ArticlePDF Available

Effects of short-term exposure to mobile phone radiofrequency (900 MHz) on the oxidative response and genotoxicity in honey bee larvae

Authors:

Abstract and Figures

Exposure of different animal species to radiofrequency electromagnetic fields (RF-EMF) could cause various biological effects such as oxidative stress, genotoxic effects and dysfunction of the immune system. However, there are a lack of results on oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. This study was performed to investigate the effects of exposure to RF-EMF on the activity of catalase, superoxide dismutase, glutathione S-transferase, lipid peroxidation level and DNA damage in honey bee larvae. Honey bee larvae were exposed to RF-EMF at 900 MHz and field levels of 10, 23, 41 and 120 V m−1 for 2 h. At a field level of 23 V m−1 the effect of 80% AM 1 kHz sinusoidal and 217 Hz modulation was investigated as well. Catalase activity and the lipid peroxidation level decreased significantly in the honey bee larvae exposed to the unmodulated field at 10 V m−1 compared to the control. Superoxide dismutase and glutathione S-transferase activity in the honey bee larvae exposed to unmodulated fields were not statistically different compared to the control. DNA damage increased significantly in honey bee larvae exposed to modulated (80% AM 1 kHz sinus) field at 23 V m−1 compared to the control and all other exposure groups. These results suggest that RF-EMF effects in honey bee larvae appeared only after exposure to a certain EMF conditions. The increase of the field level did not cause a linear dose-response in any of the measured parameters. Modulated RF-EMF produced more negative effects than the corresponding unmodulated field. Although honey bees in nature would not be exposed to such high field levels as used in our experiments, our results show the need for further intensive research in all stages of honey bee development.
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=tjar20
Download by: [37.244.251.35] Date: 28 July 2017, At: 02:52
Journal of Apicultural Research
ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: http://www.tandfonline.com/loi/tjar20
Effects of short-term exposure to mobile phone
radiofrequency (900MHz) on the oxidative
response and genotoxicity in honey bee larvae
Marinko Vilić, Ivana Tlak Gajger , Perica Tucak, Anamaria Štambuk , Maja
Šrut , Göran Klobučar , Krešimir Malarić , Ivona Žura Žaja, Ana Pavelić ,
Marin Manger & Mirta Tkalec
To cite this article: Marinko Vilić, Ivana Tlak Gajger , Perica Tucak, Anamaria Štambuk , Maja
Šrut , Göran Klobučar , Krešimir Malarić , Ivona Žura Žaja, Ana Pavelić , Marin Manger & Mirta
Tkalec (2017) Effects of short-term exposure to mobile phone radiofrequency (900MHz) on the
oxidative response and genotoxicity in honey bee larvae, Journal of Apicultural Research, 56:4,
430-438, DOI: 10.1080/00218839.2017.1329798
To link to this article: http://dx.doi.org/10.1080/00218839.2017.1329798
Published online: 04 Jul 2017.
Submit your article to this journal
Article views: 67
View related articles
View Crossmark data
ORIGINAL RESEARCH ARTICLE
Effects of short-term exposure to mobile phone radiofrequency (900 MHz) on the
oxidative response and genotoxicity in honey bee larvae
Marinko Vilic
´
a
, Ivana Tlak Gajger
b
, Perica Tucak
c
, Anamaria S
ˇtambuk
d
, Maja S
ˇrut
d
,Go
¨ran Klobuc
ˇar
d
,
Kres
ˇimir Malaric
´
e
, Ivona Z
ˇura Z
ˇaja
a
, Ana Pavelic
´
d
, Marin Manger
d
and Mirta Tkalec
d
*
a
Faculty of Veterinary Medicine, Department of Physiology and Radiobiology, University of Zagreb, Zagreb, Croatia;
b
Faculty of
Veterinary Medicine, Department for Biology and Pathology of Fish and Bees, University of Zagreb, Zagreb, Croatia;
c
Ministry of Agriculture Veterinary and Food Safety Directorate, Zagreb, Croatia;
d
Faculty of Science, Department of Biology,
University of Zagreb, Zagreb, Croatia;
e
Faculty of Electrical Engineering and Computing, University of Zagreb, Zagreb, Croatia
(Received 4 January 2017; accepted 9 May 2017)
Exposure of different animal species to radiofrequency electromagnetic fields (RF-EMF) could cause various biological effects
such as oxidative stress, genotoxic effects and dysfunction of the immune system. However, there are a lack of results on
oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. This study was per-
formed to investigate the effects of exposure to RF-EMF on the activity of catalase, superoxide dismutase, glutathione S-trans-
ferase, lipid peroxidation level and DNA damage in honey bee larvae. Honey bee larvae were exposed to RF-EMF at 900 MHz
and field levels of 10, 23, 41 and 120 V m
1
for 2 h. At a field level of 23 V m
1
the effect of 80% AM 1 kHz sinusoidal and
217 Hz modulation was investigated as well. Catalase activity and the lipid peroxidation level decreased significantly in the
honey bee larvae exposed to the unmodulated field at 10 V m
1
compared to the control. Superoxide dismutase and glu-
tathione S-transferase activity in the honey bee larvae exposed to unmodulated fields were not statistically different compared
to the control. DNA damage increased significantly in honey bee larvae exposed to modulated (80% AM 1 kHz sinus) field at
23 V m
1
compared to the control and all other exposure groups. These results suggest that RF-EMF effects in honey bee lar-
vae appeared only after exposure to a certain EMF conditions. The increase of the field level did not cause a linear dose-re-
sponse in any of the measured parameters. Modulated RF-EMF produced more negative effects than the corresponding
unmodulated field. Although honey bees in nature would not be exposed to such high field levels as used in our experiments,
our results show the need for further intensive research in all stages of honey bee development.
Efectos a corto plazo de la exposicio
´n a la radiofrecuencia de los tele
´fonos mo
´viles (900 MHz) sobre la
respuesta oxidativa y la genotoxicidad en las larvas de abejas
La exposicio
´n de diferentes especies animales a campos electromagne
´ticos de radiofrecuencia (CEM-RF) podrı
´a causar diver-
sos efectos biolo
´gicos tales como estre
´s oxidativo, efectos genoto
´xicos y disfuncio
´n del sistema inmunolo
´gico. Sin embargo,
hay una falta de resultados sobre la respuesta al estre
´s oxidativo y la genotoxicidad en la abeja de la miel (Apis mellifera)
despue
´s de la exposicio
´n a CEM-RF. Este estudio fue realizado para investigar los efectos de la exposicio
´n a CEM-RF sobre la
actividad catalasa, supero
´xido dismutasa, glutatio
´n S-transferasa, nivel de peroxidacio
´ndelı
´pidos y dan
˜o al ADN en larvas de
abejas. Las larvas de abejas fueron expuestas a CEM-RF de 900 MHz e intensidades de campo de 10, 23, 41 y 120 V m
1
dur-
ante 2 h. A 23 V m
1
de intensidad de campo, se investigo
´tambie
´nelefectodel80%demodulacio
´ndeamplitudde1kHzsinu-
soidal y 217 Hz de modulacio
´n. La actividad catalasa y el nivel de peroxidacio
´nlipı
´dica disminuyeron significativamente en las
larvas de abejas expuestas al campo no modulado de 10 V m
1
en comparacio
´n con el control. La actividad supero
´xido dismu-
tasa y glutatio
´n-S-transferasa en las larvas de abejas expuestas a campos no modulados no fue estadı
´sticamente diferente en
comparacio
´n con el control. El dan
˜o en el ADN aumento
´significativamente en larvas de abejas expuestas a campos modula-
dos (80% AM 1 kHz sinusoidal) de 23 V m
1
en comparacio
´n con el control y todos los dema
´sgruposdeexposicio
´n. Estos
resultados sugieren que los efectos de CEM-RF en larvas de abejas de miel aparecieron so
´lo despue
´s de la exposicio
´naciertas
condiciones de los campos electromagne
´ticos. El aumento de la intensidad del campo no causo
´una dosis-respuesta lineal en
ninguno de los para
´metros estudiados. El CEM-RF modulado produjo ma
´s efectos negativos que el campo no modulado cor-
respondiente. Aunque las abejas me
´feras en la naturaleza no esta
´an expuestas a intensidades de campo tan altas como las
utilizadas en nuestros experimentos, nuestros resultados indican la necesidad de una mayor investigacio
´n en todas las etapas
del desarrollo de las abejas melı
´feras.
Keywords: antioxidative enzymes; DNA damage; genotoxicity; honey bee larvae; lipid peroxidation; oxidative damage;
radiofrequency electromagnetic fields
Introduction
The use of devices that emit radiofrequency electro-
magnetic fields (RF-EMF), such as mobile phones, has
arisen rapidly in recent years. In 2015, there were
more than 7 billion mobile cellular subscriptions esti-
mated, with a tendency of further growth worldwide
(The International Telecommunication Union [ITU],
2016). With increasing usage of mobile phones, public
*Corresponding author. Email: mirta.tkalec@biol.pmf.hr
©2017 International Bee Research Association
Journal of Apicultural Research, 2017
Vol. 56, No. 4, 430–438, https://doi.org/10.1080/00218839.2017.1329798
concern about possible adverse health effects from
exposure to mobile phone signals have occurred.
