indicator for possible action of the EMFs directly related with the gonad development, ... 5.5.2: Drosophila pupae immobilized on the walls of the glass tube.
5.5 Effects of Electromagnetic Fields on the Reproductive Capacity of Drosophila Melanogaster Dimitris J. Panagopoulos and Lukas H. Margaritis University of Athens, Faculty of Biology, Department of Cell Biology and Biophysics
5.5.1 Introduction In the present section, we describe a set of simple experiments with very good reproducibility, concerning the effects of different types of electromagnetic fields, (EMFs), on the reproductive capacity of the insect Drosophila melanogaster. In the discussion of the experimental results we suggest a possible explanation on biochemical basis, which can be connected to the possible action mechanism at the biophysical level, that we presented in our theoretical paper. In our experiments, we used three different kinds of Electromagnetic Fields, with criteria, to cover as large as possible part of the electromagnetic spectrum, from ELF, (Extremely Low Frequencies), to RF, (Radio Frequencies), to be as much as possibly related to the fields to which the average person is exposed daily, and to be suspected for possible biological effects. These fields are: a) Alternating Magnetic Field, 50Hz, b) Pulsed Electric Field, with damping alternating pulses, with 10kHz carrier frequency and 50Hz repetition frequency, c) RF pulsed field from GSM, (“Global System for Mobile Telecommunications”), mobile cellular phone, with 900MHz carrier frequency. The GSM field is commercially available from mobile cellular phone devices, whereas for the generation of the magnetic and the pulsed electric fields, we constructed specific devices. For this we designed, according to theoretical calculations, and built pairs of coils with well-standardized operation characteristics, to satisfy the demands of biological experiments. The good quality of the constructions was reassured with field measurements. We also designed and built a high-voltage pulsed electric current generator, with well-standardized and controllable operation characteristics, as well as parallel plate capacitor devices with controllable capacity, to be connected to the generator, for electric field production. We chose the insect Drosophila melanogaster, Oregon R, wild type, as the experimental animal, since in our laboratory there is extended knowledge of its reproductive biology. In addition, the fruit fly is in general a very well studied experimental animal, with well-known genetic background and many advantages, including its short life circle and the good timing of its metamorphic stages and developmental processes, under constant conditions. As a properly sensitive stage from the insect’s life circle, we chose the stage of gonad development, with emphasis in oogenesis. We thought that since the insect’s oogenesis and spermatogenesis, are biologically very active and sensitive to different 545
external factors, they would also be sensitive to EMFs. As a proper biological indicator for possible action of the EMFs directly related with the gonad development, we chose the insect’s Reproductive Capacity, as this is defined by the number of F1 pupae which under the conditions of our experiments, corresponds to the insect’s oviposition. Finally, a simple experimental protocol, developed by us, related to the insect’s reproductive capacity, has been proven to be a very sensitive index, for the biological effectiveness of all the types of electromagnetic fields that we have used in our experiments. As is well known, the basic cellular processes are identical in insect and mammalian cells. Additionally, insects, (especially Drosophila), are much more resistant than mammals, at least to ionizing radiation, with which comparative studies have been carried out, [30], [32], [31], [1]. Thereby we thought that a simple and easy to use experimental protocol related to the Reproductive Capacity of Drosophila could be very useful in assessing the biological effectiveness of electromagnetic fields in general. In our experiments, the insects were held in glass bottles and kept in incubator at 25 C, with 12-h periods of light and darkness.
Fig. 5.5.1 The insects inside a glass tube with food.
5.5.2 Materials and Methods 5.5.2.1 Description of the Basic Experimental Protocol In every experiment, we collected newly emerged flies from the stock, we anaesthetised them very lightly and separated males from females. We put the collected flies in groups of ten in standard laboratory 50-ml cylindrical glass tubes with 2.5cm diameter and 10cm height, with standard food, which forms a smooth plane surface, 1cm thick at the bottom of the tubes. The glass tubes were closed with cotton, (Fig. 5.5.1). The food consisted of 450ml water, 4gr agar, 13gr yeast, 32gr rice flour, 16gr sugar, 25gr tomato pulp. The mixture was boiled for over 10min to ensure sterility, which was preserved by the addition of 2ml propionic acid and 2ml ethanol. This food quantity was enough for 25-30 glass tubes. The glass tubes were first sterilized. 546
In every experiment we separated the insects in two groups: a) G1 the exposed group and b) G2 the control, (sham exposed), group. The sham exposed groups had identical treatment with the exposed ones, except that there was no field exposure. In each group we kept in separate glass tubes the males and the females for the first 48h of each experiment. The process of oogenesis in Drosophila starts in the last stages of pupae and at eclosion, in the ovaries of newborn female flies, there are already eggs in the first preyolk stages. The eggs develop through 14 distinct stages, until they are ready to be fertilized and laid and this process for every egg lasts about 48h. So, at the end of the second day of their adult life, the female flies have in their ovipositor the first egg of stage 14, ready to be fertilized and laid. At the same time male flies are also sexually mature, (about 12h after eclosion when the necessary amount of paragonial substances-sex peptide, has been composed). Keeping males separately from females for the first 48h of each experiment ensures that the flies are in complete sexual maturity and ready for immediate pairing and laying of fertilized eggs. After the first 48h of the experiment, we placed together the male and female flies of each group in another glass tube with fresh food and we let them pair and lay eggs for 72h. During these three days, the daily egg production of Drosophila is at its maximum, (from the 3rd until the 5th day of its adult life), then stays at a plateau or declines slightly for the next 5 days and diminishes rapidly after the 10th day of adult life, [12], [56], [53]. After five days from the beginning of the experiment, we took off the paternal flies from the glass tubes and left the tubes in the incubator for six days without any further exposure.
Fig. 5.5.2: Drosophila pupae immobilized on the walls of the glass tube. Black pupae are the most developed ones, ready for eclosion as adult flies, within the next few hours.
