Radiat Environ Biophys (2000) 39:131–136
© Springer-Verlag 2000
O R I G I N A L PA P E R
Carmela Marino · Giovanni Cristalli · Paolo Galloni Patrizio Pasqualetti · Marta Piscitelli Giorgio A. Lovisolo
Effects of microwaves (900 MHz) on the cochlear receptor: exposure systems and preliminary results Received: 16 March 1999 / Accepted: 1 March 2000
Abstract The purpose of this paper is to present the experimental device and the work in progress performed in search for objective organic correlation of damage to hearing, examining possible acoustic otofunctional effects on the cochlear epithelium of the rat due to exposure to microwaves (900 MHz). Two experiments using male Sprague-Dawley rats were carried out with a farfield exposure in a cubic chamber. No statistically significant evidence was obtained at both specific absorption rate (SAR) values. The exposure system and the diagnostic apparatus are extremely useful to investigate a potential effect on the auditory system: however, with the parameters applied in these experiments, no evidence was observed.
Introduction Wireless communication is a major growth area in the global telecommunications sector, and the general public should have the best possible assurance that there are no health risks involved in using wireless, portable phones: it is fair to say that data available so far are insufficient for proper risk assessment. It is a well-established fact that people with normal hearing can perceive pulse-modulated radiofrequency (RF) and microwave (MW) radiation [1]: volunteers and patients have described the electromagnetic tinnitus in different ways e.g. as a buzzing, clicking, hissing or popping noise, according to the various characteristics of the C. Marino (✉) · P. Galloni · M. Piscitelli · G.A. Lovisolo Department of Environment, ENEA, C.R. Casaccia, Via Anguillarese 301, 00060 Rome, Italy e-mail:
[email protected] Tel.: +39-06-30486556, Fax: +39-06-30486559 G. Cristalli ENT Head and Neck Surgery Division, S.Giovanni Calibita Fatebenefratelli Hospital, Isola Tiberina, 00186 Rome, Italy P. Pasqualetti AFaR-IRCCS “San Giovanni di Dio”, 25100 Brescia, Italy
field modulation [2]. Moreover, some physiological changes have been observed in rats after exposure to low levels of microwaves [3]. It appears most likely that the sound (radiofrequency hearing) results from the thermoelastic expansion of brain tissue following a minor but rapid increase in temperature on the absorption of the incident energy [4]. Thus, the effects will be limited to frequencies which penetrate the skull and are significantly absorbed by brain tissue. The expansion causes an acoustic pressure wave which is transmitted through the skull to the cochlea, where the receptors respond as they would do to acoustic stimuli generated by normal means. Observations were reported in a study which measured auditory brainstem responses of guinea-pigs [5] exposed to pulsed 918 MHz electromagnetic (EM) radiation. It was concluded that the threshold for microwave hearing is related to the incident energy density per pulse for pulses shorter than 30 ms and is related to the peak power for longer pulses (up to 500 ms). Otoacoustic emissions (OAE) are acoustic signals coming spontaneously from the cochlea or in response to external stimuli. The discovery in 1978 by D. Kemp [6] gave rise to a revision of the classical view of the cochlea as a passive peripheral transducer. To summarize, the outer hair cells contain several proteins with contractile properties (similarly to muscular structures) and act as prime movers into the organ of Corti, thus producing a backward propagation of mechanical energy along the cochlea towards the stapes and the external auditory meatus. This kind of signal can be recorded from the ear canal in animal and all normal adult, childhood and neonate ears, and is increasingly used as a clinical test to assess the integrity of the peripheral organ and as a toxicological index of the exposure of the hearing system to a variety of exogenous and endogenous agents [7, 8, 9]. In particular, distortion-product otoacoustic emissions (DPOAE) are evoked by two pure tones at different frequencies (f1 and f2), and the response consists of the acoustic distortion product 2f1–f2. DPOAEs are generally believed to be an index of the status of the outer hair cells of the co-
132 Fig. 1 Scheme of the phantom radiated by the horn antenna (940±10 MHz) in the cubic chamber and the six measurement points (+): four points are shown, the other two (overlapped) are positioned aligned with the point in the middle
chlea at, or in connection with, the frequency corresponding to 2f1–f2. The relationship between cochlear hearing loss and DPOAE amplitude is not completely known; the DPOAE growth rate (input/output function) can be used to assess the physiological character of the response. Small but significant changes in the characteristics of otoacoustic emissions may serve as an indicator of outer hair cell subclinical or clinical pathology. The influence of the status of the cochlea on DPOAE properties has been discussed for rabbits [10, 11, 12], and it was demonstrated that a level of hearing loss of the subject examined equal to or greater than 30 dB can be considered sufficient to suppress the evoked response. The aim of this study was to assess potential changes occurring in the cochlear receptor after exposure to electromagnetic fields of a given frequency (around 900 MHz) and of low intensity: a cubic chamber was developed and a routine acoustic apparatus employed to observe otofunctional effects. In this paper, the methodological approach and the experimental set-up are described, and the first preliminary results of the cochlear receptor functionality are reported.
