Nanometer-scale elongation rate fluctuations in the Myriophyllum aquaticum (Parrot feather) stem were altered by radio-frequency electromagnetic radiation

Abstract

The emission of radio-frequency electromagnetic radiation (EMR) by various wireless communication base stations has increased in recent years. While there is wide concern about the effects of EMR on humans and animals, the influence of EMR on plants is not well understood. In this study, we investigated the effect of EMR on the growth dynamics of Myriophyllum aquaticum (Parrot feather) by measuring the nanometric elongation rate fluctuation (NERF) using a statistical interferometry technique. Plants were exposed to 2 GHz EMR at a maximum of 1.42 Wm⁻² for 1 h. After continuous exposure to EMR, M. aquaticum plants exhibited a statistically significant 51 ± 16% reduction in NERF standard deviation. Temperature observations revealed that EMR exposure did not cause dielectric heating of the plants. Therefore, the reduced NERF was due to a non-thermal effect caused by EMR exposure. The alteration in NERF continued for at least 2.5 h after EMR exposure and no significant recovery was found in post-EMR NERF during the experimental period.

Keywords: :

• electromagnetic radiation
• growth dynamics
• non-thermal
• plant stress
• statistical interferometry
• wireless communication

Introduction

Various gas, liquid, and solid contaminants are known to pollute the environment. In many cases, these pollutants can accumulate in the environment and cause considerable damage before their effects are identified and control measures are undertaken (e.g., cadmium,¹ arsenic,² O₃,³ and CO₂). It is therefore of great importance to study the possible impacts of potential contaminants that are not yet considered to be pollutants.

The utilization of radio-frequency electromagnetic radiation (EMR) is increasing with the extensive deployment of mobile technology base stations, particularly mobile phone and wireless broadband access base stations. Furthermore, in some locations, there can be 4 or more mobile phone service providers covering a single geological area,⁴ leading to increased EMR densities in those environments. EMR exposure guidelines are based on the heating effect of EMR on biological tissues,⁵ and effects of EMR on humans and other animals are areas of concern. To date, the effect of EMR on plants has not been considered to be problematic. This may be due to the assumption that environmental exposure does not have a thermal effect on plants, and that plants are less sensitive to environmental stressors than are animals. A few studies discussing EMR effects on plants have been published, but the data are insufficient to allow general conclusions to be reached. Nevertheless, some evidence suggests that plants can experience non-thermal stress as a result of exposure to EMR.^6-11

Plants alter their growth and development when exposed to various environmental stressors and stimuli, such as toxic gases (e.g., O₃ and SO₂),^12,13 magnetic fields,^14,15 electric fields,¹⁶ and vibrations.¹⁷ EMR exposure may also act as an environmental stress or stimulus for plants. For example, reactive oxygen species formation increases in plant tissues after exposure to EMR.^6,10 This has the potential to increase plant oxidative stress, which can result in a decreased growth rate.^18,19 Investigation of the growth dynamics of plants after EMR exposure would provide a good indication of the effects of EMR on plants. As yet, however, no studies have been performed examining the effect of EMR on plant growth dynamics during or immediately after exposure. This may be due to the difficulties encountered when attempting to quantify plant growth parameters over short time periods using non-invasive techniques. To overcome these limitations, we used statistical interferometry techniques (SIT) to measure the growth dynamics of Myriophyllum aquaticum (Parrot feather) plants before and after exposure to EMR. Using this technique, we measured changes in the standard deviation of nanometric fluctuation in the M. aquaticum stem length. In stable conditions, this fluctuation remains largely unchanged for several hours. The nanometric fluctuations of the lengths of some plant parts (leaves and roots) change in response to environmental stresses, and these nanometer-scale changes can be monitored using SIT.^20,21 To investigate any thermal effects, which can be caused by dielectric heating due to EMR exposure,²² the temperature of plants before and during EMR exposure was observed using thermographic images.