After the last six days, most born embryos are in the stage of pupation, where they can be seen and counted clearly on the walls of the glass tubes, (Fig. 5.5.2). Of course there will be few embryos still in the last stages as larvae, which are big enough and ready for pupation, (on the surface or already away from the food), so that they can be counted easily. Also there may be already some newborn flies of the F1 generation, which can also be counted easily. During the last six days, we inspect the surface of the food in the glass tubes, under the stereo-microscope, to see if there are any non-developed laid eggs, or 547
dead larvae, something that we did not see in our experiments, (of course, empty egg-shells can be seen after larvae hatching). The exceptions from this rule, in the exposed and the control groups were always within Standard Deviation of progeny number. Thus, under the conditions of our experiments, the number of pupae, corresponds to the oviposition. Thereby, counting pupae, we can estimate oviposition without any error at all, whereas counting the number of laid eggs under a stereomicroscope, encounters considerable error. In the way we described above, we can see, if there are any changes, due to the EMF exposure, in the oviposition of Drosophila, during the first three days of its maximum daily oviposition. Any changes in the normal oviposition of the insect, during the first three days of its maximum oviposition, reflect changes of the normal function of the insect. This simple experimental protocol, which we designed, has been proved to be a very reliable index for biological alterations due to different kinds of electromagnetic fields.
5.5.2.2 Specific characteristics of the EMFs and Protocol Alterations for each field Sinusoidal-Alternating Magnetic Field, 50 Hz Electric energy is usually produced and transferred as high-voltage, (about 104 V), three-phase alternating current, 50-60 Hz, through power lines, that sometimes pass upon houses. Then it is transformed to 110-220V and distributed in residential environments. Thus, all the electrical devices and building wirings are flowed by ELF currents. Modern man is exposed on a 24h basis to ELF electric and magnetic fields.
Field Characteristics: Intensity – Magnetic Flux Density, (rms): Β = 70G Direction: Vertical Shape: Sinusoidal-alternating Frequency: 50 Hz Temperature in the center of the coils: θΜ = 26 0.5 οC The circuit for magnetic field exposure, that we designed and constructed in our laboratory, is shown in Fig. 5.5.3. The coils L1 and L2 have exactly the same characteristics. The only difference between them is that the turns of L1 are all parallel between them, while in L2 half of the turns are antiparallel, so that the magnetic (and the induced electric), field over a region in the middle of the coil, is zero. Thereby, given that the same current passes through the two coils, the coil L1, is used for magnetic field exposure, while the coil L2 is the corresponding control.
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Fig. 5.5.3: The circuit for magnetic field exposure. The exposure coil L1, produces magnetic field with standardized characteristics, while in the center of the control coil L2, the intensity of magnetic field is zero.
The two coils during the exposures are connected in series and the temperature in the center of both coils is the same. According to the characteristics of the coils, theoretical calculation for the intensity of the magnetic field in the center of the air-core of L1, gives:
B=
o IN 4a 2 l 2
(1)
where: I the intensity of electric current that passes through the coil, N the number of turns, l the length of the coil and a the radius of the turns. The induced electric field in L1, is given by the relation: Eind(rms) = NBrms
(2)
and it is in the order of 10V/m. [The induced electric field in L2, is zero]. The Exposure Limits for 50Hz Magnetic Fields, (for rms magnetic field intensities), are 1G, (24h exposure), for the general public and 10G, (exposure of few hours during the working day), for occupational exposure, [25]. In our experiments with the magnetic field, we exposed the insects to higher field intensities than those of the corresponding Exposure Limits, in order to record more easily, biological effects. In our experiments with the magnetic field, the glass tubes with the insects were suspended in the center of the air-cores of the coils L1, L2, (Fig. 5.5.4).
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Exposure started 1h after the insects awoke from the first anesthesia. The insects were exposed to the magnetic field continuously during the five days, as described in the experimental protocol.
Fig. 5.5.4: The glass tube with the insects, suspended inside the coil, for magnetic field exposure.
Pulsed Electric Field Pulsed fields seem to be so far, more bioactive than continuous fields. Additionally, they can be transformed to high voltages, while their average power remains low.
Field Characteristics: Intensity amplitude: E = 400 kV/m = 4105 V/m = 4 kV/cm Shape: Pulsed, with exponentially damping, sinusoidal alternating pulses. Polarity: Bipolar pulses, with positive 1st semiperiod. Carrier frequency: 10 kHz Pulse repetition frequency: 50 Hz Pulse duration: δt = 0.5 msec Pulsed current duration: Δt = 30min per 2h Exposure and Control Temperature: θΕ = θcontrol = 25 0.5 oC
550
Fig. 5.5.5: The capacitor C1, for electric field exposure, connected to the high-voltage pulsed electric current generator.
For exposure to electric field, we placed the glass tubes with the insects, in the center of the capacitors, (between the metal plates), (Fig. 5.5.5). Two identical capacitors were constructed, C1 and C2. The groups of insects exposed to the electric field were placed in C1, which was connected to the high-voltage pulsed electric current generator, while the corresponding Control Groups were placed in C2, which was not connected to any voltage. The induced magnetic field in C1, is actually negligible, (in the order of 10-8G). The exposure to the pulsed electric field, started 1h after the insects were awaken from the first anesthesia and took place for 30min, repeatedly every 2h, with the aid of a timer, for the five days of exposure as described in the experimental protocol.
Fig. 5.5.6: The glass tube with the insects, placed between the metal plates of the capacitor, for exposure to electric field.
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The Exposure Limits for 50Hz Electric fields, (for rms electric field intensities), are: a) For the General Public: 5 kV/m, for 24h exposure, or 10 kV/m, for few hrs exposure per day. b) For Occupational Exposure: 10 kV/m, for 24h exposure, or exposure to field strengths between 10 and 30 kV/m, provided the intensity (kV/m) times the duration of exposure (hours) does not exceed 80 for the whole working day, [25]. The Exposure Limits for 10kHz electric fields, are: a) 0.4 kV/m, independently of exposure duration, for the general public and b) 1 kV/m, independently of exposure duration, for occupational exposure, (European Prestandard, ENV 50166-1, 1995), [51]. As we can see, in our experiments with pulsed electric field, we used field intensities higher than those of the Exposure Limits, in order to be able to easily record biological effects and also to be in agreement with electric field intensities that have been used in other published experiments, [54], [47].
