Control

191

175

100

150

**

125

50

100

25

75

0

50

Control

15

25

** * **

Midbrain Width

125

75

**

60 µT

** * * * **

1

2

** * **

3

dpf

Fig. 3. Endpoints significantly affected by the EMF during embryonic development in medaka. The number of somites was counted at 1 dpf, and the eye density measured at 3 dpf. Data were expressed as mean 7 standard error; dpf, day postfertilization; nsignificantly different from the control, p o0.05; nnp o 0.01.

Table 2 Changes of endpoints examined in medaka embryos exposed to the electromagnetic field. Dpf, day post fertilization; , increase; , decrease; , no change; –, not applied. Endpoint

dpf

EMF intensity (mT)

Number of somites Heart rate

1 2 3 1 2 3 1 2 3 3 1 2 3 1 2 3 – – –

15, 25, 25 – 15, 25, 15, 25, – 25, 60 15, 25, 15, 25 15, 25, – 15, 60 15, 25, 15, 60 15, 25, 15, 25, – 60 –

Eye width

Eye length

Eye density Eye distance

Midbrain width

Hatching rate Day to hatch Hatchling body length

Change

60

60 60

60 60

60 60 60

(Fig. 4). The groups exposed to the EMF at 60 mT, after being released in the black area, crossed significantly more quickly to the white area than the control and the 25 mT group did (Fig. 4A). The hatchlings from the group of 15 mT also crossed to the other side in less time, though not statistically significant from the control. In fact, 56 percent and 67 percent of the 15 and 60 mT groups, respectively, swam to the white area within 5 s, while only 24 percent of the control hatchling did so. Therefore, during the initial 15 s, the groups of 15 and 60 mT spent significantly less time in the black area, and significantly more time in the white area, compared to the control did (Fig. 4B). The majority (53 percent) of the 25 mT group also swam to the white area in 5 s, but a small

number of them moved slowly, making the averaged time comparable to that of the control. At the time of 30 s, the time distribution in the black/white areas of the 60 mT group continued to be significantly different from that of the control, but not that of the 15 mT group (data not shown). Interestingly, at this point the group of 25 mT started to show a significant difference in this distribution of time from that of the 60 mT group. Overall, during the 2-min test period, all of the hatchlings spent far more time in the white area. The hatchlings exposed to the EMF at 60 mT spent significantly less time in the black area, compared to the control and 25 mT groups did (Fig. 4C). As few hatchlings swam back to the black area after the initial crossing to the white area, their total time in the black area remained short at the end of the 2-min period. As to the swimming distance, the group exposed to the EMF at 60 mT also entered significantly more squares than the control and the 25 mT groups did during the initial 15 (Fig. 4D), 30 (Fig. 4E), and 45 s (data not shown). However, this difference disappeared after 60 s. At the end of the 2-min test period, the total swimming distance of the four groups was not significantly different (Fig. 4F). Similar to time distribution in the black/white areas, the swimming distance of the 25 mT group at 15, 30, and 45 s was also comparable to that of the control, and was significantly shorter than that of the 60 mT group. Interestingly, when the hatchlings were in the white area, they spent 7273% of the time moving along the sides of the box; when they were in the black area, they spent 8973% of the time swimming around the center. However, there was no significant difference in this behavior among the control and EMF-exposed groups.

4. Discussion The results of the present study indicate that the EMF with 3.2 kHz had biological effects on development of medaka embryos.

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40

a

a

15 ab

10

b

5

10

40

10

b

ab

a

ab

b

5

b

b

No. squares

20 a

150

50 25 0

a

Control

ab

15

a

25

b

60 µT

a ab

a

b a

30 sec

b

ab ab

b

a

a

a

10

150

75

a

a

20

0

100

ab

30

0

125

b

20

0

15

15 sec

30

0

No. squares

Time (sec)

Time (sec)

Time (sec)

20

No. squares

192

125

2 min

100 75 50 25 0

Control

15

25

60 µT

Fig. 4. Effects of the EMF exposure during embryonic development on anxiety-like behavior in medaka hatchlings. (A) Latent time for hatchlings first crossing from the black to white area; (B) distribution of time spent in the black and white areas during the initial 15 s; (C) 2-min test period; (D) number of squares hatchlings entered within the black or white areas during the initial 15 s, (E) 30 s, and (F) 2-min test period. Data were expressed as mean 7 standard error. Different letters indicate significant difference from the control, p o0.05. Letters on top of the columns indicate a comparison of total values from the black and white areas among groups, except (A); letters on the side of the columns indicate a comparison of values from the black or from the white areas.