Results

M. aquaticum stems exposed to EMR had significantly smaller standard deviations (SD) of nanometric elongation rate fluctuations (NERF) after exposure than prior to exposure (1.51 ± 0.43 vs. 3.24 ± 0.96 nm s⁻¹ mm⁻¹, P < 0.05). This represents a reduction in SD of 51 ± 16% from the pre-EMR exposure. By contrast, the average difference between control-plant NERF SDs before and after placement in the EMR exposure chamber was 11 ± 7%, which was not statistically significant (1.89 ± 0.89 vs. 1.71 ± 0.86 nm s⁻¹ mm⁻¹, P > 0.05; Fig. 1). To determine whether NERF recovered toward the pre-EMR exposure state, the growth rate data for both pre- and post-EMR exposure measurements were divided into 2 30-min segments and the SD of each segment was calculated separately. The NERF SDs for the 2 pre-EMR 30-min exposure measurement periods were not significantly different from one another (3.29 ± 0.93 and 3.14 ± 0.99 nm s⁻¹ mm⁻¹, P > 0.05). Similarly, the NERF SDs for the 2 post-EMR exposure measurement periods were likewise not significantly different from one another (1.68 ± 0.76 and 1.89 ± 0.97 nm s⁻¹ mm⁻¹, P > 0.05; Fig. 2).

Figure 1. Relative changes in the standard deviation of elongation rate fluctuation in M. aquaticum stems. Plants were either exposed to 2-GHz radio-frequency electromagnetic radiation (EMR; 2 GHz) or ambient-only EMR (control). Pre and Post represent pre- and post- EMR exposure, respectively. Error bars represent standard deviations.

Figure 2. Standard deviation of 2 consecutive 30-min measurements of length fluctuation in M. aquaticum stems before and after exposure to 2-GHz radio-frequency electromagnetic radiation (EMR). “Seg 1” and “Seg 2” represent 2 consecutive 30-min time segments. “Pre” and “Post” represent pre- and post-EMR exposure measurements. Error bars represent standard deviations.

Exposure to EMR did not alter the temperature of plants, even after 1 h continuous exposure. The temperature at a reference point in the EMR exposure chamber and on the plant both increased by 0.1 °C during EMR exposure. The initial temperature of the plant was 23.2 °C, which rose to 23.3 °C after 1 h continuous EMR exposure (Fig. 3). The temperature of the vinyl tube also increased by 0.1 °C after 1 h of continuous exposure, from 23 °C before EMR exposure to 23.1 °C after exposure (Fig. 4).

Figure 3. Thermal images indicating M. aquaticum plant temperatures. (A) Image taken before exposure to 2-GHz radio-frequency electromagnetic radiation (EMR), (B) image taken of the same plant on completion of 1 h continuous EMR exposure. Within each image, the letter “b” indicates the measuring point on the plant canopy, and the letter “e” indicates the temperature measurement position on a 5-mm-thick rigifoam sheet.

Figure 4. Thermal images indicating the temperature of a vinyl tube filled with distilled water. (A) Image taken before exposure to 2-GHz radio-frequency electromagnetic radiation (EMR), (B) image taken of the same tube on completion of 1 h continuous EMR exposure. Within each image, the letter “b” indicates the measuring point on the plant canopy, and the letter “e” indicates the temperature measurement position on a 5-mm-thick rigifoam sheet.

Discussion

Non-stressed plants at vegetative growth phase can show growth during the light phase.^23,24 However, while growth proceeds, there is an observed fluctuation in the length of the plants; this continues even without any measurable overall growth.²⁵ These fluctuations may result from complex mechanistic interactions within plants. For example, an exchange of ions (Ca²⁺, K⁺, Cl^-) between the symplast and apoplast spaces²⁶ and changes in cell turgor^27,28 are needed for long-distance signal transmission through the phloem. This changing cell turgor could cause nanometric fluctuations in plant lengths. Furthermore, the variations in transpiration rate over time can also lead to plant length fluctuations at the micrometer scale,²⁵ and, when the fluctuation in the stem length is measured at 3-mm sections as in the present study, this can be observed at the nanometer scale.

