Chick embryo development can be irreversibly altered by early exposure to weak extremely-low-frequency magnetic fields

Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 μT peak-to-peak amplitude, and 500 μs pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 μs (PMF-A) or 2.1 μs (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure. © 1994 Wiley-Liss, Inc.     

Development of chick embryos in 1 Hz to 100 kHz magnetic fields

Summary Chick embryos were exposed during their 48 first hours of development to sinusoidally oscillating magnetic fields. The frequencies 1 Hz, 10 Hz, 16.7 Hz, 30 Hz, 50 Hz, 1 kHz, 10 kHz and 100 kHz, and the field strengths 0.1, 1, 10 and 100 A/m were used. Each exposure group consisted of 20 eggs. After the exposure, the embryos were examined for abnormalities and classified by the developmental stage. The percentage of abnormal embryos (%AE) was significantly increased at frequencies from 16.7 Hz to 100 kHz. Above a threshold field strength of about 0.1 to 1 A/m, %AE was rather independent of the field strength, varying from 16% to 56% in different exposure groups. 13% of the sham-exposed control embryos (n = 150) were abnormal. Only the 0.1 A/m exposure group differed significantly from the controls at 1 Hz, and no significant effect was found at 10 Hz. The developmental stage was in general not affected by the magnetic fields, but some abnormal embryos showed retarded development.     

磁场对小鼠两种迷宫学习记忆的影响

据发现,磁场对生物体有一定作用,但是磁场对于人类或实验动物的学习记忆是否有影响,目前的报道结果很不一致。本实验采用实验小白鼠,给予不同强度（65高斯／50Hz,35高斯/25Hz）的低频磁场照射（每天1小时,持续25天）。磁场照射后,采用旷场行为测试、Y-迷宫和Morris水迷宫,检测小鼠的活动性、空间辨别、空间学习记忆和非空间学习记忆能力。结果表明：65高斯／50Hz磁场显著增高小鼠的活动性,并损伤小鼠Y-迷宫的空间辨别能力,但对Morris水迷宫的空间、非空间学习记忆无明显影响。35高斯／25Hz磁场处理动物行为在三个指标上均接近对照组。提示：长期的磁场照射可能会给动物,甚至人类造成一些影响。     

Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture. The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells. Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment. The cell cultures were exposed to sinusoidal 50 Hz 100 μT (root mean square) AC magnetic field during the entire time of a 48-h incubation. Testosterone content of the culture media was measured by radioimmunoassay. In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG. These findings demonstrate that sinusoidal 50 Hz 100 μT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered. Bioelectromagnetics 19:429–431, 1998. © 1998 Wiley-Liss, Inc.     

The superposition of a temporally incoherent magnetic field inhibits 60 Hz-induced changes in the ODC activity of developing chick embryos

Previously, we have shown that the application of a weak (4 μT) 60 Hz magnetic field (MF) can alter the magnitudes of the ornithine decarboxylase (ODC) activity peaks which occur during gastrulation and neurulation of chick embryos. We report here the ODC activity of chick embryos which were exposed to the superposition of a weak noise MF over a 60 Hz MF of equal (rms strength). In contrast to the results we obtain with a 60 Hz field alone, the activity of ODC in embryos exposed to the superposition of the incoherent and 60 Hz fields was indistinguishable from the control activity during both gastrulation and neurulation. This result adds to the body of experimental evidence which demonstrates that the superposition of an incoherent field inhibits the response of biological systems to a coherent MF. The observation that a noise field inhibits ODC activity changes is consistent with our speculation that MF-induced ODC activity changes during early development may be related to MF-induced neural tube defects at slightly later stages (which are also inhibited by the superposition of a noise field). Bioelectromagnetics 19:53–56, 1998. © 1998 Wiley-Liss, Inc.     

