ACTA BOT. CROAT. 79 (2), 2020 193 Acta Bot. Croat. 79 (2), 193–200, 2020 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-022 ISSN 0365-0588 eISSN 1847-8476 Growth inhibition of the toxic cyanobacterium Cylindrospermopsis raciborskii by extremely low-frequency electromagnetic fields Zakaria Mohamed1*, Fadel Ali2, Medahat Abdel-Lateef3, Asmaa Hosny3 1 Sohag University, Faculty of Science, Department of Botany and Microbiology, Sohag, Egypt 2 Cairo University, Faculty of Science, Biophysics Department, Cairo, Egypt 3 Sohag University, Faculty of Science, Department of Physics, Sohag, Egypt Abstract – This study investigates the effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on the growth and antioxidant defence enzymes of the toxic cyanobacterium Cylindrospermopsis raciborskii (Wolo- szynska) Seenayya et Subba Raju. To determine resonance frequency of growth inhibition of C. raciborskii, cells were subjected to ELF square amplitude modulated waves (QAMW) with a range of frequencies (0.1, 0.3, 0.5, 0.7, 0.9 Hz) at single intensity of 100 V m–1 for 30 minutes. The results revealed that the highest growth inhibition of Cylindrospermopsis occurred upon exposure to 0.7 Hz QAMW for 30 min. ELF-EMF-exposed cultures exhibited a marked decrease in cell number, chlorophyll-a content and activity of antioxidant enzymes compared to control cultures, and this effect increased with the prolongation of exposure time. Moreover, ELF-EMF induced morpho- logical changes in Cylindrospermopsis cells upon exposure to 0.7 Hz QAMW for 120 min, including shrinking and disintegration of cytoplasmic contents, and thickening of the cell wall. Changes in dielectric properties, as a meas- ure of interaction of cellular constituents (e.g., plasma membrane, cell wall and cytoplasm), with electromagnetic fields were also observed for treated cells. Our results provide a new possibility for using ELF-EMFs to eliminate toxic cyanobacteria from drinking and recreational water sources. Keywords: cyanobacteria, Cylindrospermospsis, electromagnetic fields, growth inhibition, water treatment * Corresponding author e-mail: mzakaria_99@yahoo.com Introduction Environmental exposure to non-ionizing, non-thermal, extremely low frequency (<300 Hz) electromagnetic fields (ELF-EMF) is becoming increasingly widespread in many countries due to the frequent use of electric appliances, elec- tronic devices, communication systems and electric trans- mission lines (Zhu et al. 2016, Bodewein et al. 2019). Despite their extremely low frequency and low energy, ELF-EMFs have a number of different biological effects and bring about cellular changes (Sienkiewicz et al. 2005, Santini et al. 2009). Many studies have reported the effects of ELF-EMFs on the growth and cell functions of microorganisms including bac- teria (Inhan-Garip et al. 2011, Fadel et al. 2014, Oncul et al. 2016), yeasts (Malıkova et al. 2015) and fungi (Potenza et al. 2012). However, the results are still controversial. The inhib- itory or stimulatory effect of ELF-EMFs on microorganisms has been shown to be dependent on the strength and fre- quency of the electromagnetic field applied, and strain used (Justo et al. 2006). Therefore, many studies are required to evaluate the influence of different EMF frequencies and in- tensities on different species. However, little is known about the effects of ELF-EMFs on cyanobacteria (Fadel et al. 2018). Cyanobacteria are pro- karyotic oxygen-evolving photosynthetic microorganisms that live in a wide range of marine and freshwater habitats (Zanchett and Oliveira-Filho 2013). Although they serve as a food source for most organisms in the aquatic environment (Mohamed and Al-Shehri 2013), they represent an ecological problem when their numbers increase and they form harmful blooms under eutrophic conditions (i.e. high nutrient concen- trations) (Mohamed 2016a). These blooms can cause the for- mation of hypoxic dead zones in lakes, leading to the suffoca- tion of aquatic animals (Zhu et al. 2015). Furthermore, many MOHAMED Z, ALI F, ABDEL-LATEEF M, HOSNY A 194 ACTA BOT. CROAT. 