RF - GSM Field, from the antenna of GSM Cellular Mobile Phone During the last years the use of cellular mobile phones has rapidly increased and at the same time there are indications for possible biological effects. Thereby we thought it would be simple and very interesting to use a GSM cellular phone itself to expose insects to 900 MHz RF field and see what happens. Besides, Mobile Phones are the most powerful RF transmitters in our proximate daily environment. During our experiments with the GSM field, we noticed that the intensity, (power density), of the radiation emitted by GSM mobile phones, increases considerably, when the user speaks on the phone during connection, than when there is no speaking.
GSM Field Characteristics Power Density, (mean value for 6 min). Average from four successive, mean value measurements Standard Deviation: A) “Non-Modulated”- or “Non-Speaking” emission: 0.037 0.007 mW/cm2, B) “Modulated” or “Speaking” emission: 0.618 0.070 mW/cm2 GSM Pulses Carrier Frequency: 890-915 MHz, [60] Pulse repetition frequency: 216.7 Hz Modulation type: “Gaussian Minimum Shift Keying Modulation”-(GMSK) Temperature: θ = 25 0.5 οC The above given Power Density values, were measured within a similar glass tube as the ones we used for the insects in our experiments and thus represent the GSM field intensities to which the insects were exposed during our experiments. In the experiments with the RF - GSM field, the glass tubes with the exposed and the control insects, were kept in the incubator, except for the intervals of the exposures. The exposures, as well as the field measurements, were carried out in 552
the same, quiet but not sound isolated room of our laboratory, with the same positions of all items around the experimental bench. After each exposure, the glass tubes with the insects were put back in the culture room. Each time before irradiation, the cotton plugs in the vials were pushed down, in order to confine the flies to a small area of about 1cm height between the cotton and the food, so as to provide, roughly even exposure to all flies, (Fig. 5.5.7). After irradiation, the cotton plugs were pulled back to the top of the vials and the vials were put back in the culture room. We exposed the flies in the glass tubes by placing the antenna of the mobile phone, outside of the glass tubes, in contact with the glass wall and parallel to the vial’s axis, (Fig. 5.5.7).
Fig. 5.5.7: The antenna of the mobile phone and the glass tube with the insects, during exposure.
Fig. 5.5.8: The experimenter’s position in relation to the antenna and the glass tube, during exposure.
The sham exposed-control groups had identical treatment as the exposed ones, except that the mobile phone was turned off. During the exposures, the glass tubes with the control insects were in the same room, but at a distance at least 3m from the mobile phone. The insects during the exposures were away from any other sources of electromagnetic fields. The total duration of irradiation was 6min per day in one dose and we started irradiating from the first day of the experiment, (day of eclosion). The daily exposure
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duration of 6min, was chosen in order to have exposure conditions that can be compared with the established “exposure limits”. The experimenter could speak on the mobile phone while this was emitting, (this we called, “Modulated” or “Speaking” emission), or could just make no sound, (this we called “Non-Modulated” or “Non-Speaking” emission). As we found out during the experiments, the radiation intensity of “speaking emission” is increased by more than one order of magnitude, in relation to the intensity of “non-speaking emission”]. In both cases, the experimenter’s position in relation to the mobile phone and the glass tubes was the same, (Fig. 5.5.8). In the diagram of Fig. 5.5.9, we can see the power density levels for “Speaking” and “Non-Speaking” emission, of a GSM mobile phone at 1cm distance from its antenna. In the diagram of Fig. 5.5.10, we can see the power density for “Speaking” emission, in the air, in relation to the distance from the antenna. In the diagram of Fig. 5.5.10, we can see that for distances, greater than 10cm from the antenna, (far field), the intensity decreases linearly in relation to the distance, whereas, in the near field decreases exponentially, as we would expect. As we can also see, the intensity of the radiation emitted by a GSM mobile phone, is actually zero for distances greater than 40 cm. We used a very common model of GSM mobile phone device in our experiments. We measured the mean power density emitted by the antenna of the GSM mobile phone, with the field-meter: “RF Radiation Survey Meter, NARDA 8718”, with its probe inside a similar glass tube with the ones we used for the insects in our experiments.
Intensity, (mW/cm^2)
Radiation Intensity of "Modulated" and "Non-Modulated" Emission from GSM Mobile Phone 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0
"Modulated" "Non-Modulated"
1
2
3
4
Measurements Fig. 5.5.9: Power Density levels for “Modulated” και “Non-Modulated” radiation emitted by the antenna of a typical GSM mobile phone. Power density values, (in mW/cm2), are measured at 1cm distance from the antenna. The diagram shows the values from four separate measurements, in the air. On each point is denoted the Standard Deviation.
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The measured, mean power density for 6min of “Modulated” emission, (speaking emission), with the antenna of the mobile phone in contact to the wall of the glass tube and parallel to its axis, was 0.6180.070 mW/cm2, with a maximum value of 2.96 mW/cm2. The “Non-Modulated”, (NM), corresponding measured values, were 0.0370.007 mW/cm2 with maximum 1.2 mW/cm2. [These values for emitted power density, are about the same with the corresponding values a mobile phone user’s head is exposed to, without the use of “hands free” optional device, (headphone), when the antenna of the mobile phone is at 1cm distance from the user’s head. These values are typical for all the commonly used GSM cellular phone devices, emitting at 900MHz, with 2W peak power output.].
Intensity, (mW/cm^2)
Intensity of "Modulated" Radiation from GSM Mobile Phone 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 0
10
20
30
40
Distance, (cm)
Fig. 5.5.10: Power density of “Modulated” radiation, (in mW/cm2), emitted by the antenna of a typical GSM mobile phone, in relation to the distance from the antenna, in the air.