Although the absorbed energy from mobile phones
cannot break chemical bonds, the results of many stud-
ies have shown that exposure to radiofrequency radia-
tion at the operating frequency of mobile phones can
induce various biological effects. These include oxida-
tive stress in human cells (Luukkonen, Hakulinen, Ma
¨ki-
Paakkanen, Juutilainen, & Naarala, 2009; Moustafa,
Moustafa, Belacy, Abou-El-Ela, & Ali, 2001), plants (Tka-
lec, Malaric
´, & Pevalek-Kozlina, 2007), earthworms
(Tkalec, S
ˇtambuk, S
ˇrut, Malaric
´, & Klobuc
ˇar, 2013),
genotoxic effects in human lymphocytes (Esmekaya
et al., 2011), earthworms (Tkalec et al., 2013), plants
(Tkalec, Malaric
´, Pavlica, Pevalek-Kozlina, & Vidakovic
´-
Cifrek, 2009) and other ecological effects in different
organisms (for review see Cucurachi et al., 2013). Fur-
thermore, alterations of antioxidant defense system
parameters such as glutathione (GSH) concentration or
glutathione peroxidase (GSH-Px), superoxide dismutase
(SOD) and catalase (CAT) activity, have also been doc-
umented in human cells (Moustafa et al., 2001) and rats
(Aydin & Akar, 2011), earthworms (Tkalec et al.,
2013), plants (Tkalec et al., 2007).
However, it is very important to emphasizes that
the mentioned effects of RF-EMF are still controversial
because there are several studies that have not found
biological effects after exposure to RF-EMF or have
some shortcomings, especially in studying genotoxic
effects (for reviews see Ruediger, 2009; Verschaeve,
2009; Vijayalaxmi & Prihoda, 2012).
Previous studies regarding effects of RF-EMF at fre-
quencies from 900 MHz to 2.4 GHz in honey bee colo-
nies investigated mostly adult honey bees. Mall & Kumar
(2014) found no effect on brooding, honey production
and foraging behavior, but other studies have shown
decreased colony strength and egg laying rate of the
queen (Sharma & Kumar, 2010), induction of “worker
piping” which is associated with swarming (Favre, 2011)
and the alteration of some biochemical parameters (total
carbohydrates, glucose, glycogen, total lipids, cholesterol,
protein, hexokinase, alkaline phosphatase, glucose 6-
phosphatase and free amino acids) in the hemolymph of
the drone (Kumar, Neha, & Preeti, 2013). However,
according to our knowledge, studies on the influence of
the RF-EMF on oxidative stress and/or genotoxicity in
honey bee larvae after exposure to radiofrequency at
900 MHz are not yet available.
The effects of RF-EMF on biological systems may
depend on physical properties of radiation such as fre-
quency, straight, modulation, exposure time (Kwee &
Raskmark, 1998; Luukkonen et al., 2009; Sarimov, Malm-
gren, Markova
`, Persson, & Belyaev, 2004) as well as on
biological parameters such as species or development
stage (Cucurachi et al., 2013). Therefore, in this study
we wanted to investigate the oxidative stress parame-
ters and genotoxicity in honey bee larvae, a vulnerable
stage of development, after short-term exposure to RF-
EMF under controlled laboratory conditions at the
operating frequency of mobile phones (900 MHz) with
different field levels and modulation.
We have used the field level of 10 V m
1
as a stan-
dard value of operating mobile devices, then 23 V m
1
as
the value of the electric field when establishing the con-
nection of mobile devices, 41 V m
1
as the maximum
value when dialing, and 120 V m
1
as a limit value (taking
factor 100) above the value (1 V m
1
) that honey bees
could be exposed in the natural environment. Although
values of field strength used in this study were higher
than those under natural conditions, we used them to
trace a linear dose-response relationship of RF radiation
and/or possible occurrence of an increased bioactivity of
RF-EMF under specific conditions. In addition, based on
results of our previous investigations on the earthworms
and plants (Tkalec, Malaric
´, & Pevalek-Kozlina, 2005;
Tkalec et al., 2007; Tkalec et al., 2009; Tkalec et al.,
2013), we compared the effects of unmodulated and
modulated wave of RF radiation at 23 V m
1
as the most
common field level. Namely, RF signal having a carrier
frequency of 900 MHz and amplitude modulated at
217 Hz is similar to that used by the global system for
mobile communication (GSM) telephone system.
The objective of this study was therefore to answer
the questions: (a) could exposure to RF-EMF at
900 MHz cause oxidative stress and genotoxic effect in
larval stage of honey bees under laboratory conditions;
and (b) is there a possibility of a different bioactivity of
RF-EMF after honey bee larvae exposure to RF-EMF at
specific field levels and modulation.
Material and methods
Honey bees (Apis mellifera)
Honey bee larvae were used in the experiment. Frames
with combs containing young honey bee brood were
taken from one hive, type Langstroth – Root. After
frames were taken from hives they were wrapped with
moist flannel and placed in a carton box. Frames with
honey bee brood were transported from bee yard to the
laboratory every day under the same conditions. Upon
arrival in the laboratory, prior to exposure, the honey
bee larvae approximately five to six days old, were ran-
domly collected from delivered combs, using entomologi-
cal tweezers, stored on a moist filter paper in Petri dishes
with perforated lids and immediately placed in a Gigahertz
transversal electromagnetic (GTEM) cell for irradiation
for 2 h. All sampled larvae had approximately equal
weight (about 30 g). The study was reviewed and
approved by the Ethics Committee of the Faculty of
Veterinary Medicine University of Zagreb. (Class: 640-01/
13-17/76; Record Number: 251/61-01/139-13-9).
Exposure
Exposure of honey bee larvae to a homogeneous elec-
tromagnetic field was carried out in a GTEM cell as
Short-term exposure to mobile phone radiofrequency on honey bee larvae 431
previously described (Tkalec et al., 2005). The larvae
were exposed to RF-EMF at 900 MHz and field levels of
10, 23, 41 and 120 V m
1
for 2 h. Corresponding power
flux densities were 0.3, 1.4, 4.2 and 38.2 W m
2
, respec-
tively. At field level of 23 V m
1
the effect of 80% AM
1 kHz sinusoidal and 217 Hz modulation was investi-
gated as well. The measurements lasted for a week.
One exposure session was displayed over the day i.e.,
one exposure condition lasted 2 h and was repeated
two times. For each exposure condition eight Petri
dishes (PD) with four larvae each were used. Of the
eight exposed PD, 6 PD were used for the analysis of
oxidative stress parameters and 2 PD for genotoxicity
analysis. For each exposure condition PD were placed
at the center of the GTEM cell, in the same plane, but
perpendicular to the electric field (Tkalec et al., 2005;
Tkalec et al., 2009). The area where Petri dishes were
placed had the most uniform field distribution
( ± 0.1 dB) as measured with the electric probe (Hola-
day HI-4455) and verified with the finite element
method (Malaric
´, Bartolic
´, & Malaric
´,2005). Unexposed
larvae kept in the GTEM cell under the same conditions
as the experimental groups but without exposure to RF
radiation were used as a control. For control, three
repetitions with in total 14 PD were done and as no
statistical difference among repetitions was found for
any investigated parameters we pooled the results of
control groups together. The exposure was performed
at the microwave laboratory of the Faculty of Electrical
Engineering and Computing in Zagreb at the room tem-
perature of 23 ± 1 ˚C. The temperature measurement
in the GTEM cell was done on the surface of the larva
prior to and immediately after the exposure with K2 K/J
Thermometer, Fluke. The difference in temperature
which was measured immediately before and after the
exposure was less than 0.1 ˚C.
Oxidative stress parameters
Tissue extracts were prepared from a composite sample
of four larvae from one Petri dish. Tissue was homoge-
nized in cold 50 mM potassium phosphate buffer (pH
7.0) containing 0.5 mM EDTA with TissueLyser II (QIA-
GEN) for 60 s at 15 Hz. The homogenate was cen-
trifuged twice at 15,000 g for 15 min at 4 ˚C. The
protein concentration in the supernatant was deter-
mined according to Bradford (1976), using bovine serum
albumin as a standard. Obtained supernatants were used
for biochemical assays.
The level of lipid peroxidation was measured as the
formation of thiobarbituric acid reactive substance
(TBARS), a by-product of lipid peroxidation that reacts
with thiobarbituric acid (Legeay, Achard-Joris, Baudri-
mont, Massabuau, & Bourdineaud, 2005). Supernatants
(300 μl) were mixed with 200 μl of cold 20% (w/v)
trichloracetic acid (TCA) to precipitate proteins. The
precipitate was pelleted by centrifugation (10,000 g for
15 min at 4 ˚C) and the obtained supernatant was
reacted with 400 μl of 1% (w/v) thiobarbituric acid pre-
pared in 20% TCA. After heating at 95 ˚C for 30 min,
the mixture was cooled in an ice bath. The absorbance
of the supernatant was measured at 532 nm and correc-
tion for unspecific turbidity was done by subtracting the
absorbance at 600 nm. The content of TBARS was cal-
culated using an extinction coefficient of
155 mM
1
cm
1
and expressed per mg of proteins.