79 (2), 2020 species of cyanobacteria can produce different kinds of toxins (hepatotoxins, neurotoxins, irritants and gastrointestinal tox- ins) that adversely affect animal and human health, particular- ly in drinking water sources (Codd et al. 2005). Such toxigenic species in drinking water sources can impair the water qual- ity (Dittmann and Wiegand 2006, Mohamed and Al-Sheh- ri 2009) and should thus be eliminated properly in drinking water treatment plants. Different conventional methods have been applied to remove cyanobacterial cells from drinking water including chlorination, coagulation, sand filtration and sonication (de la Cruz et al. 2011). However, the main draw- backs of these methods are that they are expensive, complex and cause cell lysis leading to the release of intracellular tox- ins into the surrounding water (Zanchett and Oliveira-Filho 2013, Mohamed et al. 2015). Most studies on the biological effects of electromagnetic fields yielded promising results for the application of ELF-EMFs as potential means for control or killing pathogenic and toxic microorganisms (Bodewein et al. 2019). Our previous study tested the potential inhibitory effect of ELF-EMF on the growth of the cyanobacteria Micro- cystis aeruginosa (Kützing) Kützing and Anabaena circinalis Rabenhorst ex Bornet & Flahault (Fadel et al. 2018). The re- sults revealed different effects of ELF-EMF on the two species, strongly inhibiting the growth of A. circinalis with no remark- able effect on M. aeruginosa. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju is among the toxic cyanobacterial species fre- quently found in drinking water sources and linked to harm- ful blooms in freshwater sources over the world (Rzymski and Poniedzialek 2014). It has been associated with the pro- duction of water-soluble toxins including the hepatotoxic alkaloid, cylindrospermopsin (CYN), and the neurotoxic al- kaloid, saxitoxin (STX) (Carneiro et al. 2013). Therefore, the present study investigates in details the effects of ELF-EMFs on growth and antioxidant defence system of the toxic cya- nobacterium C. raciborskii. The results of this study could be useful for controlling the growth of toxic cyanobacteria in drinking and recreational water sources. Materials and methods ELF-EMF exposure system Square amplitude modulated waves (QAMW) were gen- erated by an arbitrary function generator (BK Precision 4085 40 MHz) as described with Sketch diagram by Fadel et al. (2018). The carrier frequency was a 10 MHz sine wave with amplitude ± 10 Vpp, and modulation depth of ± 2 Vpp. Cya- nobacterial cultures in sterilized tubes were exposed to QA- MW through two parallel copper electrodes connected to the output of the generator. The temperature inside the coil was maintained at 30 ± 0.5 °C (the organism's optimal growth temperature). Experimental organism and culture conditions Cylindrospermopsis raciborskii strain (LC107906) used in this study was obtained from the microalgal culture col- lection of Botany and Microbiology Department, Faculty of Science, Sohag University, Sohag, Egypt. This species was isolated from fish ponds in Sohag region, Egypt, and re- ported as cylindrospermopsin producer (Mohamed 2016b). The strain was grown in BG-11 liquid medium with low ni- trogen (3% NaNO3, pH = 8) (Ripka et al. 1979) under con- trolled conditions of light (50 µmol m–2 s–1) and tempera- ture (30 ± 0.5 °C) in a climate-controlled chamber. Based on the growth curve, the doubling time of this strain was 15.33 h (Fig.1). One ml of exponentially growing C. raci- borskii cells (8 × 105 cells mL–1) was then inoculated into 25 mL-glass test tubes containing modified BG-11 medium (i.e. without nitrogen), and used to determine resonance frequency of growth inhibition of C. raciborskii. The inocu- lated cells were further grown for 24 h for adaptation in the same growth chamber. Thereafter, 30 tubes with cyanobac- terial cells were connected to output of the generator at a single intensity of 100 V m–1 and different frequencies (0.1– 1.0 Hz) for 30 minutes. Other 3 tubes were used as control and kept in the same conditions as the exposed ones (at 50 µmol m–2 s–1 light intensity and temperature