The above measured intensity values, represent the intensities to which the insects inside the glass tubes were exposed during our experiments. According to these values, the electric and the magnetic component of the RF field in our experiments, were: α) For “Non-Modulated” radiation: JNM = 0.037mW/cm2 = 0.37W/m2 and from equations [20], [22], it comes that: ENM = 11.8V/m and BNM = 0.4mG b) For “Non-Modulated” radiation: J = 0.618mW/cm2 = 6.18W/m2, we get: E= 48.3V/m and B = 1.6mG As we see, the intensity values for the electric and the magnetic component of the RF field are small in relation to intensity values of EMFs at lower frequencies. Nevertheless, RF and microwave fields, seem to be very bioactive, as reported in many experiments, [18] and also become evident from our experiments as described 555
50
below. This might be an indication that perhaps RF fields interact with living matter in a more complicated way than EMFs of lower frequencies and these yet unknown mechanisms are responsible for the usual appearance of non-linear biological effects of RF fields, like the so called “window effects”. The most stringent “exposure limits” for RF radiation at 900MHz, were established in 1988 by IRPA, (International Radiation Protection Association) and for occupational exposure, they refer a power density value of 2.25 mW/cm2, or wholebody mean Specific Absorption Rate, (SAR), value of 0.4 W/Kg, both values averaged over any 6 min period during the working day. The corresponding general population limits, are a power density value of 0.45 mW/cm2, or whole-body mean SAR of 0.08 W/Kg, both averaged over any 6min period during the 24-h day, [26]. In 1998 ICNIRP, (International Commission on Non-Ionizing Radiation Protection), recommended the same exposure limits, [24]. Other exposure limits proposed by ANSI, (American National Standards Institute), by IEEE, (Institute of Electrical and Electronics Engineers) and by NCRP, (National Council on Radiation Protection and Measurements), refer a little higher values of power density and SAR, than the above values of IRPA-ICNIRP, [2], [45], [23]. The above measured exposure values, are well below all the occupational “exposure limits”, and only the power density of speaking emission exceeds the IRPA limit for the general population. The first exposure to the GSM field took place 1h after the insects awoke from the first anesthesia. The total duration of irradiation was 6min per day in one dose for the first five days of every experiment, as described in the experimental protocol. The daily exposure duration of 6min, was chosen in order to have exposure conditions that can be compared to the established “exposure limits”. [In the guidelines for limiting exposure to RF fields, the exposure limits are given in average values for any 6-min exposure period], [26], [24], [2], [45], [23].
5.5.2.3 Statistical Analysis-Single Factor Anova Test The statistical method, called Analysis of Variance Test, compares the variance of a certain factor inside the different groups, (sum of squares within groups), with the variance between the groups, (sum of squares between groups). The value of this quotient is related to the Probability of the Null Hypothesis, Po , (that the groups differ from each other because of random variations). As is evident, for a statistically valid experiment, the value of Po , must be as small as possible. In any case, it must be: Po 0.05. In our experiments we have two kinds of groups, (the exposed and the control ones) and the factor to be compared between them, is the oviposition. AnoVa Test, does not demand a minimum number of statistical data, (number of experiments). This parameter is related to the number of degrees of freedom. If the number of experiments is not enough, then the ratio of variance between the groups will be greater than the permitted value for the validity of the test and also Po will be 556
large. An explanation of the different parameters in the Analysis of Variance, (AnoVa), statistical test, is given below:
n =total number of pieces of sample data, k = total number of groups, (in our experiments k =2) i = 1, 2, the different groups of data j = 1, 2, …, , the different data of each group n1 = n2 = = number of pieces in each group, (corresponds to the number of experiments of each kind) df: degrees of freedom df Between Groups = k-1 df Within Groups = n - k
x i : Average of each Group, (Group Means) x : Overall Mean SS: sum of squares k
SS Between Groups =
ni ( x i - x )2 = n1 ( x1 - x )2 + n2 ( x 2 - x )2
i 1
k
SS Within Groups =
( ni - 1) Si2
i 1
ni
(x Si = Si 2 =
1 ni 1
j
x )2
j 1
ni 1
, Sample Standard Deviation
ni
(xj - x )2 , Sample Variance
j 1
MS = SS/df F = MS Between Groups / MS Within Groups The necessary assumptions for the use of one-way Anova test are the following: 1) The samples of the (two) groups under consideration are independent of one another, 2) the populations under consideration are normally distributed, (the test is robust to moderate deviations of this assumption), 3) the Standard Deviations of the populations under consideration, are equal. The last assumption is considered satisfied, if the ratio of the largest to the smallest sample Standard Deviation is less than 2: Si max/ Si min 2 , (or if the largest to the smallest sample Variance is less than 4), [63], [42].
5.5.3 Experimental Results 5.5.3.1 50Hz, Alternating Magnetic Field We carried out several experiments with Magnetic Field Exposure, according to the described experimental protocol. We use the following symbols for the insect groups:
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M(G1): Groups of insects exposed to Magnetic Field, M(G2): Control Groups for Magnetic Field The results of the Effect of Alternating Magnetic Field on the insect’s reproductive capacity, are given in Table 5.5.1. Table 5.5.1: Mean Oviposition of Groups Exposed to Magnetic Field and Control Groups
Experiment Νο
1 2 3 4 5 6 7 8 9 Average Standard Deviation
Mean Oviposition per Female Paternal Insect
Group
Deviation from Control
M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control M(G1) M(G2) Control
11 11.7 10.8 11.5 12.4 13.1 12.1 12.9 11.3 12 10.9 11.7 11.6 12.5 11.3 11.9 12.1 13
-5.98%
M(G1) M(G2) Control
11.5 0.58 12.255 0.62
-6.16%
-6.08% -5.34% -6.20% -5.83% -6.84% -7.2% -5.04% -6.92%
In Table 5.5.1, we can see the Mean Oviposition, (number of laid eggs), per female paternal insect during the first three days of the insect’s maximun daily oviposition, (3rd, 4th and 5th day of adult life), for the Exposed, M(G1) and the corresponding Control Groups, M(G2), for 9 experiments. The Oviposition was defined by the number of progeny pupae, since during the experiments we did not record any statistically important percentage of nondeveloped laid eggs, or dead larvae, in the Exposed or in the Control Groups. As we can see, in nine experiments, the alternating magnetic field, caused an average decrease by 6.16% in the insect’s reproductive capacity. This result is graphically represented in Fig. 5.5.11. The statistical analysis of the results is given at Table 5.5.2. 558
All the necessary assumptions for the validity of the Anova test are satisfied: 1) The samples of the two groups are independent of one another, since every experiment is independent of the others, (although all the experiments were made as much as possible, under the same conditions) and the oviposition of each group is not dependent on the oviposition of the other. 2) Indeed we can accept that the mean oviposition of each group is a function, normally distributed around some theoretical value of mean oviposition. The more we keep constant the conditions between different experiments, the less variance we get around this theoretical value of mean oviposition. 3) In the above experiments with the magnetic field, the ratio of largest to smallest sample Standard Deviation is 0.62 / 0.58 = 1.07, (less than 2). The results of the statistical analysis are given in Table 5.5.2.