Materials and methods Animals For the experiments, 10-week-old male Sprague-Dawley rats (n=8) from Harlan-Nossan were used: feeding was ad libitum with standard diets, (mean body weight for the group at 0.2 W/kg was 263.125 g, SD±6.937218; for the group at 1 W/kg 299 g, SD±8.734169; for the group of the second experiment 262.875 g, SD±8.078852). The animals were maintained according to the “Interdisciplinary principles and guidelines for the use of animals in research, testing, and education” [13]. Exposure device and dosimetry The exposure system [14] consisted of a shielded (copper sheets) cubic chamber (side 1.2 m) lined with absorbing material (AN-79, ECCOSORB) only minimally reflective (-20 dB) in the frequency range of interest. A double-ridged wave-guide horn antenna (Gant=3.67), operating at around 940 MHz, was used as the standard source. The antenna was chosen in order to obtain the farfield condition exposure: far-field zone begins at distance d=(2D2/λ) for horn antennas (where D=0.24 m is the largest dimension of the antenna). Measurements were performed in air and in tissue-equivalent material using a E-field isotropic probe [15] along the horn antenna axis (Fig. 1). The specific absorption rate
Fig. 2 Relative SAR measurements performed in the conic tip, medium and bottom of cylinder of a rat phantom when the head was positioned at 65 cm distance from the source: ◆) one phantom alone, ■) one phantom among the other two
(SAR, W/kg), i.e., the rate of the electromagnetic energy absorption, was determined and checked by means of dosimetry on dielectric homogeneous phantoms (300 g each) exposed to around 940 MHz. The measurements were performed in brain equivalent tissue by a calibrated E-field probe [16]. The SAR distribution along the longitudinal axis of a conical-ended cylindrical homogeneous phantom irradiated by a plane wave at 940±10 MHz (the interval of frequency is due to different tuning) was simulated by the HFSS code and verified by SAR measurements: the qualitative evidence of a preferential deposition of electromagnetic power in the conical end (head) of the phantom was verified (Fig. 2). In order to define the exposure conditions for in vivo experiments, the attenuation effects of the jig (Lucite material) were tested and SAR values were measured in the conic tip, medium and bottom of cylinder points of the phantoms, according to the positioning of the animals immobilized in the jigs. The rat phantoms with a cylinder of Lucite (Φ=4.5 cm, l=12 cm), ending with a cone, were filled with 300 g of homogeneous semisolid tissueequivalent material (εreal=41.4, εimag=22.6). The SAR value in the phantom head (the conic end) was obtained as the average of six measurement points (see Fig. 1). A total of eight jigs (two arrays of four jigs at 50 and 60 cm from the ground over an acrylic support) were positioned at 65 cm distance from the mouth of the antenna: the longitudinal axis of animals was oriented along the direction of propagation; the heads of the rats faced the antenna. The animals were irradiated by the above-mentioned doubleridged wave-guide horn antenna at 940±10 MHz CW fed by a power input of 6 W and 30 W with vertical polarization to obtain 0.2 W/kg and 1 W/kg body weight, respectively, in the head. The exposure level was selected in consideration of the safety level for human localized absorption (or slightly lower) which currently is up to 1.6–2 W/kg averaged over 1 or 10 g [17, 18].
133 DPOAE test DPOAEs were recorded by an ILO92 (Otodynamics, UK) apparatus, with a dual sine wave stimulus generator. The gain of the microphone amplifier system before the signal is routed to a 12-bit A/D converter. Data were collected and processed and the results were stored on a disk. The stimulus section comprised two time-locked oscillators where the frequency ratio (f2:f1) was fixed at 1.22. The stimulus generator provides a synchronization signal for triggering the A/D converter. The oscillators are provided with additional tracking filters in order to reduce total harmonic distortion. Each oscillator signal is fed to a digitally controlled attenuator circuit with 1 dB resolution, and the attenuated signal is passed to a receiver via a power amplifier. The stimulator provides intensity levels in the range 0–80 dB SPL (sound pressure level), as measured in the ear canal. The stimulus level is adjusted automatically by the computer after positioning the probe in the ear canal, using the probe microphone as a reference. Two separate receivers are applied, and the signals are mixed acoustically in the connecting sound tubes. The two stimulus tones may be presented at individual intensities. The probe consists of an ear speculum in which a miniaturized electro-acoustic transducer and electric microphone are assembled. The microphone is provided with a 2-cm long metal tube terminating at the orifice of the speculum. After FFT analysis of the recorded data, the response is equalized by subtraction of the microphone response from FFT result, in order to obtain a linear frequency response within 1 dB accuracy (in the acoustic frequency range from 0.3 to 9 kHz). An HC probe SN:920309 A/B (Otodynamics, UK) was used. The probe was adapted to the external auditory canal using a silicon plug. The effective volume of the external auditory canal is assumed to be influenced by its physical dimensions and by the position of the probe within the external auditory canal. This value is critical in the threshold estimation. The ILO 92 DPOAE instrument allows the probe fitting to be tested by the stimulus frequency response in the external canal.