Catalase (CAT) activity (EC 1.11.1.6) was assayed by
measuring the decrease in absorbance at 240 nm
(ε=36mM
1
cm
1
) according to Aebi (1984). The reac-
tion mixture consisted of 50 mM potassium phosphate
buffer (pH 7.0), 10 mM H
2
O
2
and 25 μl of sample. CAT
activity was expressed in units per mg of proteins. One
unit was defined as the amount of enzyme that hydro-
lyzes 1 μmol of H
2
O
2
per minute, at 25 ˚C and pH 7.0.
Glutathione S-transferase (GST) activity (EC 1.8.1.7)
was determined at 340 nm using 1-chloro-2,4-dini-
trobenzene (CDNB) according to a modified method of
Bocchetti & Regoli (2006). The reaction mixture con-
tained 100 mM potassium phosphate buffer (pH 6.5),
2 mM CDNB, 2.5 mM glutathione (GSH), and 50 μlof
sample. GST activity was expressed in units per mg of
proteins, where one unit is defined as the amount of
enzyme producing 1 μmol of GS-DNB conjugate per
min under the conditions of the assay.
The activity of superoxide dismutase (SOD) (EC
1.15.1.1) was assayed by the xanthine oxidase/cy-
tochrome cmethod modified according to McCord and
Fridovich (1969). The reaction mixtures contained
0.01 mM cytochrome c and 0.5 mM xanthine in 50 mM
potassium phosphate buffer (pH 7.8) with 0.1 mM
EDTA. Reactions were started by adding xanthine oxi-
dase in an amount sufficient to cause change in the
absorbance of 0.025 per min. One unit of SOD inhibits
the rate of reduction of cytochrome c by 50% in a cou-
pled system, using xanthine and xanthine oxidase at pH
7.8 at 25 ˚C.
Comet assay
The alkaline Comet assay (single cell gel electrophoresis
assay) was performed according to the basic procedure
of Singh, McCoy, Tice, and Schneider (1988) with slight
modifications. To obtain the cell suspension, each larva
was placed in 1.5 ml Eppendorf tubes and dilacerated
with a Potter–Elvehjem tissue homogenizer (Braun Bio-
tech, Sartorius, Goettingen, Germany) in 500 μlof
phosphate buffer saline (PBS; 1.45 M NaCl, 60 mM
Na
2
HPO
4
,40mMKH
2
PO
4
; pH 7.0). The homogenates
obtained in this manner were filtered through a 70 μm
sieve, centrifuged at 200 g for 10 min at 4 ˚C, resus-
pended in 400 μl of PBS, centrifuged again at 180 g for
10 min at 4 ˚C and finally resuspended in 60 μl of PBS.
50 μl of a cell suspension was mixed with 50 μl of 0.5%
low melting point (LMP) agarose and transferred to
432 M. Vilic
´et al.
microscope slides precoated with 1% normal melting
point (NMP) agarose. After solidification for 2.5 min in
a freezer, a third layer consisting of 80 μl of 0.5% LMP
agarose was added and left to solidify as described
above. The cells were lysed in freshly made lysis solu-
tion (2.5 M NaCl, 100 mM ethylenediaminetetraacetic
acid (EDTA), 10 mM Tris–HCl, 10% dimethyl sulfoxide,
1% Triton X-100, pH 10) for 1 h at 4 ˚C. After rinsing
with redistilled water, the slides were placed in a hori-
zontal gel box, covered with the cold alkaline buffer
(0.3 M NaOH, 1 mM EDTA pH > 13) and left for
15 min. Electrophoresis was performed in the same buf-
fer at 35 V (1.16 V cm
1
) and 300 mA for 15 min at
4 ˚C. After electrophoresis, the slides were neutralized
in cold neutralization buffer (0.4 M Tris–HCl, pH 7.5)
for 2 ×5 min, then fixed in methanol: Acetic acid (3:1)
for 5 min and stored in the dark at room temperature.
Prior to examination, the slides were rehydrated,
stained with 10 μg/ml ethidium bromide and examined
using a Zeiss Axioplan epifluorescence microscope. For
each slide 100 nuclei were analyzed. The extent of
DNA migration was determined as the percentage of
DNA in the tail (% tDNA) using the Comet five image
analysis system (Kinetic Imaging Ltd.; UK).
Statistical analysis
All results were expressed as means followed by corre-
sponding standard errors (SE). The oxidative stress
parameter analysis was made from a composite sample
of four larvae from one Petri dish and in total there
were 6 PD for each exposure group (n= 6) and 12 PD
for control group (n= 12). Exception was exposure
group at 120 V m
1
for TBARS measurement in which
we only analyzed 4 PD (n= 4). Comet test was made
on each larva and in total there were 2 PD with four
larvae each, for each exposure and control group
(n= 8). Statistical analysis was performed using Statistica
12 (StatSoft, Inc.; USA) software package. After testing
for normal distribution (Kolmogorov–Smirnov test of
normality), the results were tested by the analysis of
variance (ANOVA) to determine the differences
between the groups and multiple comparisons between
means were determined by Tukey HSD test. Statistical
difference was considered significant at p< 0.05. The
ANOVA result is reported as an F-statistic and its asso-
ciated degrees of freedom and p-value.
Results
Oxidative stress parameters
The GST activity in the honey bee larvae exposed to
unmodulated RF-EMF at frequency of 900 MHz and field
levels of 10, 23, 41, 120 V m
1
was not statistically different
when compared to the control (Figure 1). The lowest GST
activity was measured in the larvae exposed to modulated
(80% AM 1 kHz sinus) field at 23 V m
1
, and it was signifi-
cantly lower (p<0.05, F
(6,41)
= 2.435) than in the larvae
exposed to unmodulated field at 23 V m
1
(Figure 1).
The CAT activity was significantly decreased
(p< 0.05, F
(6,41)
= 3.416) in the honey bee larvae
exposed to unmodulated RF-EMF at field level of
10 V m
1
when compared to control group, as well as
with group exposed to unmodulated RF-EMF at field
level of 23, 41, 120 V m
1
and modulated (217 Hz) field
at 23 V m
1
(Figure 2).
The SOD activity in the honey bee larvae exposed to
RF-EMF at frequency of 900 MHz and field levels of 10,
23, 41, 120 V m
1
was not statistically different compared
Figure 1. GST activity in the honey bee larvae exposed to unmodulated RF-EMFs at 900 MHz and field levels of 10, 23, 41,
120 V m
1
, as well as modulated field (80% AM 1 kHz and 217 Hz) of 23 V m
1
for 2 h. Results are presented as mean ± SE (n=6
for exposure groups and n= 12 for control). Columns with different letters are significantly different according to the Tukey HSD
test at p< 0.05.
Short-term exposure to mobile phone radiofrequency on honey bee larvae 433
to control (Figure 3). However, the lowest SOD activity
was measured in the larvae exposed to unmodulated RF-
EMF at field level of 10 V m
1
and it was significantly
lower (p< 0.05, F
(6,41)
= 2.789) than in the larvae
exposed to a modulated (217 Hz) field at 23 V m
1
.
The TBARS concentration was significantly
decreased (p< 0.05, F
(6,39)
= 3.155) in honey bee larvae
exposed to unmodulated RF-EMF at a field level of
10 V m
1
when compared to the control group
(Figure 4). Although the content of TBARS in all other
exposed groups was lower than in the control
group, there was no significant difference between the
groups.
Comet assay
DNA damage was significantly increased (p< 0.05,
F
(6,49)
= 17.304) in honey bee larvae exposed to modu-
lated (80% AM 1 kHz sinus) field at 23 V m
1
in com-
parison to the control and all other exposure groups
(Figure 5). Other treatments did not trigger significant
DNA damage in comparison to the control.
Figure 2. CAT activity in the honey bee larvae exposed to unmodulated RF-EMFs at 900 MHz and field levels of 10, 23, 41,
120 V m
1
, as well as modulated field (80% AM 1 kHz and 217 Hz) of 23 V m
1
for 2 h. Results are presented as mean ± SE (n=6
for exposure groups and n= 12 for control). Columns with different letters are significantly different according to the Tukey HSD
test at p< 0.05.
Figure 3. SOD activity in the honey bee larvae exposed to unmodulated RF-EMFs at 900 MHz and field levels of 10, 23, 41,
120 V m
1
, as well as modulated field (80% AM 1 kHz and 217 Hz) of 23 V m
1
for 2 h. Results are presented as mean ± SE (n=6
for exposure groups and n= 12 for control). Columns with different letters are significantly different according to the Tukey HSD
test at p< 0.05.
434 M. Vilic
´et al.