Average Mean Oviposition
Reproductive Capacity of Exposed and Control Groups 14 12 10 8 6 4 2 0 M(G1)
M(G2) Groups
Fig. 5.5.11: Effect of sinusoidal-alternating magnetic field, on the reproductive capacity of Drosophila Melanogaster. Average Mean Oviposition per maternal insect Standard Deviation, during the first three days of the insect’s maximum oviposition, for the Exposed, M(G1) and the corresponding Control Groups, M(G2), in 9 experiments. Table 5.5.2: Alternating Magnetic Field. Statistical Analysis. Anova: Single Factor
SUMMARY
Groups
Count
M(G1) M(G2)
9 9
Sum 103.5 110.3
Average 11.5 12.25556
Variance 0.34 0.390278
ANOVA
Source of Variation Between Groups Within Groups Total
SS 2.568889 5.842222 8.411111
df
MS
F
P-value
F crit
1 2.568889 7.035375 0.017383 4.493998 16 0.365139 17
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The statistical analysis, gives that, the possibility that the two groups differ from each other because of random variations, is P = 0.017383. Just because the decrease in oviposition caused by the magnetic field is small, in the order of 6%, the necessary number of valid experiments, (9), was relatively large in order for the P-value to become less than 0.05. The above experimental results are absolutely reproducible and each time we repeat the experiment, the P-value decreases significantly. Thereby it is proved that the exposed insect groups differ in offspring production from the controls, due to the effect of the alternating magnetic field. The number of F1 adult insects in the exposed and in the control groups, in all the experiments was the same as the number of F1 pupae. The number of exceptions from this rule was always within Standard Deviation of progeny number. During the experiments, we did not observe any morphological malformations, caused by the exposure in the paternal, or in the F1 insects.
5.5.3.2 Pulsed Electric Field We carried out 5 experiments with exposure of insects to pulsed electric field, according to the described experimental protocol. We use the following symbols: E(G1): Insect groups exposed to the electric field E(G2): Control groups for the electric field The effects of pulsed electric field exposure on the insect’s reproductive capacity, are given in Table 5.5.3. In Table 5.5.3, we can see the Mean Oviposition, (number of laid eggs), per female paternal insect during the first three days of the insect’s maximum daily oviposition, (3rd, 4th and 5th day of adult life), for the Exposed, E(G1) and the corresponding Control Groups, E(G2), for 5 experiments. [The Oviposition was again defined by the number of F1 progeny pupae, since during the experiments we did not record any statistically important percentage of non-developed laid eggs, or dead larvae, in the Exposed or in the Control Groups].
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Table 5.5.3: Mean Oviposition of Groups Exposed to Pulsed Electric Field and Control Groups Experiment Νο
Mean Oviposition
Group
Deviation from Control
per Female Paternal Insect 1 2
E(G1)
15.9
E(G2) Control
11.6
E(G1)
15.5
E(G2) Control
11.2
E(G1)
17.6
E(G2) Control
12.9
4
E(G1)
17.7
E(G2) Control
13.2
5
E(G1)
17.4
E(G2) Control
12.7
Average
E(G1)
16.82 1.04
Standard Deviation
E(G2) Control
12.32 0.87
3
+37.07% +38.39% +36.43% +34.09% +37.01% +36.5%
As we can see, in five experiments, the pulsed electric field caused an average increase in the insect’s reproductive capacity, by 36.5%. This result is graphically represented in Fig. 5.5.12. All the necessary conditions for the validity of Anova Test are satisfied, for the same reasons as in the experiments with the magnetic field. The ratio of largest to smallest Standard Deviation between the exposed and the control groups, is: 1.04/ 0.87 = 1.195 , (smaller than 2). The statistical analysis of the experimental results is given in Table 5.5.4. As we can see, the probability that the two groups differ from each other because of random variations, is P = 7.410-5. The probability of null hypothesis is considerably small in this case, only with 5 experiments, because the increase in oviposition caused by the electric field is large. The above results have very good reproducibility and every time we repeat the experiment, P-value decreases considerably. Thereby, the statistical analysis shows that the exposed groups differ in offspring production from the controls, due to the effect of the pulsed electric field. Table 5.5.4: Pulsed Electric Field. Statistical analysis. Anova: Single Factor SUMMARY Groups
Count
Sum
Average
Variance
E(G1)
5
84.1
16.82
1.077
E(G2)
5
61.6
12.32
0.757
ANOVA Source of Variation
SS
df
MS
F
P-value
F crit
Between Groups
50.625
1
50.625
55.2072
7.4E-05
5.317645
Within Groups
7.336
8
0.917
Total
57.961
9
561
Average Mean Oviposition
Reproductive Capacity of Exposed and Control Groups 18 16 14 12 10 8 6 4 2 0 E(G1)
E(G2) Groups
Fig. 5.5.12: Effect of pulsed electric field, on the reproductive capacity of Drosophila Melanogaster. Average Mean Oviposition per maternal insect, (number of laid eggs) Standard Deviation, during the first three days of the insect’s maximum oviposition, for the Exposed, E(G1) and the corresponding Control Groups, E(G2), in 5 experiments.
The number of F1 adult insects in the exposed and in the control groups, in all the experiments was the same as the number of F1 pupae. The number of exceptions from this rule was always within Standard Deviation of progeny number. During the experiments, we did not observe any morphological malformations, caused by the exposure in the paternal, or in the F1 insects.