of 30 °C), but outside the EMF generating instrument (i.e. without field application). To further confirm the effect of ELF-EMFs on the C. raciborskii, additional experiments were conduct- ed by exposing cyanobacterial cells to the electromagnetic field at the frequency showing the strongest inhibitory ef- fect (0.7 Hz) in the former experiment, for different expo- sure times (15, 30, 60, 120 min). The experiment for each exposure time was run in 9 tubes, 3 tubes were incubated for 4 h, 3 tubes incubated for 24 h and 3 tubes for 48 h. Three tubes were used as a control for each incubation period. Both treated and control cultures of C. raciborskii were in- cubated under controlled conditions of light (50 µmol m–2 s–1) and temperature (30 ± 0.5 °C) in a climate-controlled chamber. An aliquot (2 mL) from the cultures was taken under aseptic conditions for analysis. Subsamples of treat- ed and control cultures were collected after 4, 24 and 48 h for antioxidant enzyme activities and after 24 and 48 h for analysis of cell number and chlorophyll-a. Each experiment was done in triplicate. Fig. 1. The growth curve of Cylindrospermopsis raciborskii under optimal growth conditions. Each value is presented as mean ± standard deviation of three measurements. INHIBITORY EFFECTS OF ELECTROMAGNETIC FIELDS ON CYANOBACTERIA ACTA BOT. CROAT. 79 (2), 2020 195 Cell abundances Cell abundances (cells mL–1), as an estimator of Cylindro- spermopsis raciborskii growth, were estimated using a Sedge- wick Rafter chamber under a light microscope following the method of Hötzel and Croome (1999). The morphotype of the strain is a coiled trichome with an average length of 180 µm. Therefore, C. raciborskii was first counted as trichomes. The total abundance of C. raciborskii in the samples was cal- culated by multiplying the number of trichomes (trichomes mL–1) by the average number of cells per trichome (22 cells) estimated in our study. Chlorophyll-a determination Chlorophyll-a concentration (Chl-a, pg cell–1) as a mea- sure of biomass and an indicator of the physiological status of C. raciborskii, was determined spectrophotometrically at 665 and 750 nm (Appota 6200 UV/Vis spectrophotometer) in a methanol extract prepared by homogenizing cyanobac- terial cells of a known volume (2 mL) of cultures according to Tailing and Driver (1963). Chl-a was calculated using the equation: Chl-a = 13.9 (A665-A750) × v/V.N, where A665 and A750 correspond to the absorbance of methanol ex- tracted supernatant at 665 nm and 750 nm wavelength with a 1 cm pathway cuvette, 13.9 is the extinction coefficient, v is volume of extract (mL), V is volume of culture sample (mL) and N is the number of cells present in the culture sample. Assay of antioxidant enzymes To determine the activity of antioxidant enzymes, sam- ples of treated and control cultures (2 ml) of C. raciborskii were harvested by centrifugation at 10,000 g for 10 min at 4 °C and the pellets obtained were washed with 10 mM Na2- EDTA (ethylene diamine tetraacetic acid), then twice with distilled water. The algal pellets were added to 3 mL of 50 mM phosphate buffer (Na2PO4/K2HPO4), pH = 7.0, con- taining 0.1 mM Na2-EDTA and 1% of polyvinylpyrrolidone (PVP) and ground by sonication in ice-water bath. The ho- mogenate was centrifuged at 13,500 g for 20 min at 4 °C. The supernatant was re-centrifuged at 13,500 g for 15 min at 4 °C, and the resultant supernatant was collected and stored at –20 °C for analysis of catalase (CAT) and peroxidase (POD). CAT activity was measured according to the method of Aebi (1984) with minor modifications. The reaction mixture (3.0 mL) consisted of 50 mM phosphate buffer (pH = 7.0), 0.1 mM EDTA, 45 mM H2O2 and 50 μL enzyme extract). The reaction was started by addition of the extract. The activity of catalase was estimated by the decrease of absorbance at 240 nm for 1 min as a consequence of H2O2 consumption. Catalase activity was calculated based on an extinction coef- ficient 36 M–1 cm–1 and expressed as nmole H2O2 min–1 cell–1. Total POD activity was determined as described by Macad- am et al. (1992) by the oxidation of guaiacol in the