Genotoxic and cytotoxic effects of 50 Hz 1 mT electromagnetic field on larval rainbow trout (Oncorhynchus mykiss), Baltic clam (Limecola balthica) and common ragworm (Hediste diversicolor)

Aquatic Toxicology 208 (2019) 109–117

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Genotoxic and cytotoxic effects of 50 Hz 1 mT electromagnetic field on larval rainbow trout (Oncorhynchus mykiss), Baltic clam (Limecola balthica) and common ragworm (Hediste diversicolor)

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Milda Stankevičiūtėa, , Magdalena Jakubowskab, Janina Pažusienėa, Tomas Makarasa, Zbigniew Otrembac, Barbara Urban-Malingab, Dariusz P. Feyb, Martyna Greszkiewiczb, Gintarė Sauliutėa, Janina Baršienėa, Eugeniusz Andrulewiczb a b c

Nature Research Centre, Akademijos St. 2, LT-08412, Vilnius, Lithuania National Marine Fisheries Research Institute, Kołłątaja 1, 81-332, Gdynia, Poland Gdynia Maritime University, 81-225, Gdynia, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Genotoxicity and cytotoxicity assay Underwater cables Fish Marine invertebrates

The aim of this research was to assess genotoxicity and cytotoxicity responses in aquatic animals exposed to 50 Hz 1 m T electromagnetic field (EMF). Rainbow trout (Oncorhynchus mykiss) at early stages of development were exposed to EMF for 40 days, whereas marine benthic invertebrates – the common ragworm Hediste diversicolor and the Baltic clam Limecola balthica – for 12 days. To define genotoxicity and cytotoxicity responses in selected animals, assays of nuclear abnormalities in peripheral blood erythrocytes of O. mykiss, coelomocytes of H. diversicolor and gill cells of L. balthica were performed. Induction of formation of micronuclei (MN), nuclear buds (NB), nuclear buds on filament cells (NBf) and cells with blebbed nuclei (BL) were assessed as genotoxicity endpoints, and 8-shaped nuclei, fragmented (Fr), apoptotic (Ap) and binucleated (BN) cells as cytotoxicity endpoints. Exposure to EMF affected all studied species but with varying degrees. The strongest responses to EMF treatment were elicited in L. balthica, in which six out of the total eight analyzed geno- and cytotoxicity endpoints were significantly elevated. Significantly induced frequencies of MN were detected in O. mykiss and H. diversicolor cells, NBf and BL only in gill cells of L. balthica, and NB in analyzed tissues of all the test species. As cytotoxicity endpoints, a significant elevation in frequencies of cells with 8-shaped nuclei was found in O. mykiss and L. balthica, while Ap and BN was observed only in L. balthica. EMF exposure did not induce any significant cytotoxic activity in H. diversicolor coelomocytes. The present study is the first to reveal the genotoxic and cytotoxic activity of 1 m T EMF in aquatic animals, and, consequently, the first one to report the adverse effect of this factor on common marine invertebrates and early life stages of fish.

1. Introduction Artificial magnetic field (MF) and electromagnetic field (EMF) are emitted by many installations (wind farms, transmission cables, hydrokinetic turbines) in the marine environment (Petersen and Malm, 2006; Cada, 2009; Andrulewicz and Otremba, 2011; Cada and Bevelhimer, 2011; Cada et al., 2011). Depending on the technique applied for transmission of electricity, the cables which are laid on the seabed modify the natural geomagnetic field (in case of direct current (DC) solutions) or produce a low-frequency EMF (in case of alternating current (AC) solutions). The intensity of MF depends on the distance from cables, their relative position and intensity of the electric current