The reduced NERF SDs observed in the present experiment suggest that exposure to 2 GHz EMR caused a change in the physiology of M. aquaticum plants. This level of exposure is less intense than the maximum public exposure guidelines for humans (9.87 Wm^-2).⁵ Reduced NERF can indicate that plants are experiencing stress.²¹ The observed reduction in NERF could be explained by the accumulation of H₂O₂ and oxidative stress caused by EMR exposure, as reported by Tkalec, Malaric, and Pevalek-Kozlina;⁶ Tkalec, Malaric, and Pevalek-Kozlina;⁹ and Sharma, Singh, Kohli, and Batish.¹⁰ Increases in ROS activity after EMR might be due to either an effect on the photosynthetic process^29,30 or non-photosynthetic ROS formation in cells,³¹ but no clear explanation has yet been determined. Oxidative stress and accumulation of ROS in plants negatively affects growth³² and cellular activity, and may lead to cell death.³³ Furthermore, if H₂O₂ were generated as a consequence of exposure of the photosystem to EMR, H₂O₂ would be expected to induce stomatal closure.^34,35 This would lower transpiration rates, explaining the reduction of NERF in EMR-exposed plants. In the current study, NERF was recorded 1.5 h after EMR exposure. The similarity of the NERF SDs for the 2 30-min segments of post-EMR exposure measurement indicates that the plants did not exhibit any recovery, even 2.5 h after exposure. Plants can take 5–18 h to recover from oxidative stress,^36,37 and a longer recovery period is therefore likely to be required before restoration of normal NERF patterns can be observed. Future studies should investigate prolonged recovery times after exposure to EMR.

Plants can also be affected by temperature stress,³⁸ and EMR may increase plant temperature by dielectric heating.²² Thermal images indicated that 1 h of continuous exposure to EMR did not alter the temperature of plants. However, physiological processes within the plant may have served to regulate the temperature,^39-41 rendering it difficult to assess whether dielectric heating occurred. Nevertheless, the temperature of a vinyl tube filled with distilled water increased by only 0.1 °C after 1 h EMR exposure at a power density more than 4-fold that used for the NERF experiment, suggesting minimal dielectric heating effects. As the EMR power density was lower in the NERF experiments (1.42 Wm⁻²) than for the temperature experiments (6 Wm⁻²), any temperature increase in M. aquaticum during EMR exposure in the NERF experiment should have been less than 0.1 °C. An increase of at least 1 °C is required to generate a thermal effect in biological tissue.⁴² Therefore, in the present study, M. aquaticum plants did not experience heat stress caused by EMR exposure.

Plant growth is stimulated by exposure to stationary magnetic fields or very low frequency electromagnetic fields.^14,15,43,44 However, calculations using conversion formulas for continuous wave (CW) EMR reveal that the maximum magnetic flux density was approximately 5.9 × 10⁻² µT (Magnetic flux density (µT) = Wm^–2/239). This magnetic flux density is relatively small when compared with the magnetic flux densities in the above plant growth experiments (1.5 µT–0.5 T) and the magnetic flux density of the earth’s magnetic field (around 50 µT).⁴⁵ Therefore, the magnetic flux density of EMR in the present study was unlikely to directly affect the plants. However, the very high frequency of the EMR magnetic field used in the present study might have had a hitherto unknown effect on plant growth. Although the magnetic field of EMR is the same as the electric field of EMR (Electric field (Vm⁻¹) or Magnetic field (Am⁻¹)=$\sqrt{W{m}^{-2}/377}$) (1), most researchers consider the effects of EMR on plants in terms of the electric field and the effects of dielectric activity. However, because the reported effects of EMR on plants are non-thermal, we cannot determine whether the observed effects were caused by the high-frequency EMR electric field or the magnetic field, and this should be studied further.

Plants adapt to their growing environment.^46-48 Plants may thus have adapted to EMR exposure as they are continuously exposed to EMR in the natural environment due to the continuous operation of communication transmitters. Therefore, it is important to study plants exposed to EMR for long periods of time to understand any mechanisms used by plants to adapt to EMR exposure. However, plants have different levels of sensitivity to EMR, depending on the frequency and power density to which they are exposed,^49,50 and so it is expected that plants growing in the natural environment would vary in their levels of adaptation according to location and local EMR intensity.