presence of H2O2 in a reaction mixture (3.0 mL) containing 0.1 M phosphate buffer (pH = 6.5), 0.1 mM EDTA, 0.2 M guaia- col, 0.03 M H2O2 and 50 μL enzyme extract. The increase in the absorbance due to oxidation of guaiacol was measured at 470 nm. The enzyme activity was calculated in terms of nmole of tetraguaiacol oxidized per min per cell at 25 ± 2 °C using the absorbance coefficient 26.6 mM–1 cm–1.All spectro- photometric analyses of enzyme activities were performed on a spectrophotometer Appota 6200 UV/Vis spectropho- tometer. Dielectric measurements The dielectric parameters were determined for cya- nobacterial cultures exposed to 0.7 Hz square amplitude modulated waves for 2 hours, after a 24 h incubation peri- od. These measurements were carried out in subsamples of these cultures containing a fixed cell number of C. raciborskii (105 cells mL–1), which was monitored using a Sedgewick Rafter chamber as described above. The cell number was ad- justed by the dilution of the subsamples in sterile deionized water. Cyanobacterial cells in the subsamples (1 mL) were separated from the medium by centrifugation at 14,000 g at 4 °C for 15 min. The pellet was re-suspended in a 1 mL vol- ume of sterile deionized water. This step was repeated twice. The dielectric measurements were carried out in cyanobacte- rial cell suspensions in the frequency (f) range 42–100000 Hz using a loss factor meter (LCR Hi TESTER 3532, HIOKI, Ueda, Nagano, Japan) with a sample cell (PW 9510/60, Phil- ips Weisshausstrasse, Aachen, Germany) according to Fadel et al. (2017). The relative permittivity ɛo, dielectric constant ε, conductivity σ and relaxation time (t) of the samples were calculated according to the equations outlined by Fadel et al. (2017). Cylindrospermopsin analyses To detect and quantify cylindrospermopsin (CYN) toxin within Cylindrospermopsis cells as well as toxin potentially released into the medium, an aliquot (2 mL) of 48 h cul- tures (i.e. control and ELF-treated cells) was filtered through GF/C filters. The filters with retained cells were extracted in methanol (50%) for intracellular toxin, while the filtrate was used for the determination of extracellular CYN potentially released into the medium. CYN concentration was deter- mined by ELISA using commercial kits for this toxin pur- chased from Abraxis (54 Steamwhistle Drive Warminster, PA 18974). Briefly, a 100 µL culture filtrate or toxin extract, toxin standard, calibrator or negative control was added to 96-microplate wells and incubated for 30 min at room tem- perature. A 100 µL aliquot of a microcystin-enzyme con- jugate solution was then added and incubated for another 30 min at room temperature. The wells were emptied and washed four times with phosphate buffer saline and distilled water. A 100 µL aliquot of substrate was added to each well and incubated for 30 min at room temperature. The absor- bance of the colour generated from the transformation of a substrate by the enzyme was measured spectrophotometeri- cally at 450 nm using the microplate reader. The concentra- tion of CYN toxin in the culture filtrate was directly calculat- ed from standard curves drawn with the standard solutions provided in kits. Detection limit for CYN according to the kit's manufacturer is 0.04 ng mL–1. MOHAMED Z, ALI F, ABDEL-LATEEF M, HOSNY A 196 ACTA BOT. CROAT. 79 (2), 2020 Statistical analysis Differences in growth and physiological parameters be- tween treated and control cultures of C. raciborskii were de- termined by one-way ANOVA (P < 0.05) and Tukey’s post- hoc multiple range test using the software SPSS ver.16.0 (SPSS Inc. Released 2007). Results The growth response of Cylindrospermopsis raciborskii exposed to 0.1–1.0 Hz amplitude modulating frequencies for 30 min is shown in Fig. 2. The exposure of C. raciborskii to 0.7 Hz for 30 min after 24 h of incubation caused a more sig- nificant decrease (F = 201.2, P = 0.001) of cell number than other frequencies (Fig. 2). The effect of exposure time at 0.7 Hz on the growth and physiological functions of the micro- organism was also evaluated. The results showed that after 24 h of incubation, a