Based on the atlas described by Iwamatsu (2004), the EMFexposed embryos at 1 dpf developed about 2–4 h faster than the control did. This pace of development must have accelerated even further, so that the group of 60 mT could hatch in average 4.5 days ahead of the control. As reviewed by Juutilainen (2005), experimental studies on ELF and RF EMFs at non-thermal levels do not consistently result in adverse effects on embryonic development in birds and mammals, except subtle variations in skeletal formation. In fish, Cameron et al. (1985) found that development was delayed when newly fertilized medaka embryos were exposed to an ELF EMF (60 Hz, 0.1 mT) for 48 h. Skauli et al. (2000) also reported a delayed development in zebrafish if the exposure (50 Hz, 1 mT) started 48 h after fertilization, but not so if it started 2 h after fertilization. On the other hand, in our study, medaka embryos developed faster when they were exposed to an EMF at a lower intensity (15–60 mT) but with a higher frequency (3.2 kHz), and the exposure started within 5 h after fertilization. These differences suggest that developmental toxicity of EMFs may vary with species and depend on the frequency and intensity of the EMFs, as well as the time and duration of exposures. One of the consequences of these developmental effects of EMFs on the nervous system can be elucidated with the white preference test. As demonstrated in the present study, the EMFexposed hatchlings swam more energetically, and they crossed to the white area within seconds. This suggests a heightened whitepreference response, i.g., an anxiety-like behavior. Balassa et al. (2013) have reported a similar result, in which brain slices from young rats exposed in utero to an ELF EMF (50 Hz, 0.5 mT) were significantly more excitable. It is not clear whether these differences produce any long-lasting effects in affected animals. But, since anxiety-like behavior costs more energy, it would not be advantageous for long-term survival.

Such neurological and behavioral consequences of EMF exposures during critical developmental stages have been observed in several studies. For example, Huang et al. (2013) have reported that long-term but low-leveled EMF exposure from high voltage transmission lines might have a negative impact on neurobehavioral function in children. In mice, the ELF EMF exposure has been found to lower memory consolidation in adults (8 mT; Foroozandeh et al., 2013), but improve the spatial learning acquisition and memory retention in adolescents (2 mT, Wang et al., 2013). The RF EMF exposure may also reverse cognitive impairment in Alzheimer's transgenic mice (Arendash et al., 2012). These results suggest that the nervous system is a target of EMF biological effects, and the effects are variable depending on the change being studied. The mechanisms for these effects are still under investigation. The thermal mediated effect is a known property of RF EMF in which absorption of energy results in increased temperature in biological tissues. For our study, this effect seems a possible explanation for accelerated development of EMF-exposed groups, as medaka embryos in general hatch faster in higher temperature. Though the thermal effect was reported to occur only at the radiofrequency ranging from 1 MHz to 300 GHz (D’Andrea et al., 2003), we can not rule out the possibility that the EMF at 3.2 kHz might subtly raise the temperature in some sensitive cells of the embryos. Another possible mechanism involves melatonin production, which has been shown to be affected by EMFs. But Touitou and Selmaoui (2012) in their review study found that the results from human and animal studies were contradictory, and no biological effect was linked to ELF EMF exposures in humans. Lerchl et al. (2008) also found that RF EMFs with frequencies up to 1800 MHz had no effect on melatonin levels in Djungarian hamsters. The level of melatonin production during embryonic development in medaka is unknown. In zebrafish, melatonin is not yet

W. Lee, K.-L. Yang / Ecotoxicology and Environmental Safety 108 (2014) 187–194

detectable within 1 dpf (Kazimi and Cahill, 1999). This is likely to be true for medaka, as they develop much slower than zebrafish. Since the EMF biological effects in our study were already significant at 1 dpf, the involvement of melatonin, at least initially, should be minimal. Other likely targets/causes of EMFs effects are the voltage gated calcium channels (Pall, 2013), neuronal sodium current (He et al., 2013), oxidative stress (Ciejka et al., 2011; Selaković et al., 2013), DNA damage (Panagopoulos et al., 2013), altered vascular formation (Costa et al., 2013), and disturbed free radical homeostasis (Mattsson and Simkó, 2012). Interestingly, Mo et al. (2012) have demonstrated that morphological abnormalities can be induced by a brief exposure to a hypogeomagnetic field at early cleavage stages of the clawed frog embryo, in which the mitotic spindle acts as a sensor for the sublevel magnetic field. If so, the same sensor might also detect higher EMFs and cause morphological changes accordingly. Whether developmental toxicity of the EMF shown in this study applies to human is unclear. As we are constantly exposed to not just EMFs but also other contaminants in our environment, it is possible that biological effects of EMFs may interact with these environmental factors. For example, in an in vitro study, coexposure to an ELF EMF at 3 mT would alter the effect of genotoxins (Villarini et al., 2005). Since inconsistencies also exist in toxicological studies of chemical substances, we are facing a tremendous task if we want to tackle the problems. We will continue using medaka embryos as an in vivo model system to investigate developmental toxicity of EMFs and the mechanisms involved.

5. Conclusions Using the medaka embryo as a model system, we have found that development of the EMF-exposed embryos was accelerated and the hatchlings exhibited heightened anxiety-like behavior. These results demonstrate that the medaka embryo is a simple, sensitive, and cost-efficient in vivo model system for studying the EMF effects on development and behavior. This model system can also be used to investigate the potential ecological impact of the EMFs on fish, which at present is poorly understood.

Acknowledgment We thank Mr. Art Wang for building the electromagnetic field generator.

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