Environmental magnetic fields: Influences on early embryogenesis

A 10-mG, 50 to 60-Hz magnetic field is in the intensity and frequency range that people worldwide are often exposed to in homes and in the workplace. Studies about the effects of 50- to 100-Hz electromagnetic fields on various species of animal embryos (fish, chick, fly, sea urchin, rat, and mouse) indicate that early stages of embryonic development are responsive to fluctuating magnetic fields. Chick, sea urchin, and mouse embryos are responsive to magnetic field intensities of 10–100 mG. Results from studies on sea urchin embryos indicate that exposure to conditions of rotating 60-Hz magnetic fields, e.g., similar to those in our environment, interferes with cell proliferation at the morula stage in a manner dependent on field intensity. The cleavage stages, prior to the 64-cell stage, were not delayed by this rotating 60-Hz magnetic field suggesting that the ionic surges, DNA replication, and translational events essential for early cleavage stages were not significantly altered. Studies of histone synthesis in early sea urchin embryos indicated that the rotating 60-Hz magnetic field decreased zygotic expression of “early” histone genes at the morula stage and suggests that this decrease in early histone production was limiting to cell proliferation. Whether these comparative observations from animal development studies will be paralleled by results from studies of human embryogenesis, as suggested by some epidemiology studies, has yet to be established.     

Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field

The effects of intermittent exposure (2 h on/22 h off) to a 200 μT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 μT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs. © 1996 Wiley-Liss, Inc.     

Mouse early embryos obtained by natural breeding or in vitro fertilization display a differential sensitivity to extremely low-frequency electromagnetic fields

We have investigated the sensitivity of pre-implantation embryos obtained by natural breeding (NB) or in vitro fertilization (IVF) to extremely low-frequency magnetic fields (ELF-MF). Fertilized eggs obtained by NB were removed from mothers 12h after mating and cultured in vitro for 5 days under continuous ELF-MF exposure (constant strength of 50Hz and various intensities, i.e. 60, 120 and 220 microT). Alternatively, zygotes obtained by IVF were subjected to ELF-MF exposure (50Hz, 60 microT), starting 12h after IVF for 5 days. We found that ELF-MF exposure causes a small yet significant (P<0.05) decrease in the survival rate of NB-derived embryos at the latest stages of pre-implantation development, i.e. the eight cell-to-blastocyst transition. In embryos exposed to the highest field intensity (220 microT), the effect became apparent somewhat earlier. When IVF-derived embryos were exposed to ELF-MF, the reduction in the rate of embryo survival was more pronounced and the difference from controls was more significant (P<0.01). Moreover, the decreased survival rate in IVF embryos became apparent as early as the first cleavage and persisted throughout pre-implantation. These results suggest that IVF-derived embryos are more sensitive than NB-generated embryos to ELF-MF, and that this sensitivity occurs earlier in development.     

Retardation of embryogenesis by extremely low frequency 60 Hz electromagnetic fields

Fertilized Medaka fish eggs were used to determine if electromagnetic fields, designed to simulate those beneath a high voltage power line, have biological effects on vertebrate embryo development. The newly fertilized eggs were exposed to a 60 Hz electrical field of 300 mA/m2 current density, a 60 Hz magnetic field of 1.0 gauss RMS, or to the combined electric plus magnetic fields for 48 hours. No gross abnormalities were observed in any of the embryos as they developed, but significant development delays were seen in those embryos exposed to either the magnetic or to the combined electromagnetic fields; delays were not seen in the embryos exposed to the electrical field. Thus, a 60 Hz magnetic field like that encountered in a man made powerline environment was shown to retard development of fish embryos.     

Teratogenic effects of sinusoidal extremely low frequency electromagnetic fields on morphology of 24 hr chick embryos