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flowing through them (Otremba and Andrulewicz, 2014). Transmission of alternating current induces a low-frequency EMF, intensity of which is determined by the values of its effective component – an alternating magnetic field (Otremba and Andrulewicz, 2014). EMFs that we face in the case of electricity transmission systems have a frequency of 50 Hz in Europe and 60 Hz in North America and can be classified as the socalled extremely-low-frequency EMF (ELF-EMF) (1–300 Hz) (Touitou and Selmaoui (2012)). The close proximity of conductors in many AC three-core cables (in which all the three insulated conductors are placed into a single cable) almost completely nullifies EMF (Meißner et al., 2006; Öhman et al., 2007). However, the AC systems intended for transmission of larger amounts of power are composed of three separate

Corresponding author. E-mail addresses: [email protected], [email protected] (M. Stankevičiūtė).

https://doi.org/10.1016/j.aquatox.2018.12.023 Received 12 October 2018; Received in revised form 22 December 2018; Accepted 31 December 2018 Available online 02 January 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.

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of EMF was assessed by a comet assay, a sister chromatid exchange or a micronucleus test (McCann et al., 1998). In their review of genotoxic and carcinogenic EMF effects, Kocaman et al. (2018) drew a conclusion that EMF may be hazardous to living organisms. However, geno- and cytotoxicity studies were not performed on aquatic organisms. Generation of micronuclei in terrestrial organisms as an indication of genotoxicity was evaluated in several experimental studies on EMF (Erdal et al., 2007). Other nuclear abnormalities, such as nuclear buds, blebbed nuclei, 8-shaped nuclei, binucleated and fragmented-apoptotic cells, were not assessed. Nuclear abnormalities are reliable indicators of genotoxic and cytotoxic damage (Gomes et al., 2015). In this study, the formation of micronuclei (MN), nuclear buds (NB), nuclear buds on filament (NBf) and cells with blebbed nuclei (BL) were assessed as genotoxicity endpoints, and 8-shaped nuclei, fragmented (Fr), apoptotic (Ap) and binucleated (BN) cells as cytotoxicity endpoints. Micronuclei and other associated nuclear abnormalities (NB, NBf and BL) are biomarkers of genotoxic events and chromosomal instability (Fenech et al., 2011). Fragmented-apoptotic cells are indicators of programmed cell death. A higher rate of cytokinesis failure can result in an increase of binucleated and 8-shaped nucleus cells. Frequencies of binucleated and 8-shaped nuclei cells were specified as indicators of cytotoxicity in previous studies (Sannino et al., 2017; Baršienė et al., 2014; Toy et al., 2014; Cavas et al., 2005; Tolbert et al., 1992). The majority of the data available regarding EMF effects on organisms are based on short-term experiments, which mimic environmental exposure scenarios in a very limited way (Simkó, 2007). For this reason, in this study, long-term experiments were carried out to assess EMF effects. In this study, two experiments were designed for assessing long-term effects of EMF exposure. The first experiment focused on geno- and cytotoxicity responses in marine invertebrates, while another experiment on the assessment of EMF impacts on geno- and cytotoxicity endpoints in early development stages of teleost fish. Two marine benthic invertebrates: the polychaete Hediste diversicolor and the bivalve Limecola balthica, and the teleost fish species Oncorhynchus mykiss were selected as bio-indicators for assessing EMF geno- and cytotoxicity. It might be assumed that benthic organisms, especially the infauna characterized by relatively limited mobility, and fish which might be attracted to EMF, are particularly vulnerable to EMF generated by underwater cables. Therefore, two key benthic invertebrates dominating shallow soft bottoms of the Baltic Sea (deep bottoms also, in the case of bivalves) were chosen for the experiments (Laine, 2003; Aarnio et al., 2011; Gogina et al., 2016). Both species are important components of marine ecosystems, occupying significant positions in a food web (Mattila and Bonsdorff, 1998; Obolewski et al. (2009)) and playing an irreplaceable role in sediment bioturbation and enrichment processes (Reise, 1983; Christensen et al. (2000); Michaud et al., 2006; Pischedda et al., 2012). H. diversicolor and L. balthica are considered to be appropriate species for genotoxicity evaluation (Saez et al., 2015; Butrimavičienė et al., 2018). Moreover, changes observed in both species are commonly used as indicators of environmental conditions (Bonsdorff et al., 1995; Durou et al., 2007; Smolarz and Bradtke, 2011; Maranho et al., 2014). O. mykiss is commonly used as a good model fish species in a wide range of research areas, including genotoxicity and cytotoxicity research, where it is used as a bio-indicator (Thorgaard et al., 2002; Gomiero et al., 2018; Stankevičiūtė et al., 2016). Moreover, among teleosts, both, diadromous and nonmigrating salmonids have a well-documented ability of magnetoreception at different life stages (Quinn, 1980; Ueda et al., 1986; Formicki et al., 2004; Lohmann et al., 2008), including O. mykiss (Haugh and Walker, 1998; Hellinger and Hoffmann (2012). The aim of the present study was to assess the potential genotoxic and cytotoxic effects of 50 Hz 1 m T EMF on the coelomocytes of H. diversicolor and on the gill cells of L. balthica after 12 days of exposure, and on peripheral blood erythrocytes of rainbow trout O. mykiss fry exposed to this factor for 40 days. The 40-day long period was selected so as to study the whole early life period of fish from eyed-egg stage to