5.5.3.3 RF-GSM Field According to the basic experimental protocol we have described, we carried on four experiments with “Non-Modulated” GSM field, (the experimenter was not making any sound during the exposure) and four experiments with “Modulated” GSM field. We use the following symbols: GSM(G1)NM: Groups exposed to “Non-Modulated” GSM field GSM(G2)NM: Control groups for the “Non-Modulated” GSM field GSM(G1): Groups exposed to “Modulated” GSM field GSM(G2): Control groups for the “Modulated” GSM field
Experiments with “Non-Modulated” GSM-Field In Table 5.5.5 we can see the mean oviposition, (number of laid eggs), per female maternal insect, during the first three days of the insect’s maximum oviposition, (3rd, 4th andι 5th day of adult life), for the groups exposed to “NonModulated” GSM field, GSM(G1)NM and for the corresponding control groups, GSM(G2)NM, in four experiments. The insect’s oviposition was again determined by the number of F1 progeny pupae. 562
As we can see the “Non-Modulated” GSM field, caused a considerable decrease in the insect’s reproductive capacity, as this is defined by the oviposition. The average decrease in four experiments was 18.24%. These results are graphically represented in Fig. 5.5.13. Table 5.5.5: Mean Oviposition of Groups Exposed to “Non-Modulated” GSM Field and corresponding Control Groups
Experiment Νο
1 2 3 4 Average Standard Deviation
Mean Oviposition per Female Paternal Insect
Group
Deviation from Control
GSM (G1)NM GSM (G2)NM Control GSM (G1)NM GSM (G2)NM Control GSM (G1)NM GSM (G2)NM Control GSM (G1)NM GSM (G2)NM Control
9.7 11.6 10 11.9 9.8 12.4 10.4 12.9
-16.38%
GSM (G1)NM GSM (G2)NM Control
9.975 0.31 12.2 0.57
-18.24%
-15.96% -20.16% -19.38%
All the necessary conditions for the validity of Anova test are satisfied. The ratio of largest to smallest Standard Deviation for the “Non-Modulated” GSM field is: 0.57/0.31 = 1.84 , (smaller than 2). The results of the statistical analysis are given in Table 5.5.6.
Table 5.5.6: “Non-Modulated” GSM field. Statistical analysis. Anova: Single Factor
SUMMARY
Groups
Count
GSM(G1)NM GSM(G2)NM
4 4
Sum
Average
39.9 48.8
9.975 12.2
Variance 0.095833 0.326667
ANOVA
Source of Variation Between Groups Within Groups Total
SS 9.90125 1.2675 11.16875
df
MS
1 6 7
9.90125 46.86982 0.21125
563
F
P-value
F crit
0.000478
5.987374
As we can see, the probability that the exposed groups differ from the control ones in oviposotion because of random variations, is P = 0.000478. The results have again, very good reproducibility.
Experiments with “Modulated” GSM field In Table 5.5.7 we can see the mean oviposition, (number of laid eggs), per female maternal insect, during the first three days of the insect’s maximum oviposition, (3rd, 4th andι 5th day of adult life), for the groups exposed to “Modulated” GSM field, GSM(G1) and for the corresponding control groups, GSM(G2), in four experiments. The oviposition was determined by the number of F1 pupae. Table 5.5.7: Mean Oviposition of Groups Exposed to “Modulated” GSM Field and corresponding Control Groups
Experiment Νο
1 2 3 4 Average Standard Deviation
Mean Oviposition per Female Paternal Insect
Group
Deviation from Control
GSM (G1) GSM (G2) Control GSM (G1) GSM (G2) Control GSM (G1) GSM (G2) Control GSM (G1) GSM (G2) Control
6.7 13.1 5.1 11.8 5.6 12.1 6 12.8
-48.85%
GSM (G1) GSM (G2) Control
5.85 0.67 12.45 0.6
-53.01%
-56.78% -53.72% -53.125%
As we can see, the “Modulated” GSM field caused a dramatic decrease in the insect’s reproductive capacity. The average decrease in four experiments was 53.01%. All the necessary conditions for the validity of the statistical test are satisfied as in the previous sets of experiments. The ratio of largest to smallest Standard Deviation between the two groups, is: 0.67/ 0.6 = 1.12 , (smaller than 2). The results of the ststistical analysis are given in Table 5.5.8.
564
Table 5.5.8: “Modulated” GSM Field. Statistical analysis. Anova: Single Factor
SUMMARY
Groups GSM(G1) GSM(G2)
Count
Sum
4 4
23.4 49.8
Average 5.85 12.45
Variance 0.456667 0.363333
ANOVA
Source of Variation
SS
df
Between Groups Within Groups Total
87.12 1 2.46 6 89.58 7
MS 87.12 0.41
F 212.4878
P-value 6.54E-06
F crit 5.987374
As we can see, the probability that the exposed groups differ in oviposition from the controls because of random variations, is found P = 1.69 10-6, (actually negligible). These results are again, absolutely reproducible. The effects of the “Modulated” and “Non-Modulated” GSM fields on the insect’s reproductive capacity are graphically represented in Fig. 5.5.13. The number of F1 adult insects in the groups exposed to either “Modulated” or “Non-Modulated” GSM field and in the corresponding control groups, in all the experiments was the same as the number of F1 pupae. The number of exceptions from this rule was always within Standard Deviation of progeny number. During the experiments, we did not observe any morphological malformations, caused by the exposure in the paternal, or in the F1 insects. The statistical analysis shows that, the Drosophila groups, exposed to the GSM field, differ in offspring production from the control groups, due to the effect of the GSM radiation. As we saw, the oviposition of the exposed groups is dramatically decreased, (up to 60%), compared to the controls.
565
Reproductive Capacity of Exposed and Control Groups
Average Mean Oviposition
14 12 10 8 6 4 2 0 GSM(G1)NM
GSM(G2)NM GSM(G1) Groups
GSM(G2)
Fig. 5.5.13: Effect of “Modulated” and “Non-Modulated” Field from the antenna of a GSM cellular phone, on the Reproductive Capacity of Drosophila melanogaster. Average Mean Oviposition per female paternal insect, (number of laid eggs), Standard Deviation, during the first three days of the insect’s maximum daily oviposition, for the groups exposed to the GSM field, GSM(G1), GSM(G1)NM and the corresponding control groups, GSM(G2), GSM(G2)NM, in four experiments with each field.