significant reduction in the cell number in treated cultures was observed compared to control, and that reduction varied significantly (F = 177.7, P = 0.0003) based on the exposure time of the cultures to the electro- magnetic field (Fig. 3). The increase in the exposure time led to a sharp reduction in microbial growth (i.e. low cell num- ber). However, at the incubation period of 24 h, no complete inhibition of cyanobacterial growth was found at different exposure times tested. On the other hand, at 48-hour incu- bation, the exposure to the electromagnetic field reduced the cyanobacterial growth sharply in comparison to 24-hour incubation, and caused complete death for 2-hour exposure cells (Fig. 3). The cyanobacterial cell number also showed a decrease in control cultures at 48 h incubation period, indi- cating that the cells entered the decline phase at that period based on growth curve (Fig .1). Alongside the cell density of C. raciborskii, concentra- tion of Chl-a, was also influenced by an ELF-EMF at 0.7 Hz. Chl-a content of treated cells exhibited a significant de- crease (F = 9.2, P = 0.002) in comparison to control at 24 hours incubation period (Fig. 4), and this decrease was ex- posure time-dependent. Two-hour exposure to electromag- netic field completely reduced Chl-a content at 48 hours in- cubation period (Fig. 4). The activity of antioxidant enzymes (CAT and POD) in C. raciborskii responded differently, when exposed to ELF- EMFs compared to that of control cultures (Fig. 5). The ac- tivity of POD in the cells of 2 h-exposure to the electro- magnetic field was enhanced after the first 4 h of incubation period (Fig. 5). This activity decreased sharply in 2-h expo- Fig. 2. The change in cell number of Cylindrospermopsis racibor- skii exposed to square amplitude modulated waves at different frequencies (0.1–1.0 Hz) for 30 minutes and grown for 24 hours. Each value is the average of three replicates ± standard deviation. Asterisk indicates significant differences (One-way Anova at P ≤ 0.05) in cell number between different exposure times and control. Fig. 4. The change in chlorophyll-a concentration of Cylindro- spermopsis raciborskii exposed to 0.7 Hz for different exposure periods, and grown for 24 and 48 h. Each value is the average of three replicates ± standard deviation. Different uppercases let- ters indicate significant differences (One-way Anova at P = 0.05) in chlorophyll-a concentrations between incubation periods (24 and 48 h) for the same exposure time. Different lowercases letters indicate significant differences (One-way Anova at P ≤ 0.05) in chlorophyll-a concentrations among different exposure times for the same incubation period. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 control 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 sllec( reb mun lle C x 10 6 m L– 1 ) Frequency (Hz) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 control 15 30 60 120 C hl or op hy ll- a (p g ce ll- 1 ) Exposure time (min) 24 hours 48 hours Aa Aa Ab Ab Ac Aa Bb Ac Bd Be Fig. 3. The growth inhibition of Cylindrospermopsis raciborskii ex- posed to 0.7 Hz square amplitude modulated waves for different ex- posure periods, and grown afterwards for 24 and 48 h. Each value is the average of three replicates ± standard deviation. Asterisks indicate significant differences (One-way Anova at P ≤ 0.05) in cell number between incubation periods (24 and 48 h) for the same exposure time. Different letters indicate significant differences (One-way Anova at P ≤ 0.05) in cell number among different exposure times for each incubation period. INHIBITORY EFFECTS OF ELECTROMAGNETIC FIELDS ON CYANOBACTERIA ACTA BOT. CROAT. 