Discussion
Our results demonstrate that 2 h-exposure to RF-EMF
at 900 MHz and field levels of 10, 23, 41, 120 V m
1
as
well as modulated field of 23 V m
1
, with the modula-
tion of 217 Hz or 80% AM 1 kHz sinus, induced alter-
ations of the antioxidant enzymes activity and lipid
peroxidation level as well as DNA damage in the honey
bee larvae. It was interesting that effects of RF-EMF
were observed only under certain conditions; they did
not follow a linear dose-response relationship, and they
strictly depended on the measured parameters, field
levels and modulation.
Although results of many studies on mammals show
a significant increase of antioxidant enzyme activity as
well as DNA damage and intensity of lipid peroxidation,
after exposure to short-term RF-EMF (see review Yaky-
menko et al., 2016), the present results could hardly be
compared with them. Firstly, it is generally known that
antioxidant protection against oxidative stress is related
to phylogenetic groups, and it differs between mammals
Figure 4. TBARS concentration in the honey bee larvae exposed to unmodulated RF-EMFs at 900 MHz and field levels of 10, 23,
41, 120 V m
1
, as well as modulated field (80% AM 1 kHz and 217 Hz) of 23 V m
1
for 2 h. Results are presented as mean ± SE
(n= 6 for exposure groups, except for group 120 V m
1
where n= 4, and n= 12 for control). Columns with different letters are
significantly different according to the Tukey HSD test at p< 0.05.
Figure 5. DNA damage (% tDNA) in the honey bee larvae exposed to unmodulated RF-EMFs at 900 MHz and field levels of 10,
23, 41, 120 V m
1
, as well as modulated field (80% AM 1 kHz and 217 Hz) of 23 V m
1
for 2 h. Results are presented as mean ± SE
(n= 8). Columns with different letters are significantly different according to the Tukey HSD test at p< 0.05.
Short-term exposure to mobile phone radiofrequency on honey bee larvae 435
and invertebrates (Nikolenko, Saltykova, & Gaifullina,
2011). The second reason relates to the contradictory
results that have been published on the antioxidant
enzyme activity and level of lipid peroxidation after
exposure to RF-EMF. Some authors reported increased
activity of antioxidant enzymes and lipid peroxidation
level after exposure to RF-EMF (Gu¨ler et al., 2012; Khi-
razova et al., 2012; Ozgur et al., 2013; Yurekli et al.,
2006), some reported a decrease in these parameters
(Akbari, Jelodar, & Nazifi, 2014; Jelodar, Nazifi, &
Akbari, 2013) or no RF-EMF effects (Avci, Akar, Bilgici,
& Tunc¸el, 2012; Dasdag et al., 2003; Shehu, Mohammed,
Magaji, & Muhammad, 2016; Stronati et al., 2006; Zeni
et al., 2005), while in some studies antioxidant enzymes
showed variation in their activity (Ayata, Mollaoglu, &
Yilmaz, 2004; Balci, Devrim, & Durak, 2007;Tu¨redi
et al., 2015).
The results of the present study indicate that a short-
term exposure of honey bee larvae to RF-EMF at
900 MHz caused a different response of investigated
parameters.The results could be divided into three
groups: (i) decreased level of some parameters after
exposure to the unmodulated RF-EMF (i.e., the CAT
activity and TBARS content were lower in larvae
exposed to the unmodulated RF-EMF at 10 V m
1
than in
the control; (ii) decreased level of some parameters at
the modulated RF-EMF compared to the unmodulated
field (i.e., the GST activity in larvae exposed to the mod-
ulated (80% AM 1 kHz sinus) RF-EMF at 23 V m
1
was
lower than in those exposed to the unmodulated field at
23 V m
1
); and (iii) increased level of some parameters at
the modulated RF-EMF compared to the unmodulated
field (i.e., DNA damage was increased in larvae exposed
to the modulated (80% AM 1 kHz sinus) RF-EMF com-
pared to the unmodulated field and the control whereas
SOD and CAT activities were increased at the
modulated (217 Hz) RF-EMF at 23 V m
1
compared to
the unmodulated field at 10 V m
1
. At the moment the
reason for such behavior is not clear. Our results are not
in accordance with the literature data on the same oxida-
tive stress parameters after short-term exposure to
unmodulated RF-EMF at 900 MHz in earthworms Eisenia
fetida (Tkalec et al., 2013). These authors showed that
CAT activity and lipid peroxidation level increased signifi-
cantly in earthworms after exposure for 2 h to RF-EMF
of 23 and 120 V m
1
. We suppose that the decreased
activity of CAT (as well as level of TBARS) observed in
the five to six days old honey bee larvae after 2 h expo-
sure to unmodulated RF-EMF of 10 V m
1
could be due
to the function of this enzyme in cell defense from oxida-
tive stress. It has been known that RF-EMF could, even at
low intensity, induce overproduction of reactive oxygen
species (Burlaka et al., 2013), which in honey bees can be
scavenged by SOD, CAT and GST, the most important
antioxidative enzymes. Farjan et al. (2012) showed that
four to six day old A. mellifera larvae have higher activity
of CAT and GST than SOD under physiological condi-
tions and that enzyme activities change during larvae
development; the activities of SOD, CAT and GST
increase slightly from day 1–6, but then decrease after
day six to the end of honey bee development. Among
these enzymes CAT activity shows the highest decrease
during the development. Therefore, according to the
CAT development profile during larval stage and the fact
that CAT is the most important enzyme decomposing
hydrogen peroxide in honey bee brood (Farjan et al.,
2012) we assume that honey bee larvae were more sen-
sitive to the exposure to the EMF level of 10 V m
1
than
to the other field levels.
This finding corresponds well with previously
reported experimental evidence on non-linear dose-re-
sponse relationship between EMF exposure and biologi-
cal effect (Panagopoulos, Johansson, & Carlo, 2013). For
instance, biological effects such as DNA damage could
show stronger effects at lower field levels of RF-EMF
than at higher field levels (Panagopoulos et al., 2010).
Specifically, several studies reported on the regions of
increased bioactivity called “windows” in which the bio-
logical effects reach a maximum compared to the effects
at smaller or larger values of a physical parameter like
the intensity or frequency of the radiation (Panagopou-
los et al., 2013 and references therein).
Our results showed that only the exposure of honey
bee larvae to modulated field (80% AM 1 kHz sinus) at
23 V m
1
led to the increase in DNA damage. Parallel
with that, there was a decrease in GST activity indicating
negative effect of modulated EMF on oxidative status in
honey bee larvae. Similar increase in DNA damage after
exposure to modulated field at 23 V m
1
was previously
reported in E. fetida earthworms (Tkalec et al., 2013). It
has already been reported that frequency modulation can
have significant effect and fields of the same specific
absorption rate (SAR) but of different carrier or modula-
tion frequencies produce different biological effects (dis-
cussed in Panagopoulos et al., 2013). However, it is
important to emphasize that RF-EMF modulated at
217 Hz, which is used in GSM communication, did not
cause any statistically significant changes in investigated
parameters when compared to the control group.
Concerning the DNA damage caused by RF-EMF,
controversial findings have been reported after in vivo
and in vitro exposures to various radiofrequency signals.
While some studies reported DNA damage or cell dam-
age induced by mobile telephony or similar RF radia-
tions at non-thermal intensity levels others did not find
such connection (reviewed in Panagopoulos & Margari-
tis, 2008; Miyakoshi, 2013). Although inconsistent, the
data imply that certain conditions of exposure to RF-
EMF can exert genotoxic properties. One plausible
mechanism for RF-EMF-induced DNA damage is free
radical damage (Burlaka et al., 2013; Phillips, Singh, &
Lai, 2009). Particularly, an extremely low frequency field
(ELF) as well as RF-EMF modulated by ELF can act as a
moderate damaging agent causing mild oxidative stress
responsible for DNA damage (Mihai, Rotinberg, Brinza,
& Vochita, 2014).
436 M. Vilic
´et al.
In conclusion, the results of our study showed that
effects of RF-EMF at 900 MHz in honey bee larvae
appeared only after exposure to the certain EMF condi-
tions. RF-EMF modulated at 1 kHz showed an increase
of DNA damage, while unmodulated RF-EMF produced
alteration in catalase activity and lipid peroxidation at
the lowest field level of 10 V m
1
. Evidently, the
increase of the field level did not cause a linear dose-re-
sponse relationship in any of the measured parameters.
Although honey bees in nature would not be
exposed to such high field levels as used in our experi-
ments, our results show the need for further intensive
research in all stages of honey bee development, as well
as the intensive research on the possible existence of a
“window” effect under natural conditions during the
annual cycling of bees.
Acknowledgements
The authors wish to thank the participating beekeeper for
sampling collections.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This work was supported by University of Zagreb, Republic of
Croatia [grant number BM1.77].
ORCID
Ivana Tlak Gajger http://orcid.org/0000-0002-4480-3599
Anamaria S
ˇtambuk http://orcid.org/0000-0002-3177-7694
Maja S
ˇrut http://orcid.org/0000-0002-3120-7843
Go¨ran Klobuc
ˇar http://orcid.org/0000-0002-0838-4593
Kres
ˇimir Malaric
´http://orcid.org/0000-0002-1255-6415
Ana Pavelic
´http://orcid.org/0000-0003-2612-7467
Marin Manger http://orcid.org/0000-0002-1644-6336
Mirta Tkalec http://orcid.org/0000-0003-4733-828X
References
Aebi, H. (1984). Catalase in vitro.Methods in Enzymology, 105,
121–126.