single-core cables, usually laid at a distance of 10–100 m from each other, which is a too large distance for reducing the magnetic field generated by each wire (Johansson et al., 2005). In the case of singlecore solutions, low-frequency EMF spreads in seawater as a static magnetic field (Otremba and Andrulewicz, 2014). Submarine cables may conduct a current reaching 1330–1600 A resulting in the magnetic induction at the surface of the cable armor, which usually ranges from 3.2 to 6.4 milliteslas (mT), and may decrease to 0.32 - 0.64 m T at 1 m distance, depending on the cable diameter (Bochert and Zettler, 2004; Meißner et al., 2006; Otremba et al. (2019)). The AC cables that are present in the environment generate an EMF that is even up to 8 m T (Cada et al., 2011), while the magnetic flux intensity (induction) of the geomagnetic field in the Baltic Sea is about 0.05 m T (Hulot et al., 2010). EMF is considered much more hazardous for biological structures than static MF (Panagopoulos et al., 2002; Directive, 2013/35/EU). According to the research data available, the effect of EMF on mutagenicity is considered to be 200 times stronger than that of static MF (Suzuki et al., 2006). Moreover, different aquatic animals including fish and invertebrate species are able to perceive geomagnetic field and use it for navigation purposes (Arendse and Kruyswijk, 1981; Lohmann, 1985; Lohmann and Willows, 1987; Kirschvink, 1997; Wiltschko and Wiltschko, 2005). However, both, static and alternating artificial fields might disturb this ability (Gill, 2005; Gill et al., 2014). Despite the fact that the European Commission Marine Strategy Framework Directive obliges member states to investigate the impacts of energies (including electromagnetic) released to the environment on aquatic organisms and to reduce them to the level that does not produce adverse effects on the marine environment (Directive, 2008/56/EC), experimental studies into effects of magnetic fields on aquatic organisms are still very scanty. They are mainly limited to the effects of static MF on fish including their early life stages (Formicki and Winnicki, 1998; Sadowski et al., 2007; Loghmannia et al., 2015; Fey et al., 2019), and a few species of marine invertebrates (Aristarkhov et al., 1988; Bochert and Zettler, 2004). As far as the research into EMF conducted to date is concerned, its findings indicate that exposure to low-frequency EMF of different induction values disturbs the mitotic cycle in embryos of sea urchin Strongylocentrotus purpuratus (Levin and Ernst, 1995), induces activation of MAP kinases and the expression of heat shock proteins in blue mussel Mytilus galloprovincialis (Malagoli et al., 2003,2004), delays the hatching period of zebrafish Danio rerio (Skauli et al., 2000), causes a pineal melatonin level increase in freshwater brook trout Salvelinus fontinalis (Lerchl et al., 1998) and alters behavior of a few freshwater fish species (Bevelhimer et al., 2013). Significant physiological effects and changes in behavior of the edible crab (Cancer pagurus) exposed to EMF of the strength predictable around sub-sea cables were reported by Scott et al. (2018). However, values of the applied magnetic induction as well as the field frequency used in the above-mentioned experiments not always correspond with the values currently observed in the environment or those predictable from planned investments. Due to limited research, the evidence regarding harmful EMF effects on marine or freshwater organisms is inconclusive, thus potential environmental effects of submarine AC cables remain mostly speculative, so far. The International Agency for Research on Cancer has classified ELFEMF under the category 2B comprising agents that are “possibly carcinogenic to humans” (IARC, 2002). However, the mechanisms underlying ELF-EMF remain uncertain (Manser et al., 2017; Kocaman et al., 2018). The scientific literature data indicate that there is still no general agreement on cellular effects of ELF-EMF (Santini et al., 2009; Mihai et al., 2014). Moreover, no consistent pattern of EMF- induced genotoxicity or cytotoxicity in cells or organisms has been determined and these findings are still considered preliminary. Previously published studies on ELF-EMF exposure reported no geno- or cytotoxic effects (Cho and Chung, 2003; An et al., 2015). However, contradictory findings on in vitro and in vivo cellular effects caused by EMF exposure have been published in literature (Kocaman et al., 2018). Genotoxicity 110