To examine the potential teratogenicity of electromagnetic fields (EMF; sinusoidal and rectangular) on development of chick embryos (white leghorn), 221 freshly fertilized chicken eggs (55-65 g) were exposed during first 24 hr of postlaying incubation (38 degrees +/- 0.5 degree C) to 24 different EMFs, with 50Hz repetition rate and 8.007-10.143 mT flux density. Following exposure, the exposed fertilized chicken eggs (n = 8-10) and sham-exposed fertilized chicken eggs (n = 15) were incubated simultaneously for 8 more days and unexposed control fertilized chicken eggs (n = 20) for 9 days in absence of EMFs. The embryos were removed from egg shells and studied blind. All 24 EMF exposed-groups (inside the coil with exposure) showed an increase in the percentage of developmental anomalies compared to sham-exposed (inside the coil with no exposure) and control groups (outside the coil). Further, egg's weight was evaluated on day 9. This variable did not show significant difference between control and exposed-groups. The investigation also covered the measurement of body weight, length of crown to rump, length of tip of the beak to occipital bone, heart and liver weight. Statistical comparison between sham-exposed and control values did not show significant differences, but comparison between 8.007, 8.453 and 8.713 mT exposed-groups and control groups showed significant differences; in other exposed-groups, the changes were not significant. These results revealed that 50 Hz electromagnetic fields can induce irreversible developmental alterations in 24 hr chick embryos and confirm that its strength could be a determinant factor for the embryonic response to extremely low frequency electromagnetic fields (window effects).     

Oxidative DNA damage in rats exposed to extremely low frequency electro magnetic fields

Extremely low frequency (ELF) electromagnetic field (EMF) is thought to prolong the life of free radicals and can act as a promoter or co-promoter of cancer. 8-hydroxy-2'-deoxyguanosine (8OHdG) is one of the predominant forms of radical-induced lesions to DNA and is a potential tool to asses the cancer risk. We examined the effects of extremely low frequency electro magnetic field (ELF-EMF) (50 Hz, 0.97 mT) on 8OHdG levels in DNA and thiobarbituric acid reactive substances (TBARS) in plasma. To examine the possible time-dependent changes resulting from magnetic field, 8OHdG and TBARS were quantitated at 50 and 100 days. Our results showed that the exposure to ELF-EMF induced oxidative DNA damage and lipid peroxidation (LPO). The 8OHdG levels of exposed group (4.39+/-0.88 and 5.29+/-1.16 8OHdG/dG.10(5), respectively) were significantly higher than sham group at 50 and 100 days (3.02+/-0.63 and 3.46+/-0.38 8OHdG/dG.10(5)) (p<0.001, p<0.001). The higher TBARS levels were also detected in the exposure group both on 50 and 100 days (p<0.001, p<0.001). In addition, the extent of DNA damage and LPO would depend on the exposure time (p<0.05 and p<0.05). Our data may have important implications for the long-term exposure to ELF-EMF which may cause oxidative DNA damage.     

Effects of pulsing electromagnetic fields on the prenatal and postnatal development in mice and rats: in vivo and in vitro studies

Electromagnetic fields (EMF) might have various biological effects on the developing embryo. We studied the effects of pulsing electromagnetic fields (PEMF) on the in vitro development of preimplantation mouse embryos and of early somite rat embryos as well as on the in vivo development of rat embryos. We used PEMF at frequencies of 1, 20, 50, 70, and 100 Hz with a tension of 0.6 V/m. The embryos were exposed to PEMF throughout the experimental period. PEMF at frequencies of 20 and 50 Hz were embryotoxic, inhibiting over 50% of blastocysts from hatching and further development, all within 72 h of culture. PEMF at frequencies of 50 and 70 Hz induced 22% and 30% incidence of malformations in 10.5 day old rat embryos after 48 h in culture. The main malformations were absence of telencephalic, optic, and otic vesicles and of forelimb buds. In addition, retarded growth and development manifested by fewer somites, reduction in crown-rump length, and retarded closure of the neural tube were found in many embryos. No significant pathological changes were found by TEM in PEMF-exposed embryos. Disappearance of microvilli and collapse of apical parts of endodermal cells were observed by SEM in many yolk sacs of embryos exposed to 50 and 70 Hz PEMF. A slightly reduced litter average, a reduction or increase of weight, and a delay in eye opening was observed among offspring of pregnant rats exposed throughout pregnancy to PEMF at frequencies of 20, 50, and 100 Hz. No malformations were observed among these offspring. The mechanism of PEMF-induced embryotoxicity and teratogeneity is unknown, as is the mechanism of the "protective effects" of the mother on the rat embryos exposed to PEMF in vivo.     