Akbari, A., Jelodar, G., & Nazifi, S. (2014). Vitamin C protects
rat cerebellum and encephalon from oxidative stress fol-
lowing exposure to radiofrequency wave generated by a
BTS antenna model. Toxicology Mechanisms and Methods,
24, 347–352.
Avci, B., Akar, A., Bilgici, B., & Tunc¸el, O
¨.K. (2012). Oxidative
stress induced by 1.8 GHz radio frequency electromagnetic
radiation and effects of garlic extract in rats. International
Journal of Radiation Biology, 88, 799–805.
Ayata, A., Mollaoglu, H., & Yilmaz, H.R. (2004). Oxidative
stress-mediated skin damage in an experimental mobile
phone model can be prevented by melatonin. The Journal
of Dermatology, 31, 878–883.
Aydin, B., & Akar, A. (2011). Effects of a 900-MHz electromag-
netic field on oxidative stress parameters in rat lymphoid
organs, polymorphonuclear leukocytes and plasma. Archives
of Medical Research, 42, 261–267.
Balci, M., Devrim, E., & Durak, I. (2007). Effects of mobile
phones on/antioxidant balance in cornea and lens of rats.
Current Eye Research, 32, 21–25.
Bocchetti, R., & Regoli, F. (2006). Seasonal variability of oxida-
tive biomarkers, lysosomal parameters, metallothioneins
and peroxisomal enzymes in the Mediterranean mussel
Mytilus galloprovincialis from Adriatic Sea. Chemosphere, 65,
913–921.
Bradford, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Analytical Biochemistry,
72, 248–254.
Burlaka, A., Tsybulin, O., Sidorik, E., Lukin, S., Polishuk, V.,
Tsehmistrenko, S., & Yakymenko, I. (2013). Overproduc-
tion of free radical species in embryonal cells exposed to
low intensity radiofrequency radiation. Experimental Oncol-
ogy, 35, 219–225.
Cucurachi, S., Tamis, W.L.M., Vijver, M.G., Peijnenburg,
W.J.G.M., Bolte, J.F.B., & de Snoo, G.R. (2013). A review
of the ecological effects of radiofrequency electromagnetic
fields (RF-EMF). Environment International, 51, 116–140.
Dasdag, S., Zulkuf Akdag, M., Aksen, F., Yılmaz, F., Bashan, M.,
Mutlu Dasdag, M., & Salih Celik, M. (2003). Whole body
exposure of rats to microwaves emitted from a cell phone
does not affect the testes. Bioelectromagnetics, 24, 182–188.
doi:10.1002/bem.10083
Esmekaya, M.A., Aytekin, E., Ozgur, E., Gu¨ler, G., Ergun, M.A.,
O
¨merog
˘lu, S., & Seyhan, N. (2011). Mutagenic and mor-
phologic impacts of 1.8 GHz radiofrequency radiation on
human peripheral blood lymphocytes (hPBLs) and possible
protective role of pre-treatment with Ginkgo biloba (EGb
761). Science of the Total Environment, 410, 59–64.
doi:10.1016/j.scitotenv.2011.09.036
Farjan, M., Dmitryjuk, M., Lipin
´ski, Z., Biernat-Łopien
´ska, E., &
Z
˙o
´łtowska, K. (2012). Supplementation of the honey bee diet
with vitamin C: The effect on the antioxidative system of Apis
mellifera carnica brood at different stages. Journal of Apicultural
Research, 51, 263–270. doi:10.3896/IBRA.1.51.3.07
Favre, D. (2011). Mobile phone-induced honey bee worker
piping. Apidologie, 42, 270–279.
Gu¨ler, G., Tomruk, A., Ozgur, E., Sahin, D., Sepici, A., Altan,
N., & Seyhan, N. (2012). The effect of radiofrequency radi-
ation on DNA and lipid damage in female and male infant
rabbits. International Journal of Radiation Biology, 88, 367–
373. doi:10.3109/09553002.2012.646349
ITU. (2016). Key ICT indicators for developed and developing coun-
tries and the world (totals and penetration rates). Retrieved
from http://www.itu.int/en/ITU-D/Statistics/Pages/stat/de
fault.aspx
Jelodar, G., Nazifi, S., & Akbari, A. (2013). The prophylactic
effect of vitamin C on induced oxidative stress in rat
testis following exposure to 900 MHz radio frequency
wave generated by a BTS antenna model. Electromagnetic
Biology and Medicine, 32, 409–416. doi:10.3109/15368378.
2012.735208
Khirazova, E.E., Baizhumanov, A.A., Trofimova, L.K., Maslova,
D.M.V., Sokolova, N.A., & Kudryashova, N.Y. (2012).
Effects of GSM-frequency electromagnetic radiation on
some physiological and biochemical parameters in rats. Bul-
letin of Experimental Biology and Medicine, 153, 817–820.
Kumar, N.R., Neha, R., & Preeti, K. (2013). Biochemical
changes in haemolymph of Apis mellifera L. Drone under
the influence of cell phone radiations. Journal of Applied and
Natural Science, 5, 139–141.
Kwee, S., & Raskmark, P. (1998). Changes in cell proliferation
due to environmental non-ionizing radiation: 2. Microwave
radiation. Bioelectrochemistry and Bioenergetics, 44, 251–255.
Short-term exposure to mobile phone radiofrequency on honey bee larvae 437
Legeay, A., Achard-Joris, M., Baudrimont, M., Massabuau, J.C.,
& Bourdineaud, J.P. (2005). Impact of cadmium contamina-
tion and oxygenation levels on biochemical responses in
the Asiatic clam Corbicula fluminea.Aquatic Toxicology, 74,
242–253.
Luukkonen, J., Hakulinen, P., Ma
¨ki-Paakkanen, J., Juutilainen, J.,
& Naarala, J. (2009). Enhancement of chemically induced
reactive oxygen species production and DNA damage in
human SH-SY5Y neuroblastoma cells by 872 MHz radiofre-
quency radiation. Mutation Research/Fundamental and Molec-
ular Mechanisms of Mutagenesis, 662, 54–58.
Malaric, K., Bartolic, J., & Malaric, R. (2005). Immunity mea-
surements of TV and FM/AM receiver in GTEM-cell. Mea-
surement, 38, 219–229.
Mall, P., & Kumar, Y. (2014). Effect of electromagnetic radia-
tions on brooding, honey production and foraging behavior
of European honey bees (Apis mellifera L.). African Journal of
Agricultural Research, 9, 1078–1085.
McCord, J.M., & Fridovich, I. (1969). Superoxide dismutase an
enzymic function for erythrocuprein (hemocuprein). Journal
of Biological Chemistry, 244, 6049–6055.
Mihai, C.T., Rotinberg, P., Brinza, F., & Vochita, G. (2014).
Extremely low-frequency electromagnetic fields cause
DNA strand breaks in normal cells. Journal of Environmental
Health Science & Engineering, 12, 15. doi:10.1186/2052-
336X-12-15
Miyakoshi, J. (2013). Cellular and molecular responses to
radio-frequency electromagnetic fields. Proceedings of the
IEEE, 101, 1494–1502.
Moustafa, Y.M., Moustafa, R.M., Belacy, A., Abou-El-Ela, S.H., &
Ali, F.H. (2001). Effects of acute exposure to the radiofre-
quency fields of cellular phones on plasma lipid peroxide
and antioxidase activities in human erythrocytes. Journal of
Pharmaceutical and Biomedical Analysis, 26, 605–608.
Nikolenko, A.G., Saltykova, E.S., & Gaifullina, L.R. (2011).
Molecular mechanisms of antioxidant protective processes
in honey bee Apis mellifera. In T. Farooqui & A.A. Farooqui
(Eds.), Oxidative stress in vertebrates and invertebrates (pp.
279–293). Hoboken, NJ: John Wiley & Sons.
Ozgur, E., Kismali, G., Guler, G., Akcay, A., Ozkurt, G., Sel, T.,
& Seyhan, N. (2013). Effects of prenatal and postnatal
exposure to gsm-like radiofrequency on blood chemistry
and oxidative stress in infant rabbits, an experimental
study. Cell Biochemistry and Biophysics, 67, 743–751.
Panagopoulos, D.J., Johansson, O., & Carlo, G.L. (2013). Evalu-
ation of specific absorption rate as a dosimetric quantity
for electromagnetic fields bioeffects. PLoS ONE, 8, e62663.
doi:10.1371/journal.pone.0062663
Panagopoulos, D.J., & Margaritis, L.H. (2008). Mobile telephony
radiation effects on living organisms. In A.C. Harper & R.V.
Buress (Eds.), Mobile telephones: Networks, applications and
performance (pp. 107–149). New York, NY: Nova Science.
Panagopoulos, D.J., & Margaritis, L.H. (2010). The identification
of an intensity ‘window’ on the bioeffects of mobile tele-
phony radiation. International Journal of Radiation Biology, 86,
358–366.