79 (2), 2020 197 sure cultures after 48 h incubation period. On the other hand CAT activity in treated cells increased along the whole incu- bation period, except cells of 1-h and 2-h exposure, which showed a marked reduction in CAT activity at 48 h incuba- tion period. Morphological changes were also noted in cells exposed to ELF-EMF at 0.7 Hz for 2 h. These changes began with shrinking of cytoplasmic contents forming amorphous ag- gregates, and thickening of the cell wall. They eventually ended with the disintegration of cytoplasmic material, but without apparent lysis in the cell wall (data not shown). Changes in dielectric properties, as a measure of interac- tion of cellular constituents (e.g. plasma membrane, cell wall and cytoplasm) with electromagnetic fields, were evident in the present study. The data shown in Table 1 revealed a sig- nificant decrease in the dielectric increment (∆ɛ) and con- ductivity (S) for exposed cyanobacteria cells to ELF-EMF at 0.7 Hz for 2 hours and incubated for 24 h, as compared with control (F = 9, P = 0.04 and F = 12.1, P = 0.02, respectively). The results of ELISA for CYN toxin revealed that CYN concentrations released naturally into the medium of control cultures were 0.2 ± 0.03 µg L–1 while the toxin was not de- tectable in the medium of ELF-treated cultures. Conversely, the toxin concentrations within cells of ELF-treated cultures (26.1–27.3 pg cell–1) were higher than those in cells of con- trol cultures (4.31–6.25 pg cell–1). Discussion The present study determined the effects of the different frequencies of ELF-EMF on the growth of Cylindrospermop- sis raciborskii. Maximum growth inhibition and decrease in Chl-a content of C. raciborskii were observed at a frequency of 0.7 Hz. The results also provided evidence for the inhibi- tory effect of ELF-EMFs at 0.7 Hz on the physiological func- tions and antioxidant system of the toxic cyanobacterium C. raciborskii. This is in agreement with the results of our previous study, which found that the growth inhibition of Anabaena circinalis by ELF-EMFs occurred at a resonance frequency 0.7 Hz (Fadel et al. 2018). For heterotrophic bacte- ria, the resonance frequency for the growth inhibitory effects of ELF-EMF seems to vary among species. For instance, the growth of Salmonella typhi was highly inhibited by an ELF- EMF at a resonance frequency of 0.8 Hz (Fadel et al. 2014), while the inhibiting resonance frequency of ELF-EMF was 1.0 Hz for Agrobacterium tumefaciens (Fadel et al. 2017), and 50 Hz for Escherichia coli and Pseudomonas aeruginosa (Se- gatore et al. 2012). The inhibition effect is due to the interfer- ence of the ELF-EMFs according to the frequency with the bioelectric signals generated from the physiological func- tions of microbial cells (Zanchett and Oliveira-Filho 2013). In this respect, Fadel et al. (2014) reported that the bioelec- tric signals generated during metabolic activities of cells are usually in the extremely low frequency range. Therefore, the applied electromagnetic wave could have a similar frequen- cy, interfering with these signals. Our data also revealed that cell proliferation of C. raci- borskii decreased sharply when exposed to the electromag- netic field compared to control cultures, and this decrease was dependent on the exposure time. Our results are thus in accordance with the results of previous studies demonstrat- ing that the inhibitory effect of ELF-EMFs on the growth of bacteria (Gaafar et al. 2006) and cyanobacteria (Fadel et al. 2018) increased with the length of exposure. Meanwhile, there was a decrease in the cell number of C. raciborskii in Tab. 1. Dielecteric parameters for Cylindrospermopsis raciborskii exposed to 0.7 Hz square amplitude modulated waves for 2 hours, as compared with the control (untreated). Asterisks indicate sig- nificant difference (One-way Anova at P ≤ 0.05) in the parameters between control and treated cultures (N=3). Relaxation time (τ µs) Conductivity 𝜎(S m–1) at 100 kHz Dielectric increment ∆ɛ(ɛ˳–ɛ͚) Control 14.05 ± 1.0 (6 ± 1.2) × 10-3 1721 ± 512.6 0.7 Hz 12.9* ± 0.97 (4.8* ± 2.1) × 10-3 1347* ± 165.3 Fig. 5. The change in the activities of the antioxidant enzymes peroxidase (A) and catalase (B) of Cylindrospermopsis raciborskii exposed to 0.7 Hz for different exposure periods, and grown for 4, 24 and 48 h. Each value is the average of three replicates ± standard deviation. Different uppercases letters indicate significant differences (One-way Anova at P = 0.05) in enzyme activity among different exposure times for each incubation period. Different lowercases letters indicate significant differences (One-way Anova at P ≤ 0.05) between incubation periods (4, 24 and 48 h) for the same exposure time. 