Experimental protocol The rats were identified by coloured spots on the tail. DPOAEs were registered, under general gas anaesthesia (70% N2O, 30% O2 and 2–3% halothane), at zero time (T0): both ears were always tested. The tests were performed under the following conditions: amplitude of f1 tone=72 dB SPL, amplitude of f2 tone=70 dB SPL, f1/f2=1.22; the noise tolerance during the recording session, expressed as S/N ratio, was 10 dB (this specifies the best S/N to be maintained during the DPOAE recording); the ratio is based on the background noise level inside, at the beginning of the test; rejection threshold was fixed at 8 mPa. These amplitudes for f1 and f2 and their ratio (1.22) were set according to typical experimental conditions for DPOAE recording [6, 19, 20, 21, 22, 23]. DPOAE results were analysed as DP-gram: four different pairs of f1 and f2 values were considered (f1=2600 Hz/f2=3174 Hz; f1=3284 Hz/f2=4004 Hz; f1=4126 Hz/f2=5042 Hz and f1 5200 Hz/f2 6348 Hz) in order to allow for the study of the cochlear response at four different frequencies 2f1–f2 (2026, 2564, 3210 and 4052 Hz, respectively), in both unexposed and exposed animals. In the first experiment, rats were exposed to electromagnetic fields with a frequency of 950 MHz, at SAR values of 0.2 W/kg and 1 W/kg, for 3 subsequent days and 3 h per day (9–12 am); while in the second experiment at 936 MHz, the exposure time, at 1 W/kg, was prolonged to 5 days, for the same number of hours per day. Sham exposure was performed in both experiments. Thermosensors (fibreoptic sensors) were placed on the skin of one rat in the jig during the first day of exposure to monitor any change of temperature. After exposure, animals were again recorded by DP-gram, following the same procedures. Thus, each group (exposed and sham) in both experiments was measured before, immediately after (t0), 24 h (t24) and 48 h (t48) after exposure (Table 1).
Table 1 Average and peak SAR values measured in head rat phantom irradiated by two levels of power Power input in antenna (W)
Power irradiated by antenna (W)
Power density(a) (W/m2)
SARav (W/kg)
SARmax (W/kg)
5.5 28
20.2 102
4.58 23
0.2 1
0.32 1.6
(a) P =power input in antenna, P = power irradiated by antenna in out (b) Power density (W/m2) measured in air at 65 cm from the source
Statistical analysis The data for the DPOAE of the rats examined (exp. I: 8 exposed for each dose group and 8 sham-exposed; exp. II: 8 exposed and 8 sham exposed) were stored in a spreadsheet, then analysed with an SPSS package. The aim of the experimental design was to evaluate the effect of the ‘SAR value’ and ‘time’ (main effects and their interaction) on the variations of DPOAE, measured before and after exposure. The measurements were related to the single ear at the selected four acoustic frequencies. The same animals were observed at different times after exposure and both ears were evaluated. A statistical analysis was carried out by means of analysis of variance for repeated measures with time as within-subject factor and SAR as a between-subjects factor. Throughout the statistical analysis, the level of significance was set at 0.05.
Results Several measurements were performed to characterize the exposure parameters (absorbed dose): mean and peak values were determined. In the first experiment the rats were exposed to 0.2 and 1 W/kg (values averaged in the head phantom, see Fig. 1) at 65 cm distance from the source, meanwhile the SAR peak values of 0.32 and 1.6 W/kg, respectively, were measured (Table 2). No temperature increase was noticed on the skin of the animals. According to the result of ANOVA (with acoustic frequency, SAR and time as factors) for repeated measurements (when the same animals were observed), none of the considered factors resulted significant (p>0.20) consistently across frequencies. It is also clear that the homogeneity of variances – an important assumption for the validity of ANOVA results – is not encountered in our data (Levene’s test, p