Phillips, J.L., Singh, N.P., & Lai, H. (2009). Electromagnetic fields
and DNA damage. Pathophysiology, 16, 79–88.
Ruediger, H.W. (2009). Genotoxic effects of radiofrequency
electromagnetic fields. Pathophysiology, 16, 89–102.
Sarimov, R., Malmgren, L.O.G., Markova, E., Persson, B.R.R., &
Belyaev, I.Y. (2004). Nonthermal GSM microwaves affect
chromatin conformation in human lymphocytes similar to
heat shock. IEEE Transactions on Plasma Science, 32,
1600–1608.
Sharma, V.P., & Kumar, N.R. (2010). Changes in honey bee
behavior and biology under the influence of cellphone radi-
ations. Current Science, 98, 1376–1378.
Shehu, A., Mohammed, A., Magaji, R.A., & Muhammad, M.S.
(2016). Exposure to mobile phone electromagnetic field
radiation, ringtone and vibration affects anxiety-like beha-
viour and oxidative stress biomarkers in albino wistar rats.
Metabolic Brain Disease, 31, 355–362.
Singh, N.P., McCoy, M.T., Tice, R.R., & Schneider, E.L. (1988).
A simple technique for quantitation of low levels of DNA
damage in individual cells. Experimental Cell Research, 175,
184–191.
Stronati, L., Testa, A., Moquet, J., Edwards, A., Cordelli, E., Vil-
lani, P., … Lloyd, D. (2006). 935 MHz cellular phone radia-
tion. An in vitro study of genotoxicity in human
lymphocytes. International Journal of Radiation Biology, 82,
339–346.
Tkalec, M., Malaric
´, K., Pavlica, M., Pevalek-Kozlina, B., & Vida-
kovic
´-Cifrek, Z
ˇ.(2009). Effects of radiofrequency electro-
magnetic fields on seed germination and root meristematic
cells of Allium cepa L. Mutation Research/Genetic Toxicology
and Environmental Mutagenesis, 672, 76–81.
Tkalec, M., Malaric
´, K., & Pevalek-Kozlina, B. (2005). Influence
of 400, 900, and 1900 MHz electromagnetic fields on
Lemna minor growth and peroxidase activity. Bioelectro-
magnetics, 26, 185–193.
Tkalec, M., Malaric
´, K., & Pevalek-Kozlina, B. (2007). Exposure
to radiofrequency radiation induces oxidative stress in
duckweed Lemna minor L. Science of the Total Environment,
388, 78–89.
Tkalec, M., S
ˇtambuk, A., S
ˇrut, M., Malaric
´, K., & Klobuc
ˇar,
G.I.V. (2013). Oxidative and genotoxic effects of 900 MHz
electromagnetic fields in the earthworm Eisenia fetida.Eco-
toxicology and Environmental Safety, 90, 7–12.
Tu¨redi, S., Hancı, H., Topal, Z., U
¨nal, D., Mercantepe, T.,
Bozkurt, I
˙., … Odacı E. (2015). The effects of prenatal
exposure to a 900-MHz electromagnetic field on the
21-day-old male rat heart. Electromagnetic Biology and Medi-
cine, 34, 390–397.
Verschaeve, L. (2009). Genetic damage in subjects exposed to
radiofrequency radiation. Mutation Research/Reviews in
Mutation Research, 681, 259–270.
Vijayalaxmi, & Prihoda, T.J. (2012). Genetic damage in human
cells exposed to non-ionizing radiofrequency fields: A
meta-analysis of the data from 88 publications
(1990–2011). Mutation Research/Genetic Toxicology and
Environmental Mutagenesis, 749, 1–16.
Yakymenko, I., Tsybulin, O., Sidorik, E., Henshel, D., Kyry-
lenko, O., & Kyrylenko, S. (2016). Oxidative mechanisms
of biological activity of low-intensity radiofrequency radia-
tion. Electromagnetic Biology and Medicine, 35, 186–202.
Yurekli, A.I., Ozkan, M., Kalkan, T., Saybasili, H., Tuncel, H.,
Atukeren, P., … Seker, S. (2006). GSM base station elec-
tromagnetic radiation and oxidative stress in rats. Electro-
magnetic Biology and Medicine, 25, 177–188.
Zeni, O., Romano
`, M., Perrotta, A., Lioi, M.B., Barbieri, R.,
d’Ambrosio, G., … Scarfı M.R. (2005). Evaluation of geno-
toxic effects in human peripheral blood leukocytes follow-
ing an acute in vitro exposure to 900 MHz radiofrequency
fields. Bioelectromagnetics, 26, 258–265.
438 M. Vilic
´et al.
... [7]. Mobile phone radiation may induce alterations in antioxidant enzyme activities and lipid peroxidation levels and cause DNA damage in the exposed larvae [8]. Such radiation significantly reduces the hatching ratio of adult queens whose larvae were exposed and may alter pupal development [9]. ...
... The investigations in the former category (e.g., [7,12]) study the effects of ambient EMR on various aspects of honey bee colonies by measuring ambient EMR with sensors without any structural modifications of hives, placement of sensors on bees, or placement of EMR sources (e.g., smartphones) directly into hives. The latter category (e.g., [5,[8][9][10][11]) includes studies that modify the hive or the bee, introduce EMR into the habitat, or expose individual bees extracted from a hive to artificially induced electromagnetic fields in the laboratory. ...
Article
Full-text available
Since bee traffic is a contributing factor to hive health and electromagnetic radiation has a growing presence in the urban milieu, we investigate ambient electromagnetic radiation as a predictor of bee traffic in the hive’s vicinity in an urban environment. To that end, we built two multi-sensor stations and deployed them for four and a half months at a private apiary in Logan, Utah, U.S.A. to record ambient weather and electromagnetic radiation. We placed two non-invasive video loggers on two hives at the apiary to extract omnidirectional bee motion counts from videos. The time-aligned datasets were used to evaluate 200 linear and 3,703,200 non-linear (random forest and support vector machine) regressors to predict bee motion counts from time, weather, and electromagnetic radiation. In all regressors, electromagnetic radiation was as good a predictor of traffic as weather. Both weather and electromagnetic radiation were better predictors than time. On the 13,412 time-aligned weather, electromagnetic radiation, and bee traffic records, random forest regressors had higher maximum R2 scores and resulted in more energy efficient parameterized grid searches. Both types of regressors were numerically stable.
... In our previous study (VILIĆ et al., 2017), we demonstrated that exposure of honey bee larvae to RF-EMF in Petri dishes only caused a change in the activity of antioxidant enzymes and DNA damage under certain conditions of RF-EMF field strength and modulation. Specifically, we found that a modulated (80% AM 1 kHz sinus) electric field, with strength at 23 Vm -1 , increases DNA damage in honey bee larvae. ...
... Namely, it is known that CAT is the most important enzyme decomposing hydrogen peroxide in honey bee broods (FARJAN et al., 2012). However, in our previous study (VILIĆ et al., 2017) we found that the same exposure conditions (modulated RF-EMF at 23 V m −1 ) increased DNA damage in honey bee larvae in comparison to the control group, while antioxidative enzyme activity remained at the control level. Differences in the observed effects could be attributed to variations in the manipulation of the larvae. ...
Article
Full-text available
Exposure to radiofrequency electromagnetic fields (RF-EMF) at the operating frequencies of different communication devices can cause various biological effects. However, there is a lack of studies on the oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. In this study, we investigated the oxidative stress and DNA damage in honey bee larvae situated in waxcomb cells, exposed to modulated RF-EMF 23 Vm-1. The glutathione S-transferase activity decreased, whereas the catalase activity increased significantly in the honey bee larvae upon RF-EMF exposure. Superoxide dismutase activity, the level of lipid peroxidation, and DNA damage were not statistically altered in exposed honey bee larvae when compared to the control group. These results suggest that the biological effects of modulated RF-EMF in honey bee larvae depend on the exposure design.
... In the multi-stress site, we wanted to evaluate the cumulative effects of two different stress factors: the exposure to pesticides (the same as the chemical site) and the presence of an electromagnetic field generated by an electric transport line located above the experimental hives. Electromagnetic fields are known to cause different biological effects such as oxidative stress, genotoxic effects and immune system dysfunctions, all observed on different animal species [109]. The negative effects of electromagnetic radiation emitted by antennas, cell phones and high voltage power lines have been studied in humans [110][111][112][113] and in animals, including mice [114], bats [115], birds [116,117] and insects [118]. ...
... The negative effects of electromagnetic radiation emitted by antennas, cell phones and high voltage power lines have been studied in humans [110][111][112][113] and in animals, including mice [114], bats [115], birds [116,117] and insects [118]. On bees, both electromagnetic fields generated by cell phones [109,119,120] and those generated by high voltage electricity transport lines [121] were studied. In this study, the cumulative effect of chemical and electromagnetic field exposure (multi-stress conditions) showed the worst general health condition, considering colony survival, pathology emergence and behavioural anomalies such as abnormal honey storage and excess of drone brood deposition. ...