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 4 24 48 P er ox id as e (n m ol m in -1 ce ll- 1 ) A control 15min. 30min. 60min. 120min. Aa Ac Aa Ab Ab Bc Ba Bb Cc Ca Cb Dc Da Db Ec 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 4 24 48 C at al as e ac tiv ity (n m ol m in -1 ce ll- 1 ) Incubation time (hours) B Aa Aa Ab Aa Ab Bc Aa Bb Cc Aa Cb Dc Ba Db Ec 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 4 24 48 C at al as e ac tiv ity (n m ol m in -1 ce ll- 1 ) Incubation time (hours) B Aa Aa Ab Aa Ab Bc Aa Bb Cc Aa Cb Dc Ba Db Ec MOHAMED Z, ALI F, ABDEL-LATEEF M, HOSNY A 198 ACTA BOT. CROAT. 79 (2), 2020 control cultures at 48 h of incubation period. This decrease may be due to the high initial cell density of inoculum we used in our experiments (Fig.1), which promoted the rapid growth of cyanobacterial cells leading to nutrient consump- tion, shortness of cell proliferation time and early decline phase. This agrees with the finding of Cheng et al. (2018) re- porting that high inoculum density of algal cells can shorten the time of cell proliferation and promote the rapid growth of cells. In the present study, ELF-EMF also reduced the concen- tration of Chl-a of this cyanobacterium. This may be due to the decrease in the cyanobacterial cell number or the inhibi- tion of Chl-a synthesis. Therefore, Chl-a is a useful indicator of the phototrophic biomass and physiological status of the organism, and has been used in bioassays of environmental stresses (Borowiak et al. 2015). The damaging effect of ELF- EMF on photosynthetic pigments may occur through active oxygen mediated peroxidation (Zeeshan and Prasad 2009). In this respect, numerous hypotheses have been proposed to explain the mechanisms of the biological effects of elec- tromagnetic fields on cells and organisms. The main theo- ries are based on fact that ELF-EMF promotes the formation of free radicals (e.g. reactive oxygen species, ROS) through changing the level of ionization of water molecules (Fernie and Reynolds 2005), which affect the synthesis of macro- molecules and relevant metabolic processes (Cabsicol et al. 2000). The other possible effects are that ELF-EMF changes the permeability of the ionic channels in the cell membrane (Oncul et al. 2016), and thus affect ion transport into the cells resulting in biological changes in the organism. Our data revealed an increase in the dielectric properties of cyano- bacterial cell constituents of treated cultures, indicating that damage and change in the permeability of cell membrane occurred. Notably, the electrical conductivity and relaxation time are directly related to the electric dipole moment of the microbial cell, and the decrease in these parameters is main- ly due to changes in the charge distribution upon the pro- tein molecules of the cell membrane. Therefore these two parameters can be indicators for structural changes in the cell membrane of an organism upon exposure to stress such as ELF-EMFs (Fadel et al. 2014). Therefore, our study sup- ports the hypothesis that the biological effect of ELF-EMFs is due the change in the permeability of the ionic channels in the membrane (Oncul et al. 2016). In this regard, it has been reported that dielectric prop- erties of a biological system reflect information about the morphology and permeability of the cellular membrane, and about possible changes in structure and composition of biological macromolecules such as protein, DNA, ami- no acids (Dopp et al. 2000). Additionally, our study found that ELF-EMFs induced morphological changes in C. raci- borskii cells including shrinking and disintegration of cyto- plasmic material. Such morphological changes in addition to filament fracture and coils dissociation were also observed for Anabaena circinalis when exposed to ELF-EMF (Fadel et al. 2018). Inhan-Garip et al. (2011) demonstrated shrink- ing and disintegration of cell contents, and cell wall lysis of Gram-negative bacteria induced by ELF-EMFs. Howev- er, the cell wall lysis of ELF-EMF-exposed