Development of preincubated chicken eggs following exposure to 50 Hz electromagnetic fields with 1.33-7.32 mT flux densities

The effects of applying extremely low-frequency (50 Hz) electromagnetic fields (ELF-EMF) for 24 hr and different densities (1.33-7.32 mT) were examined on healthy, freshly fertilized white leghorn chicken eggs (55-65 g). Results showed no increase in the rate of abnormalities in exposed groups, but were only significant in 4.19, 5.32 and 5.86, 6.65 mT densities. Alizarin red S and alcian blue 8GX staining showed some embryos with extra ribs, defects in ribs and vertebrae, anuria and abnormal beaks. Study of egg weight, after 9 days of incubation, showed no significant differences between control, sham-exposed and experimental groups. Analysis of crown-rump, beak-occipital length and weight of embryo, showed significant decrease in weight at 4.39 and 5.52 mT intensities, comparing with control and sham-exposed groups. These results revealed that 50 Hz electromagnetic fields can even induce developmental alterations in preincubated chick embryos and confirm that its strength could be a determinant factor for the embryonic response to extremely low frequency EMFs (window effects) prior to incubation.     

Effects of extremely low frequency magnetic field on fertility of adult male and female rats.

To investigate the effects of an extremely low-frequency (ELF) magnetic field on their fertility, adult male and female Sprague-Dawley rats were exposed to a 50 Hz sinusoidal magnetic field of approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. Exposure to a 50 Hz field reduced male rat fertility. The number of pregnant females was reduced when mated with exposed males, and the number of resorptions increased. The effects of magnetic field on male fertility were shown to be partly reversible, when the same exposed group of males were remated 45 and 90 days after being removed from the fields. Exposure of adult female rats to 50 Hz magnetic fields for 90 days before mating significantly reduced their fertility. The mean numbers of implantations and living fetuses per litter were statistically significantly decreased in the 50 Hz group. These results suggest that low frequency magnetic fields have some adverse effects on fertility of male and female rats.     

Influence of 50 Hz electromagnetic fields on recurrent embryogenesis and germination of cork oak somatic embryos

Plant tissue culture techniques are carried out under environmentally controlled conditions in phytotrons. However, electric components of phytotrons generate electromagnetic fields that may act as a environmental factor influencing plant growth and morphogenesis. Isolated somatic embryos of Quercus suber, picked from embryogenic lines, were chronically exposed to a 50 Hz and 15 μT electromagnetic field generated in a Helmholtz-coil system for 8 weeks, in order to examine if the extremely low frequency (ELF) magnetic field (MF) affected the morphogenic behaviour of embryogenic cultures during recurrent embryogenesis. Germination of somatic embryos from genotype G7.1 was carried out under the same electromagnetic field, and also under conditions in which the local geomagnetic field was suppressed. The ELF MF did not influence the growth of embryogenic clumps of the assayed genotypes, but reduced the number of detachable embryos produced by genotype G3.27. The ELF MF did not modify the percentages of germination or plant formation of somatic embryos. However, somatic embryos had better germination when cultured under the suppressed geomagnetic field condition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of sinusoidal electromagnetic fields on histopathology and structures of brains of preincubated white Leghorn chicken embryos

There are several reports indicating linkages between exposures to 50-60?Hz electromagnetic fields and abnormalities in the early stages of chicken embryonic development. Based on our previous published research carried out at the Department of Animal Sciences, Faculty of Biological Sciences, Shahid Beheshti University, effects of sinusoidal electromagnetic fields on histopathology and structures of brains of preincubated white leghorn hen eggs were investigated. Three hundred healthy fresh fertilized eggs (55-65?gr) were divided into three groups of experimental (n?=?50), control (n?=?75), and sham (n?=?75). Experimental eggs (inside the coil) were exposed to 3 different intensities of 1.33, 2.66, and 7.32?mT and sham groups were located inside the same coil with no exposure, for 24?h before incubation. Control, sham, and experimental groups were all incubated in an incubator (38?±?0.5(°)C, 60% humidity) for 14 days. 14-day old chicken embryos were removed by C-sections, and the brains of all embryos of all groups were fixed in formalin(10%), stained with H&E and TUNEL assay, for studying the histopatholog and process of apoptosis. The brains of other embryos were prepared for Scanning Electeron Microscope. Results showed electromagnetic fields have toxic effects on brain cells by increasing the number of apoptotic cells and degeneration of brains' tissues of exposed chicken embryos. These findings suggest that the electromagnetic fields induce brain damages at different levels.     