Article
Full-text available
Honeybee and general pollinator decline is extensively reported in many countries, adding new concern to the general biodiversity loss. Many studies were addressed to assess the causes of pollinator decline, concluding that in most cases multi-stress effects were the most probable ones. In this research, the combined effects of two possible stress sources for bees, pesticides and electromagnetic fields (multi-stress conditions), were analyzed in the field. Three experimental sites were chosen: a control one far from direct anthropogenic stress sources, a pesticide-stress site and multi-stress one, adding to the same exposure to pesticides the presence of an electromagnetic field, coming from a high-voltage electric line. Experimental apiaries were monitored weekly for one year (from April 2017 to April 2018) by means of colony survival, queen activity, storage and brood amount, parasites and pathogens, and several biomarkers in young workers and pupae. Both exposure and effect biomarkers were analysed: among the first, acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (ALP) and Reactive Oxygen Species (ROS); and among the last, DNA fragmentation (DNAFRAGM) and lipid peroxidation (LPO). Results showed that bee health conditions were the worst in the multi-stress site with only one colony alive out of the four ones present at the beginning. In this site, a complex picture of adverse effects was observed, such as disease appearance (American foulbrood), higher mortality in the underbaskets (common to pesticide-stress site), behavioral alterations (queen changes, excess of honey storage) and biochemical anomalies (higher ALP activity at the end of the season). The overall results clearly indicate that the multi-stress conditions were able to induce biochemical, physiological and behavioral alterations which severely threatened bee colony survival.
... We noted differences in the levels of neuropeptide mRNAs in the ticks' synganglion after exposure to electromagnetic radiation with different intensities. Previous studies on drosophila and honeybees have reported different effects of radiofrequency radiation on the organism depending on the radiation parameters used as well [44,[46][47][48]. Modulation, pulsed fields, intensity, and specific absorption rate have been cited as factors that enhance the biological effects of exposure [49]. ...
Article
Full-text available
Anthropogenic electromagnetic radiation is an important environmental factor affecting the functionality of biological systems. Sensitivity to various frequencies of electromagnetic radiation has been detected in ixodid ticks in the past. However, the physiological aspects of radiation effects have not yet been studied in ticks. In the presented experiment, 360 Ixodes ricinus ticks, 180 males and 180 females, were divided into 16 irradiated and 8 control groups. The irradiated groups were exposed to two different intensities of electromagnetic radiation with a frequency of 900 MHz at different lengths of exposure time. RT-PCR was utilized to determine the changes in mRNA levels in tick synganglia after irradiation. Four randomly selected neuropeptide genes were tested-allatotropin (at), FGLa-related allatostatins (fgla/ast), kinin, and arginine-vasopressin-like peptide (avpl). A significant decrease in transcript levels in all female groups exposed to higher intensity radiofrequency radiation for 1 to 3 h was found. After one hour of radiofrequency exposure, a significant downregulation in allatotropin expression in males was detected. A consistent downreg-ulation of the at gene was detected in males irradiated with at a higher intensity. Unfortunately, the specific functions of the studied neuropeptides in ticks are not known yet, so a more comprehensive study is necessary to describe the effects of EMF on observed neuropeptides. This study represents the first report on the effects of the abiotic environment on tick neurophysiology.
... By-products from pesticide manufacturing industries and residues in the environment after use have been blamed for persistent, long-lasting, and dreadful human ailments, either because of consumption through food and water or exposure on skin and eyes. It is a proven fact that most synthetic chemicals are nonbiodegradable and therefore cause pollution and contamination to soil, water, and air besides depletion of the ozone layer and a reduction in ozone thickness, with the consequence of harmful radiation (TlakGajger et al., 2019b;Vilic et al., 2017;Žura Žaja et al., 2021) reaching the surface of the earth, which strongly influences agricultural production, animal health, and vegetation. The blind application of synthetic pesticides has resulted in an unfortunate association with several burning issues in the environment and have therefore agitated and urged a call for alternative pest management strategies. ...
Chapter
Full-text available
Primary agriculture alone is not enough to sustain our agrarian economy, so secondary agriculture needs to follow up. Not only does it add value to the primary agricultural commodities but also increases their use efficiency. Moreover, it promotes entrepreneurship and makes the venture more economical. In India for years 2017–2018, 516 million tonnes of crop residues were generated, creating a huge pressure on its disposal. This chapter attempts to highlight the prospects of crop residues in secondary agriculture as a fortified animal feed, bioactive compost, vermicompost, energy source, biochar, use in mushroom cultivation, mulching, packaging and mat making. Greater incentives, technical know-how, machinery, technologies and marketing are needed to exploit the full potential of crop residues as a novel enterprise for secondary agriculture. Secondary agriculture is the need of the hour especially for the Indian scenario to benefit the farmers, already suffering due to the changing government policies and bills.
... The present study aimed to demonstrate the effect of the electromagnetic field at 50 Hz with different levels of 250, 500 & 1000 mT on the oxidative stress parameters and the morphological features of the C. pipiens larvae. No references had previously demonstrated the effect of the extremely low frequency (50 Hz) of EMF on the C. pipiens but many authors had studied the effect of the extremely low frequency of EMF on oxidative stress and DNA damage for other insects like (Maliszewska et al., 2018;Migdał et al., 2020;Vilić et al., 2017) who found that the electromagnetic field induced alterations of the antioxidant enzyme activity and lipid peroxidation level as well as DNA damage in the honeybee larvae, American cockroach, honeybee workers, respectively. ...
Article
The electromagnetic field (EMF) is a vital issue in research, but its use as an alternative technique for insect management is still in its beginnings. The study aimed to evaluate the effects of exposure to the electromagnetic field on the biochemical and morphological changes in Culex pipiens. Therefore, the third instar larvae were exposed to EMF frequency (50 Hz) at different intensities (250, 500, 1000 mT) for 1h during four successive days. After end exposure to EMF, the biochemical analysis, oxidative stress parameters, body weight, and morphological changes were investigated. The results showed that larval body weight and total protein content were decreased significantly in all treated groups compared to controls. The total lipid content significantly decreased at 500 & 1000 mT exposure, while the treatment group exposed to EMF at 250 mT was not statistically different compared to control. At a high intensity of EMF 1000 mT all oxidative stress parameters; catalase (CAT) and Glutathione S-transferase (GST) activity and lipid peroxidation level (MDA) were decreased significantly compared to the control. On the other hand, at a low intensity (250 mT), CAT and GST activity was significantly increased, while MDA content was not statistically changed. Scanning electron microscopy showed that both 500 & 1000 mT caused distinct malformations to the larval body parts, mouthparts, thorax and abdomen with last abdominal structures. In conclusion, the results demonstrated that EMF exposure is a considerable stress factor that affects oxidative state and morphological features in mosquitoes and give hope that EMF will be used as an alternative method for Culex pipiens vector control.
Article
Urbanization and the increasing use of wireless technologies lead to higher emission rates of radiofrequency electromagnetic fields (RF-EMF) in populated areas. This anthropogenic electromagnetic radiation is a form of environmental pollution and a potential stressor on bees or other flying insects. Cities often have a high density of wireless devices operating on microwave frequencies, which generate electromagnetic frequencies e.g. in the 2.4 and 5.8 GHz bands commonly used by the wireless technologies. To date the effects of nonionizing electromagnetic radiation on the vitality and behavior of insects are poorly understood. In our experiment we used honey bees as model organisms and analyzed the effects of defined exposures to 2.4 and 5.8 GHz on brood development, longevity and homing ability under field conditions. To generate this radiation, we used a high-quality radiation source which generates a consistent, definable and realistic electromagnetic radiation, engineered for this experiment by the Communications Engineering Lab (CEL) at the Karlsruhe Institute of Technology. Our results show significant effects of long-term exposures on the homing ability of foraging honey bees, but no effects on brood development and adult worker longevity. Using this novel and high-quality technical set-up, this interdisciplinary work provides new data on the effects of these widely used frequencies on important fitness parameters of free-flying honey bees.
Article
Background and Objective: Electromagnetic fields coming from electric and electronic devices, mobile telephony antennas, or electrical installations are continuously growing and are in direct relation with population growth. In that sense, the purpose of this investigation was to determine what are the effects of artificial electromagnetic fields on the behavior and viability of bees through a global perspective (1968-2022). Materials and Methods: The methodology used in this research consisted of the review of literature obtained from platforms such as Scopus, EBSCO, IEEE, Wiley, Google Scholar and Taylor & Francis. Results: It was possible to review 36 studies on the field and to state that investigations on this topic have increased in 2019, at a compounded annual growth rate (CAGR) of 6.86% (in a period of 54 years). Poland and USA are the leading countries in the number and importance of investigations on this topic. Keywords were grouped on the basis of the advancement of the research (honeybee, animals, Apis mellifera and apoideos). Conclusion: The study of the effects of electromagnetic fields on bees makes it possible to understand its impact on the metabolism and viability of bees.