The power density of EMR due to mobile phone base stations (8.6 mWm⁻²)⁵¹ is lower than the power density used in the present study and in most of the studies examining the effects of EMR on plants that are cited in this article. However, increasing power densities can be expected in the future due to the congested deployment of base stations and public exposure guidelines for frequencies above 2 GHz that are as high as 9.87 Wm^-2.⁵ The current public exposure guidelines are based on the thermal effects on biological tissues caused by dielectric heating due to EMR. However, our observations of reduced NERF alongside unchanged temperature, combined with oxidative stress data reported elsewhere, strongly suggest that plants experience non-thermal stress in response to EMR exposure.

Materials and Methods

Plant preparation

M. aquaticum stems were selected that were at least 15 cm in total length, had emergent portions at least 10 cm in length, and had roots developing on the stem in the submerged portion. The selected stems were cut from the plant stock, and 3–5 stems were planted in clean cube-shaped (15 × 15 × 15 cm) small glass tanks that had been filled to the halfway mark with cleaned fine river sand (sand was thoroughly washed 3 times with tap water and once with distilled water before use). Hoagland solution (35%) was then added to a level of 5 cm above the sand. Water levels in the tanks were maintained by adding distilled water every 2–3 d and 35% Hoagland solution once per week. Plants were grown under 55–60 µmol m⁻² s⁻¹ photosynthetically active radiation as measured at 10 cm above the tank, with a 12/12 photoperiod. Stems were allowed to grow until they naturally fell to a horizontal position from their original vertical position (around 20-cm emergent stem length), after which they were used for SIT experiments (Fig. 5). Horizontally positioned plants were used for the experiment because the probing laser beams of the SIT system were arranged horizontally to measure stem elongation.

Figure 5. Horizontally positioned M. aquaticum plant positioned within the nanometric elongation recording system. (A) Two probing beams separated by 3 mm, (B) soft clamp holding the plant in place, and (C) charge-coupled device camera.

Statistical interferometry technique

SIT is a novel optical method to measure the strain or change of an object having a rough surface. Statistical interferometry has been successfully employed to measure extremely short-term changes at the sub-nanometer scale in the lengths of plant tissues with the aim of estimating environmental conditions. Therefore, SIT can be used to measure changes in plant elongation and rates of elongation more accurately than can be achieved using conventional techniques.

To achieve measurements of an extremely high accuracy, SIT employs a simple optical system and involves no contact with the studied object. Two points on the stem of the plant under investigation were illuminated by laser beams. As a result of the strong scattering of the sample, 2 speckle fields were generated in the diffraction field of the system. The 2 fully developed independent speckle fields were superimposed, and the resulting 2 speckle fields observed through a charge-coupled device (CCD) camera placed in the diffraction field.^52,53 Elongation was determined every 0.5 s. The elongation rate (nm mm⁻¹ sec⁻¹) was obtained from the elongation over a period τ of 5.5 s and was defined by Elongation rate = Elongation / τd where d is laser beam separation. Detailed descriptions of SIT are given in Kobayashi and Kadono^21, Kadono, Bitoh, and Toyooka.⁵² Data were acquired over the whole duration of 1 h.

The NERF measurement and EMR exposure

NERF measurements were performed on EMR-exposed and control plants in the same laboratory environment. Environmental EMR exposure was not controlled in either the test or control plants. The stem of each plant was gently clamped, and loose cotton was placed around each plant stem to prevent slippage away from the laser measurement probes. Plants were provided with 55–60 µmol m⁻² s⁻¹ photosynthetically active radiation density from a halogen lamp. After a 1.5 h stabilization period, initial NERF were measured continuously for 1 h. Fluctuations were measured 8–10 cm below the top (canopy area) of each plant. After the initial measurements, plants were placed directly under an EMR antenna in an EMR exposure chamber. “EMR-exposed” plants were then subjected to 2-GHz continuous wave (CW; electromagnetic waves of constant amplitude and frequency) EMR at 1.42 Wm⁻² power density for 1 h. The power density was approximately 0.65 Wm⁻² at the canopy area of the plant, which was approximately 20 cm away from the center of the antenna (Fig. 6). Electromagnetic waves consist of a magnetic and an electric field oscillating perpendicular to one another. In this experiment, the electric field was orientated with the object, and thus EMR polarization was perpendicular to the stem during exposure. Control plants were left in the EMR exposure chamber for 1 h in the absence emission of EMR from the antenna. After 1 h, plants were removed from the EMR exposure chamber and again positioned within the NERF measuring system. After a 1.5 h stabilization period, NERF measurements were made at the plant location where the initial measurements were taken. The experiment was repeated 4 times.