The reproductive capacity is much more decreased with “Modulated” emission, (50-60%), than with “Non-Modulated” emission, (15-20%). We did not detect any temperature increase, caused by the GSM field, inside the glass tubes, (in the food). [We used an Hg-thermometer with 0.05 C accuracy]. Even if there were some temperature increase, certainly smaller than 0.1 C, this would not have any observable effect on the insect’s oviposition, [57]. Thereby, the recorded effect cannot be attributed to any possible temperature increase caused by the radiation. Actual GSM signals are never constant. There are always changes in the intensity and frequency of these signals. Electromagnetic fields with changing parameters, usually have stronger biological action than fields with constant parameters, [18], probably because it is more difficult for living organisms to get adapted to them. Experiments with constant GSM signals can be performed, but they do not simulate actual conditions. In order to simulate actual conditions, we used a common cellular phone itself, in our experiments. Usually in biological experiments with RF fields, special devices are used, in order to ensure constant exposure conditions of a biological sample, or of different samples simultaneously, like Transverse Electromagnetic field (TEM), Cells, Radial Transmission Lines etc. In our experiments we had one or mostly two, samples for simultaneous exposure and besides it wasn’t important for us to have all the flies in the tube, under a constant power density or SAR, because this would not simulate actual conditions to which a user is exposed and because the recorded effect is statistical. Of course in such a case, when the characteristics of the exposure system are not constant, a proper statistical analysis is necessary and this is actually what we did.
566
5.5.3.4
Experiments with combination of Magnetic Field and Thermal Stress, (co-stress conditions)
In a number of experiments, we exposed the newly emerged flies to combination of alternating 50Hz, 28.3G magnetic field and heat shock, (θ = 30C). The exposure to both stressors simultaneously, was continuous for 48 hours. In these experiments the ten males and the ten females of each group were put together in the same vial from the beginning of each experiment and they were exposed for 48h. Then the females were dissected and the eggs of different stages in their ovaries were examined for possible malformations.
Fig. 5.5.14a: Photograph of stage 12 Drosophila egg, with morphological malformations, from ovary of insect exposed to combination of alternating magnetic field and thermal stress. The malformations are obvious in the oocyte, in the chorion and in the epithelial sheath that surrounds the whole follicle. The stage 12 follicle is connected in the ovariole, with the next follicle of stage 8, which has normal morphology. [Magn. about 1000]
Fig. 5.5.14b: Photograph of stage 12 Drosophila egg, with normal morphology, [Magnification about 1000].
567
Fig. 5.5.15a: Photograph of stage 14B follicle, with obvious abnormalities in its whole structure, from the ovary of an insect exposed to combination of alternating magnetic field and thermal stress. [Magn. about 1000]
Fig. 5.5.15b: Photograph of a normal, stage 14B follicle. [Magn. about 1000]
568
The exposure to co-stress conditions, induced malformations to a small percentage, (about 5%), of eggs of different developmental stages, in the ovaries of the female flies, (Figures 5.5.14a, 5.5.14b, 5.5.15a, 5.5.15b, 5.5.16, 5.5.17, 5.5.18). Malformations were not observed in the control insects that were exposed only to thermal stress, neither in insects that were exposed to combination of static magnetic field 37.5G and thermal stress, 30C.
Fig. 5.5.16: Photograph of stage 13 or 14 follicle, from the ovary of an insect exposed to combination of alternating magnetic field and thermal stress, with obvious abnormalities in the shape of the oocyte and without respiratory filaments, which normally start to develop at stage 11. The malformations in the oocyte look the same kind as in Fig. 5.5.17. [Magn. about 1000]
Fig. 5.5.17: Longitudinal section from a stage 14 follicle from ovary of an insect exposed to combination of alternating magnetic field and thermal stress. Normally the oocyte should be contiguous to the vitellin membrane. The section is colored with toluidine blue. [Magn. about 5000].
569
Fig. 5.5.18: Longitudinal section from a stage 14 follicle with normal morphology. [Magn. about 3000].
5.5.4 Discussion on the Experimental Results. Possible Biochemical Explanation The experiments showed, a) small but statistically significant decrease, in the order of 6%, of the reproductive capacity, following exposure to the alternating magnetic field, b) large increase of the reproductive capacity, in the order of 40%, following exposure to the pulsed electric field and c) dramatic decrease of the reproductive capacity, up to 60%, following exposure to the GSM field. Results from additional experiments of ours, with different experimental protocols, suggest that: 1) the EMFs affect the reproductive capacity of both female and male insects. 2) the above recorded effects of the EMFs do not change, if we expose the insects during their whole adult life. In our experiments the EMFs were found to affect more the reproductive capacity of the female, than the male insects. This can be explained, since spermatogenesis in Drosophila, starts earlier than oogenesis. Spermatogenesis starts during embryogenesis and the first mature spermatozoa are developed about 6h after eclosion of adult male flies and the necessary amount of paragonial substances to stimulate oviposition, (sex-peptide), is developed about 12h, after eclosion, [3], [29], [13], [58]. In contrary, oogenesis in Drosophila starts during pupariation and the first mature follicles have developed during the second day of the adult female flies. In our experiments, the exposure to the EMFs started after the eclosion of adult flies and thereby it was expected that would affect oogenesis, more than spermatogenesis. The small decrease of the insect’s reproductive capacity caused by the ELF magnetic field in our experiments, seems to agree with the small decrease in cell proliferation rate, [55], [28], and the decrease of the cytosolic concentration of Ca2+ ions, [65], that are reported in other published experiments with ELF magnetic fields.