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the whole aquarium volume (Fig. 1). The reference aquarium is positioned in the natural geomagnetic field. The water in the experimental system is continuously aerated and pumped between aquaria and the conditioning tank at the rate of 1100 ml min−1.

larval stage, i.e. the life period that determines successful recruitment of fish to the greatest extent. For studying the response of invertebrates belonging to two different classes (polychaetes and bivalves), we chose a shorter period of exposure (12-days) as a compromise between very short (hours) and long (several weeks) duration of exposure that is usually applied in ecotoxicological studies with invertebrates (Fonseca et al., 2018; Xu et al., 2018; Rocha et al., 2014; Cong et al., 2014). Magnetic induction of 1 m T was chosen because it is within the range of values occurring in the environment in the vicinity of AC cables (at a distance of 0.3 m from the cable core with current-carrying capacity of 1600 A and at a distance of 0.25 m from the cable core – 1330 A or at the 0.1 m distance from the cable core – 500 A; the author’s calculations).

2.2. Animal collection and maintenance before the (start of the) experiment The eyed eggs (244 D°) of the rainbow trout (O. mykiss Walbaum 1792) were obtained from the Dąbie Fish Hatchery (Dąbie, Poland). In the laboratory, 1500 eggs were placed on the perforated metal sheets in the experimental aquarium and the same number in the reference tank. The system was filled with fresh, constantly aerated water. For acclimation, the eggs in both tanks were kept for 24 h under constant temperature conditions (T = 9.6 °C).

2. Material and methods 2.3. Sediment and invertebrates 2.1. The experimental system H. diversicolor (Müller, 1776) and L. balthica (Linnaeus, 1758) were collected from a depth of 70–100 cm at Kuźnica station located in the inner part of the Puck Bay (the southern part of the Baltic Sea). The sediment and fauna were transported to the laboratory within 2 h, where they were placed into tanks equipped with a flow-through water system filled with natural seawater (S = 7.2) and were kept at the constant temperature corresponding to field conditions (T = 16 °C) for the period of one week in order to acclimate. One day before starting the experiment, the sediment was sieved through a 1 mm mesh in order to exclude all macrofauna, and after homogenization was added to experimental and reference tanks. The 12 cm sediment layer in each tank was left for 24 h to stabilize, after which 25 individuals of L. balthica and 30 individuals of H. diversicolor were introduced into each tank. Only intact specimens in good condition were chosen for the experiment.