Figure 6. Perpendicular positioning of an M. aquaticum stem with respect to an electromagnetic radio-frequency antenna, resulting a gradient of power intensity along the stem. (A) Transmission antenna, (B) direct waves causing highest power density, (C) angled waves causing low power density, and (D) radio-frequency electromagnetic radiation power density decreases in the direction of the arrow.

Thermographic imaging

Exposure to EMR can increase the temperature of biological materials due to dielectric heating. Dielectric heating is a mechanism by which heat is generated due to the interaction of the alternating electric field of EMR and dipolar water molecules.⁵⁴ Temperature changes in plants exposed to EMR were assessed using infrared thermographic images with a thermal sensitivity of 0.08 °C (at 30 °C; InfReC Thermo GEAR G120, NEC). In this experiment, 10-cm-tall vertically growing M. aquaticum plants were used. Plants were exposed to 2 GHz CW EMR at a power density of 6 Wm⁻² at the canopy area for 1 h. Thermal images of the plant canopy were obtained prior to exposure and immediately before the exposure ended, 1 h later. To investigate the ability of EMR to increase the temperature of a water column with approximately the same diameter as an M. aquaticum stem, a vinyl tube filled with distilled water was subjected to EMR exposure and the changes in temperature measured. In this experiment, a 12-cm vinyl tube that had internal and external diameters of 5 mm and 6 mm, respectively, was filled with distilled water. Both ends of the tube were sealed using silicon sealant (Senedainn silicon sealant 8060). The tube was then positioned vertically, directly under an EMR transmission antenna, by connecting it to a podium. The tube was exposed to EMR of the same frequency and power density as used in the plant thermographic imaging experiments, and thermal images were taken prior to continuous exposure to EMR for 1 h, and immediately before the exposure ended. Thermal measurements of the vinyl tube were targeted 1.5–2 cm below the top of the tube.

Data analysis

The NERF of the M. aquaticum stem continued over time (Fig. 7). The power spectrum analysis was not able to clearly differentiate the effect of NERF, and thus the NERF standard deviation was calculated to characterize the fluctuations. NERF SDs of EMR-exposed and control plants were compared using 2 independent sample t-tests with the Levene test for equality of variances. Analyses were performed using SPSS 16.0 (SPSS Inc, USA). Thermal images were manually compared with evaluate changes in temperature.

Figure 7. Representative fluctuation of elongation rate of M. aquaticum for 1 h. (A) Nanometric elongation rate fluctuation before radio-frequency electromagnetic radiation (EMR) exposure, and (B) nanometric elongation rate fluctuation after EMR exposure. Vertical axis denotes rate of fluctuation in nm s⁻¹ mm⁻¹ distance of the plant stem. Horizontal axis denotes duration.

Abbreviations:
CCD	=	charge-coupled device
CW	=	continuous wave
EMR	=	radio-frequency electromagnetic radiation
NERF	=	nanometric elongation rate fluctuation
NERF SD	=	standard deviation of nanometric elongation rate fluctuation
SIT	=	statistical interferometry technique

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Prof Yuichi Kimura for helpful discussions during the preparation of this manuscript, Dr Makoto Miwa for valuable support during this study, and Mr Sakuyoshi Saito for his support in the preparation of the EMR exposure system. This study was funded by the Ministry of Education, Culture, Sports, Science, and Technology, Japan.