570
In cases where, magnetic fields are found to increase or accelerate biological functions, it seems that the induced electric field was responsible, [22], [35]. The large increase in the insect’s reproductive capacity caused by the electric field in our experiments, also seems to agree with corresponding increases in the rate of DNA synthesis, [54], [47], increases in cell proliferation rate, [55], [15], [16], [17] and increases in the cytosolic concentration of Ca2+ ions, [7], [43], [5], [38], [35], [52], reported in other experiments. Finally, the dramatic decrease of the insect’s reproductive capacity caused by the RF-GSM field in our experiments, seems to agree with the significant decreases in cell proliferation rate, [34], [62] and in the cytosolic concentration of Ca2+ ions, [4], [6], [14], [10], found by other experimenters. In our experiments with all the above EMFs, we did not observe any statistically significant mortality of laid eggs, larvae, or pupae, in the exposed or in the control groups. This is due to the fact that in our experiments we used newly emerged flies and their reproductive capacity was determined during the first three days of maximum daily oviposition. Additionally, in our experiments we put 10 pairs in the same vial. In contrary, in some other experiments in which, a significant egg mortality, (more than 5%), was reported, they collected eggs from random population, [53], [41], or eggs laid by separate pairs, [49]. The adult life of Drosophila melanogaster, lasts about 20 days and It is true that after the first days of their adult life, especially during the last 5-10 days, female flies lay eggs that in a significant percentage do not develop. Additionally, a significant percentage of male flies, do not accomplish copulation at all, while other male flies accomplish copulation with more than one female. Thereby it’s possible that separate pairs of insects may not produce fertilized eggs. This possibility was eliminated in our experiments, by placing ten pairs in the same vial. Especially for the RF-GSM field, the effect on the reproductive capacity appears to be not linearly depended on the field’s power density. While the power density of “Modulated” emission, is increased by more than ten times, in relation to “NonModulated” emission, the reproductive capacity is decreased with “Modulated” emission, (50-60%), about three times as much as with “Non-Modulated” emission, (15-20%). The results of our experiments show clearly that RF electromagnetic radiation, and especially GSM mobile phones’ radiation at 900MHz, is highly bioactive, causing significant alterations in the physiological function of living organisms. Our results imply the need of prudent avoidance of exposure to this radiation and a very reasonable use of the mobile phones. The effects of the EMFs do not seem to be genotoxic, since we did not observe any morphological changes in the paternal or the F1 insects and the sections from follicles of different developmental stages look physiological. Nevertheless, it seems that simultaneous exposure to alternating magnetic field and thermal stress, can be genotoxic, since in such experiments, we did observe abnormalities in a small percentage of eggs, in the ovaries of exposed female insects. 571
Although there isn’t any established biochemical explanation for the biological effects of electromagnetic fields, we think that such an explanation of our results, can be sought in correlation with other laboratory studies which have showed that electromagnetic fields, alter the proliferation rate of cells, as well as the rate of DNA, RNA and protein synthesis, [33], [34], [55], [16], [17], [39], [40], [54]¸ [20], [19], [21]. These biochemical processes, are strongly affected, by changes in cytosolic calcium ion concentration and such changes can be induced by electromagnetic fields, [7], [43], [5], [38], [35], [52], [65], [4], [6], [10], [11], [14], [37]. Especially for the GSM field, it is shown that RF fields modulated by extremely low frequencies, (ELF), decrease cytosolic calcium ion concentration and in some experiments, this effect was maximum, for power densities between 0.6 and 1 mW/cm2, [4], [6]. GSM signals are RF carrier signals, pulsed on ELF and the measured power density in our experiments was within these values, (0.618 0.070 mW/cm2). Besides, it is known that cell proliferation, DNA, RNA and protein synthesis are connected with increased cytosolic calcium ion concentration, [8], [9], [47], [61], [36], [50] and with depolarization of the plasma membrane, [27], [7]. Thereby, since EMFs can change the cytosolic calcium ion concentrations, they can also change the rate of DNA, RNA and protein synthesis and the cell proliferation rate. In the early stages of Drosophila oogenesis and spermatogenesis there are repeated cell divisions, accompanied by DNA, RNA and protein synthesis. Additionally, DNA, RNA and protein synthesis is continued during the whole processes. Thereby, any changes in cell proliferation rates and in the synthesis of the above biomolecules, will result to retardation or acceleration of the whole processes of oogenesis, spermatogenesis and synthesis of proteins, necessary for the oviposition, (sex peptide), with corresponding alterations of the insect’s reproductive capacity. The effect of external electromagnetic fields on the cytosolic concentration of 2+ Ca ions, seems to be connected with interaction between the external field and the cation channels of the plasma membrane, which results in irregular gating of these channels, [54], [47], [35]. A possible mechanism for this interaction has been proposed, [48] and it is described in detail in our theoretical contribution in the present edition, [Panagopoulos and Margaritis, “Theoretical Considerations for the Biological Effects of EMFs”]. Additionally, any external electromagnetic field can interact directly with the endogenous physiological electric fields, that are known to control proliferation, differentiation and movement of cells to different locations, [44], [46], [64], phenomena that dominate during Drosophila oogenesis and spermatogenesis. These endogenous electric fields are also connected with Ca2+ ion currents and membrane depolarization, [7], [43]. As it becomes clear from our experiments, Man-made EMFs, can affect significantly the reproductive capacity of insects. Our results imply the need for prudent avoidance of human exposure to all kinds of EMFs. 572
Especially about RF radiation at 900 MHz, emitted by GSM mobile phones, the results of our experiments show that this radiation, with exposure conditions similar to those a mobile phone user is subjected to, is highly bioactive, causing dramatic decrease in the reproductive capacity of insects. It is clear from our studies that full power use of GSM cellular phones, (such as happens during a normal “talking” conversation-“speaking emission”), has dramatic effects on the reproduction of the insects, even with 6min exposure daily, for only 2-5 days. On the other hand, when the mobile phones are used in “listening” mode, (“non-speaking emission”), then the effects are less dramatic, but still important. Although we cannot simply parallel our results with possible corresponding effects on humans, we think that our results imply the need for least exposure to GSM radiation and a very reasonable use of mobile phones. Since the power density of this radiation in our experiments was within “Safety Levels”, our results imply also the need for reconsideration of the corresponding Exposure Criteria. The action of artificial electromagnetic fields on living organisms, according to the experiments of the present work and according to the other published experiments, seems to be most of the times, a non-genotoxic, acceleration or retardation of the cellular processes. This action, seems that can be genotoxic, if it is combined with another stress factor, (co-stress conditions), or with a non-healthy condition of the living organism. In the present paper we described some serious biological effects, caused by different kinds of man-made electromagnetic fields. Further research is necessary, for deeper investigation of the recorded effects, in biochemical and biophysical level.
Acknowledgements This work was supported by the “Special Account for Research Grants” of the University of Athens.
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