The experimental system for the study of EMF effects on aquatic animals consists of: EMF generator, two aquaria of the same size (V = 25 dm3, 30 × 30 × 28 cm) – the experimental and the reference one – connected to a conditioning tank (V = 300 dm3) equipped with a cooling system (Titan 4000, Aqua Medic, Germany) that regulates and maintains temperature of the water at a constant level; and a closedloop pumping system with mechanical and biological filters. The EMF generator was designed and constructed at the Physics Department of Gdynia Maritime University. It consists of two identical Helmholtz coils arranged in parallel to one another. The generator is powered by an alternating current by connecting the encased regulating autotransformer KIEA 15 (Breve Tufvassons, Sweden) and, for safety reasons, two isolation transformers PFS60 (Breve Tufvassons, Sweden), which allow maintaining the electrical current in the coils up to 20 amps. The generator is equipped with AC gaussmeter GM-2 (AlphaLab Inc., USA), which allows measuring and adjusting values of magnetic flux intensity (induction). Helmholtz coils are cooled by circulating water system connected to the water cooling unit Titan 2000 (Aqua Medic, Germany). The cooling power is adjusted to the value of the electrical current in coils. The generator produces EMF with a frequency of 50 Hz with magnetic induction values in the range 0–1 m T. The experimental aquarium is positioned in the centre of the generator. The size of this aquarium is adapted to the size of the generator in such a way that the generator-produced EMF is uniform almost throughout

2.4. Exposure to electromagnetic field Both invertebrates and fish early life stages were exposed to 50 Hz EMF of 1 m T. The fish were exposed to EMF for 40 days starting with embryos at eyed-egg stage (244°D). The eggs were kept in the dark but after the hatching of larvae the day/night cycle was applied and the larvae were exposed to an artificial light for 12 h a day. The temperature was set at 9.6 °C for the entire period of the experiment. The embryos started hatching after 13 days both in the treatment and control

Fig. 1. (a) EMF distribution in the experimental aquarium. The hatched area indicates the space in which the field stream intensity ranges from 0.95 to 1.05 m T (the average induction value inside the aquarium is 1 m T). The white square shows the contour of tank walls. (b) Dimensions of space between the coils of the generator. 111

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by Heddle et al. (1991); Fenech et al. (2003), and Baršienė et al. (2014). 2.7. Data analysis and statistics Since the genotoxicity and cytotoxicity data obtained in most cases do not follow a normal distribution (Kolmogorov-Smirnov and ShapiroWilk normality test), the nonparametric Mann-Whitney U test was used to evaluate EMF-produced genotoxicity and cytotoxicity effects (GraphPad Prism® 5.01 (GraphPad Software Inc., San Diego, CA, USA)). The results were expressed as mean ± standard error (SE). The level of significance was established at p < 0.05. Fig. 2. Genotoxicity endpoints (nuclear abnormalities (NAs): micronuclei (MN), nuclear buds (NB), nuclear buds and erythrocytes with blebbed nuclei (BL)) induced in erythrocytes of O. mykiss larvae (22–26 mm TL) after exposure to EMF of 1 m T for a period of 40 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20.

3. Results 3.1. Survival The survival of hatched larvae in the treatment group (EMF-exposed group) was 99.1% and in the control group 99.5% (more details on EMF exposure effects on rainbow trout early life stages will be provided in Fey et al., submitted). The survival of H. diversicolor in treatment (EMF-exposed) and control groups were 100 and 99.3% respectively, and those of L. balthica in treatment (the EMF-exposed) and control groups were 92 and 96%, respectively. Individuals of both species spent most of the exposure time buried in the sediment. Beneath the oxidized surface zone of up to 20 mm, there was darker sediment, as determined by visual inspection. The vertical burrows, which were visible from the side of the aquaria, extended to a depth of 9 cm with a thin oxidized zone of approx. 5 mm, indicating typical burrowing behavior of a polychaete. The oxygenated surface layer of the sediment was 2 cm thick, while oxygenated burrows of polychaetes were even 9 cm long.