Gram-negative bacteria observed by Inhan-Garipet al. (2011) did not oc- cur for the cyanobacterium C. raciborskii during our study (not shown). Our observation is similar to that obtained for Gram-positive bacteria cell wall being apparently not- disrupted upon exposure to ELF-EMFs (Inhan-Garip et al. 2011). The discrepancy between gram negative bacteria and cyanobacteria can be explained by the fact that although cy- anobacteria are Gram-negative bacteria, their cell wall con- tains features of Gram-positive bacteria. These include: the peptidoglycan layer found in cyanobacteria is considerably thicker than that of most gram-negative bacteria, the degree of cross-linking between the peptidoglycan chains within the murein is more similar to that of gram-positive bacteria (56% to 63%), and the cyanobacterial peptidoglycan is com- plexed with specific polysaccharides (Litzinger and Mayer 2010). What is interesting in this study is that unlike natu- rally occurring in control cultures (i.e. toxin release), CYN toxin was found within the cells of ELF-treated cultures but it was not detectable at all in the medium of these cultures during the whole incubation period (i.e. not just a decrease in its concentration). Therefore, the presence of CYN toxin within treated cells and its absence in the medium indicates that these cells could not release the toxins into the medi- um. These results confirm that ELF did not lyse cyanobacte- rial cell wall but increased its thickness so that cytoplasmic contents including toxins cannot be released from the cells. However, the presence of CYN in the medium of control cultures can be explained by the fact that CYN is often nat- urally released from living cells into the surrounding water (Rücker et al. 2007). Furthermore, increased activities of CAT and POD en- zymes in C. raciborskii cells exposed to the electromagnetic field during the first 24 hours incubation period in com- parison to control, provide evidence that ELF-EMF induces oxidative stress and promotes the formation of ROS. POD reduces H2O2 to water using various substrates as electron donors, and CAT catalyzes the breakdown of H2O2 into wa- ter and oxygen (Wang et al. 2009). This finding supports the hypothesis that reactive oxygen species (ROS) mediate the effects of ELF-EMFs on living organisms (Fernie and Reyn- olds 2005). In this respect, it is well known that ROS are continuously produced and eliminated by living organisms normally maintaining them at certain steady-state levels (Lushchak 2011). Most organisms including cyanobacteria have developed an antioxidant defence system comprising enzymes such as CAT and POD that scavenge the resultant ROS and maintain the balance between ROS generation and elimination (Ganapathy et al. 2015). However, under some circumstances (e.g., stress intensity and duration, rate of ROS production, capacity of ROS scavenging), this balance is disturbed, leading to enhanced ROS level and oxidative damage (Lushchak 2011). Accordingly, our results showed a sharp decrease in the activity of antioxidant enzymes (CAT and POD) of cells with 1-h and 2-h exposure time after the initial increase in the activity. Decreases in CAT and POD INHIBITORY EFFECTS OF ELECTROMAGNETIC FIELDS ON CYANOBACTERIA ACTA BOT. CROAT. 79 (2), 2020 199 activities at higher exposure times (1h and 2h) to ELF-EMF may have been caused by high concentrations of free radicals generated by electromagnetic field leading to the inhibition of protein synthesis (Luna et al. 1994).The alterations in the activity of antioxidant enzymes upon exposure to ELF-EMF have been previously documented in plants (Shashurin et al. 2017), human cells (Mahmoudinas et al. 2016) and bacteria (Fadel et al. 2017). Conclusion In conclusion, the results of this study demonstrated that the 2-h exposure to an ELF-EMF at 0.7 Hz inhibited the growth of the toxic cyanobacterium C. raciborskii. The re- duction in cell number was associated with induction and subsequent decrease in the antioxidant defense enzymes, leading to changes in cell physiology and morphology. 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