Acute exposure to power-frequency magnetic fields has no effect on the acquisition of a spatial learning task by adult male mice

A series of four experiments was performed to determine whether acute exposure to a range of 50 Hz magnetic fields had any effect on a learning task in adult male CD1 mice. A radial-arm maze placed within the bore of an electromagnet was used to assess spatial discrimination learning for food reward. Subjects were reduced to 85% of their free-feeding weight and were placed in the maze for up to 15 minutes each day for 10 days. Performance of the task was measured by using maximum likelihood techniques to calculate the probability that an animal would not reenter any given arm of the maze. Experimental subjects were exposed to a vertical, 50 Hz sinusoidal magnetic field at 5 μT, 50 μT, 0.5 mT, or 5.0 mT (rms). Control subjects were exposed only to a background time-varying field of less than 50 nT and the ambient static field of about 40 μT. The variation in the applied magnetic field was less than 5% except at the ends of the arms, where it approached 10%. It was found that all eight groups of subjects (n = 10 in all cases) showed similar increases in performance with testing, and the acquisition curve for each group of experimental subjects was not significantly different from that of their control group (P > 0.05 in all cases). It was concluded that exposure had no effect on learning at any flux density. This result is contrary to the findings of a number of preliminary studies, although other studies have reported that magnetic fields do not affect spatial learning in adult male rodents. It is possible that differences between experimental conditions might explain some of this apparent discrepancy. © 1996 Wiley-Liss, Inc.     

Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos

BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities.     

Influence of weak static and 50 Hz magnetic fields on the redox activity of cytochrome-C oxidase

The effects of static and 50 Hz magnetic fields on cytochrome-C oxidase activity were investigated in vitro by strictly controlled, simultaneous polarographic measurements of the enzyme's high- and low-affinity redox reaction. Cytochrome-C oxidase was isolated from beef heart. Control experiments were carried out in the ambient geomagnetic and 50 Hz magnetic fields at respective flux densities of 45 and 1.8 μT. The experimentally applied fields, static and time-varying, were generated by Helmholtz coils at flux densities between 50 μT and 100 mT. Exposures were timed to act either on the combined enzyme-substrate interchange or directly on the enzyme's electron and proton translo-cations. Significant changes as high as 90% of the overall cytochrome-C oxidase activity resulted during exposure (1) to a static magnetic field at 300 μT or 10 mT in the high-affinity range, and (2) to a 50 Hz magnetic field at 10 or 50 mT in the low-affinity range. No changes were observed at other flux densities. After exposure to a change-inducing, static or time-varying field, normal activity returned. © 1993 Wiley-Liss. Inc.     

The effects of inverter magnetic fields on early seed germination of mung beans

The biological effects of extremely low frequency magnetic fields (ELF MFs) on living organisms have been explored in many studies. Most of them demonstrate the biological effects caused by 50/60 Hz magnetic fields or pulsed magnetic fields. However, as the development of power electronics flourishes, the magnetic fields induced are usually in other different waveforms. This study aims to assess the effects of magnetic fields generated by inverter systems on the early growth of plants using mung beans as an example. In the experiment, an inverter which can produce sinusoidal pulsed width modulation (SPWM) voltages was used to drive 3 specially made circular coils and an AC motor. Six SPWM voltages with different fundamental frequencies (10, 20, 30, 40, 50, and 60 Hz) set on the inverter drive the circuit to produce the specific kinds of MFs. The results indicate that the magnetic field induced by a 20 or 60 Hz SPWM voltage has an enhancing effect on the early growth of mung beans, but the magnetic fields induced by SPWM voltages of other frequencies (30, 40, and 50 Hz) have an inhibitory effect, especially at 50 Hz.