group. From the 30th day of the experiment, after the absorption of significant yolk-sac volume, the larvae were fed four times a day with commercial feed, which was precisely dosed by an automatic fish feeder AutoFood (JBL, Germany). Invertebrates were exposed to EMF at the constant water temperature (T = 16 °C) for 12 days. The natural seawater and sediment abounded with organic matter, thus animals could feed on both suspended and deposited material, and, therefore, additional feeding was unnecessary (Urban-Malinga et al., 2013,2014; Urban-Malinga et al., 2016). Survival rate and behaviour (the number of individuals on the sediment surface) were recorded every day. Both experiments were monitored daily to remove dead fish larvae or invertebrates from the sediment surface. 2.5. Preparation of samples

3.2. Genotoxicity and cytotoxicity responses in O. mykiss

After 40 days of exposure to EMF, blood samples were collected from 20 specimens of larval O. mykiss (total body length TL 22–26 mm) of the treatment (EMF-exposed) group and from the same number of specimens of the control group. Gill arches were collected from 20 individuals of L. balthica with the shell length of 9–13 mm of the treatment and control groups. The coelomic fluid was collected from 20 H. diversicolor specimens with the body length of 30–38 mm (measured after preservation in ethanol) per treatment. Peripheral blood of rainbow trout specimens was taken from the caudal vein with a drop of blood directly smeared on microscopic slides and air-dried. Small pieces of Baltic clam gills were dissected, softly dragged along a clean slide and allowed to dry. Coelomocytes of common ragworm specimens were obtained by puncturing the coelomic cavity. Coelomic fluid was directly smeared on microscopic slides and air-dried. Dried peripheral blood, gills and coelomic fluid smears were fixed in ethanol for 10 min. and stained with 10% Giemsa solution in phosphate buffer pH = 6.8 for 40 min.

Exposure of rainbow trout to 1 m T EMF for a period of 40 days induced formation of three out of the four analysed genotoxicity endpoints (Fig. 2). MN and NB frequencies exhibited a statistically significant elevation in peripheral blood erythrocytes (Mann-Whitney U test, p = 0.025 and p = 0.027, for MN and NB, respectively). Although the frequency of BL erythrocytes increased 4-fold compared to the control group (Fig. 2), this change was not statistically significant (Mann-Whitney U test, p > 0.05). Cytotoxicity endpoints detected in the EMF-exposed group were 8shaped nuclei, Fr and Ap erythrocytes (Fig. 3). The frequency of 8shaped nuclei erythrocytes showed the highest elevation among the endpoints analyzed and the induced frequency was statistically significant (Mann-Whitney U test, p = 0.002) compared to the control

2.6. Analysis of nuclear abnormalities (NAs) in an in vivo assay The stained slides were analyzed under bright-field Olympus BX51 microscopes (Tokyo, Japan) using an immersion objective (1000×). 4000 erythrocytes with intact cellular and nuclear membranes per fish, 1000 gill cells per mussel and 1000 coelomocytes per common ragworm were evaluated using blind scoring. Final results were expressed as the mean value (‰) of sums of the analyzed individual lesions scored in 1000 cells per organism sampled from every study group. The formation of micronuclei (MN), nuclear buds (NB), nuclear buds on filament (NBf) and cells with blebbed nuclei (BL) were assessed as genotoxicity endpoints, and 8-shaped nuclei, fragmented (Fr), apoptotic (Ap) and binucleated (BN) cells as cytotoxicity endpoints. Nuclear abnormalities were identified using criteria described

Fig. 3. Cytotoxicity endpoints (NAs: apoptotic (Ap), fragmented (Fr) and 8shaped nucleus erythrocytes) induced in O. mykiss larvae (22–26 mm TL) after exposure to EMF of 1 m T for a period of 40 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20. 112

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Fig. 4. Genotoxicity endpoints (NAs: micronuclei (MN), nuclear buds (NB), nuclear buds on filament (NBf) and cells with blebbed nuclei (BL)) induced in L. balthica gills cells after exposure to EMF of 1 m T for a period of 12 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20.