Chapter
The crisis of food to feed the increasing human population diverted scientific attention concerning manufacture and advocacy of poisonous man-made chemicals as an immediate and an efficient way of controlling various biotic factors influencing agricultural production. Nevertheless, the overdependence on artificially manufactured pesticides is demoralized by their negative impacts on living systems and more owing to the phenomenon of resistant development in pest strains. The increasing urgency for organically safe foods invigorated the exploration for other safer strategies and biopesticides are especially gaining momentum to fill this research gap for better living. Biopesticides are more or less naturally available, efficacious, cheap, easily dissociated and disintegrated, and bestowed with various modes of action and activity for effective management of agriculture pests. Their low toxicity has attracted the attention of scientists for the benefits of living and to tackle climate change, besides sustaining prolonged food production in an eco-friendly manner. Owing to the diverse attributes and qualities of biopesticides plant protection experts are motivated based other research results to incorporate them into integrated pest management (IPM) systems so as to present to imperishable crop growth. The administration of biopesticides on insect larvae significantly delayed the ecdysis and molting processes, which are based on biopesticide dose. Plant extracts and oils have diverse mechanisms of actions and symptoms caused essentially comprise growth retardation, suppression, and inhibition. Botanical extracts are rapidly degraded in the environment, besides having non-accumulation and high dissociation properties; therefore, the chances of causing various types of pollutions are nil. Nonetheless, the biopesticides have not been endorse in depth owing to difficulties in preparation, extraction, and advertisement, which are accredited to the deficiency of chemical data, structure, adverse impacts, and practical pest control. This chapter addresses the biochemistry information of some chosen plant base pesticides, their breakdown, their role in IPM, and the hurdles facing their promotion, implementation, application, and consumption for prolonged agricultural crop pest management.
Article
In this paper, we review the literature on three important exposure metrics that are inadequately represented in most major radiofrequency radiation (RFR) exposure guidelines today: intensity, exposure duration, and signal modulation. Exposure intensity produces unpredictable effects as demonstrated by nonlinear effects. This is most likely caused by the biological system's ability to adjust and compensate but could lead to eventual biomic breakdown after prolonged exposure. A review of 112 low-intensity studies reveals that biological effects of RFR could occur at a median specific absorption rate of 0.0165 W/kg. Intensity and exposure duration interact since the dose of energy absorbed is the product of intensity and time. The result is that RFR behaves like a biological "stressor" capable of affecting numerous living systems. In addition to intensity and duration, man-made RFR is generally modulated to allow information to be encrypted. The effects of modulation on biological functions are not well understood. Four types of modulation outcomes are discussed. In addition, it is invalid to make direct comparisons between thermal energy and radiofrequency electromagnetic energy. Research data indicate that electromagnetic energy is more biologically potent in causing effects than thermal changes. The two likely functionthrough different mechanisms. As such, any current RFR exposure guidelines based on acute continuous-wave exposure are inadequate for health protection.
Article
Full-text available
The effects of Electromagnetic radiations (EMR) are being felt by wildlife and the environment as a whole, birds, bees, worms, trees are being affected. So the main focus of present study was carried out to analyse the influence of cell phone radiations on the biochemical aspects of drone of Apis mellifera L. The drone was exposed for 30 mins to radiations using live cell phones kept in working mode with tape recorder at the speaker end and positive response at the receiver's end. The results of the treatment were analyzed and compared with the control. The concentration of various biomolecules increased from 1.65 mg/ml to 2.75 mg/ml for carbohydrates , 3.74 mg/ ml to 4.85 mg/ml for proteins and from 0.325 mg/ml to 1.33 mg/ml for lipids under the influence of EMR.
Article
Full-text available
An enzyme which catalyzes the dismutation of superoxide radicals (O2·⁻ + O2·⁻ + 2H⁺ → O2 + H2O2) has been purified by a simple procedure from bovine erythrocytes. This enzyme, called superoxide dismutase, contains 2 eq of copper per mole of enzyme. The copper may be reversibly removed, and it is required for activity. Superoxide dismutase has been shown to be identical with the previously described copper-containing erythrocuprein (human) and hemocuprein (bovine). Stable solutions of the superoxide radical were generated by the electrolytic reduction of O2 in an aprotic solvent, dimethylformamide. Slow infusion of such solutions into buffered aqueous media permitted the demonstration that O2·⁻ can reduce ferricytochrome c and tetranitromethane, and that superoxide dismutase, by competing for the superoxide radicals, can markedly inhibit these reactions. Superoxide dismutase was used to show that the oxidation of epinephrine to adrenochrome by milk xanthine oxidase is mediated by the superoxide radical. An assay of several tissues indicates that superoxide dismutase is widely distributed within mammalian organisms.
Article
Full-text available
Research on the effects of Mobile phone radio frequency emissions on biological systems has been focused on noise and vibrations as auditory stressors. This study investigated the potential effects of exposure to mobile phone electromagnetic field radiation, ringtone and vibration on anxiety-like behaviour and oxidative stress biomarkers in albino wistar rats. Twenty five male wistar rats were randomly divided into five groups of 5 animals each: group I: exposed to mobile phone in switched off mode (control), group II: exposed to mobile phone in silent mode, group III: exposed to mobile phone in vibration mode, group IV: exposed to mobile phone in ringtone mode, group V: exposed to mobile phone in vibration and ringtone mode. The animals in group II to V were exposed to 10 min call (30 missed calls for 20 s each) per day for 4 weeks. Neurobehavioural studies for assessing anxiety were carried out 24 h after the last exposure and the animals were sacrificed. Brain samples were collected for biochemical evaluation immediately. Results obtained showed a significant decrease (P < 0.05) in open arm duration in all the experimental groups when compared to the control. A significant decrease (P < 0.05) was also observed in catalase activity in group IV and V when compared to the control. In conclusion, the results of the present study indicates that 4 weeks exposure to electromagnetic radiation, vibration, ringtone or both produced a significant effect on anxiety-like behavior and oxidative stress in young wistar rats.
Chapter
Full-text available
Evolutionary development by a honeybee of an ecological niche has defined a special role of molecular mechanisms of oxidative stress regulation in the processes defining preservation of a homeostasis by the separate individual and a honeybee colony. The balance of oxygen active forms and antioxidants is especially important in winter in the north of a specific area. High life span of winter generation is connected with influence of vitellogenin and hormonal balance on stability to free-radical oxidation that conducts to delay of aging of an organism. Control of toxic processes in intestines at accumulation of vital functions waste for half a year of non-flying period isn't less important. Change of hormonal balance during metamorphosis, occurrence of the phagocytes participating in histolysis processes, also is accompanied by oxidative explosion and an induction of its restraint molecular mechanisms. The oxidative stress is universal reaction of a honeybee organism to negative factors influence. In structure of a bee genome there aren’t enough the genes connected with innate immunity and a detoxification proteins. It is compensated by nonspecific reaction of phagocytes to negative environment influences and is accompanied by oxidative explosion with formation of melanin quinoid intermediates, superoxide anion, hydrogen peroxide, nitrogen oxide and peroxinitrite with high cytotoxic action. The system of free-radical oxidation control mechanisms is basic in formation of social insects resistance.
Article
Full-text available
This review aims to cover experimental data on oxidative effects of low-intensity radiofrequency radiation (RFR) in living cells. Analysis of the currently available peer-reviewed scientific literature reveals molecular effects induced by low-intensity RFR in living cells; this includes significant activation of key pathways generating reactive oxygen species (ROS), activation of peroxidation, oxidative damage of DNA and changes in the activity of antioxidant enzymes. It indicates that among 100 currently available peer-reviewed studies dealing with oxidative effects of low-intensity RFR, in general, 93 confirmed that RFR induces oxidative effects in biological systems. A wide pathogenic potential of the induced ROS and their involvement in cell signaling pathways explains a range of biological/health effects of low-intensity RFR, which include both cancer and non-cancer pathologies. In conclusion, our analysis demonstrates that low-intensity RFR is an expressive oxidative agent for living cells with a high pathogenic potential and that the oxidative stress induced by RFR exposure should be recognized as one of the primary mechanisms of the biological activity of this kind of radiation.
Article
Full-text available
The growing spread of mobile phone use is raising concerns about the effect on human health of the electromagnetic field (EMF) these devices emit. The purpose of this study was to investigate the effects on rat pup heart tissue of prenatal exposure to a 900 megahertz (MHz) EMF. For this purpose, pregnant rats were divided into experimental and control groups. Experimental group rats were exposed to a 900 MHz EMF (1 h/d) on days 13-21 of pregnancy. Measurements were performed with rats inside the exposure box in order to determine the distribution of EMF intensity. Our measurements showed that pregnant experimental group rats were exposed to a mean electrical field intensity of 13.77 V/m inside the box (0.50 W/m(2)). This study continued with male rat pups obtained from both groups. Pups were sacrificed on postnatal day 21, and the heart tissues were extracted. Malondialdehyde, superoxide dismutase and catalase values were significantly higher in the experimental group rats, while glutathione values were lower. Light microscopy revealed irregularities in heart muscle fibers and apoptotic changes in the experimental group. Electron microscopy revealed crista loss and swelling in the mitochondria, degeneration in myofibrils and structural impairments in Z bands. Our study results suggest that exposure to EMF in the prenatal period causes oxidative stress and histopathological changes in male rat pup heart tissue.