Fig. 6. Genotoxicity endpoints (NAs: micronuclei (MN), nuclear buds (NB), nuclear buds on filament (NBf) and cells with blebbed nuclei (BL)) induced in H. diversicolor coelomocytes after exposure to 1 m T EMF for 12 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20.

group. The frequencies of Fr and Ap nuclei erythrocytes were low (< 0.1‰), while induction of binucleated erythrocytes was not observed at all.

NB frequencies (Mann-Whitney U test, p = 0.0498 and p = 0.02 for MN and NB, respectively). Other genotoxicity endpoints, such as NBf and BL increased approximately 1.8-fold and 1.3-fold, respectively, but the changes were not statistically significant (Mann-Whitney U test, p > 0.05 for both NBf and BL). Fig. 7 shows the frequencies of cytotoxicity endpoints in coelomocytes of H. diversicolor. Exposure to EMF did not cause statistically significant changes in the analyzed cytotoxicity endpoints (MannWhitney U test, p > 0.05 for all endpoints). However, the frequencies of Ap, Fr, and 8-shaped nuclei and BN cells were approximately 1.8fold, 1.9-fold, 1.4-fold and 6-fold, respectively, higher than control values.

3.3. Genotoxicity and cytotoxicity responses in L. balthica Fig. 4 shows frequencies of genotoxicity endpoints in gill cells of L. balthica exposed to 1 m T EMF for 12 days. A statistically significant increase in formation of NBf, NB, and BL cells were observed in the treatment group compared to the control group (Mann-Whitney U test, p = 0.002, p = 0.002, and p = 0.003 for NBf, NB, and BL, respectively). The frequencies of NBf, NB, and BL increased approximately 10-fold, 2fold and 1.7-fold compared to control levels, respectively. The frequency of MN did not increase significantly compared to the control level (Mann-Whitney U test, p > 0.05). Exposure to EMF induced the formation of all analyzed cytotoxicity endpoints in L. balthica gill cells (Fig. 5). The frequencies of cytotoxicity endpoints followed the sequence 8-shaped > Fr > Ap > BN. A statistically significant increase in formation of Ap, 8-shaped, and BN cells in the EMF-exposed group was observed in comparison to the control group (Mann-Whitney U test, p = 0.017, p = 0.004, and p = 0.046 for Ap, 8-shaped, and BN cells, respectively). Although the frequency of fragmented nuclei cells increased 2 times, it was not statistically significant (Mann-Whitney U test, p > 0.05).

4. Discussion

The effect of 1 m T EMF on genotoxicity endpoints in coelomocytes of H. diversicolor after a 12-day exposure is presented in Fig. 6. H. diversicolor exposure to EMF resulted in a significant elevation of MN and

This is the first research to evaluate genotoxic and cytotoxic properties of EMF in aquatic biota. The present study demonstrated that 50 Hz 1 m T EMF can increase frequencies of genotoxicity or cytotoxicity endpoints in cells of both marine (invertebrates) and freshwater (early life stages of fish) organisms. Significantly elevated frequencies of 2 (MN, NB), 3 (MN, NB and 8-shaped) and 6 (NBf, NB, BL, Ap, 8shaped and BN) nuclear abnormalities out of the 8 analyzed were detected in H. diversicolor, O. mykiss and L. balthica cells, respectively. Significant induction of MN was detected in O. mykiss erythrocytes and H. diversicolor coelomocytes after exposure to EMF for 40 and 12 days, respectively. The mechanism of MN formation is well described (Fenech et al., 2003; Hintzsche et al., 2017). They are induced by clastogens or aneugens during cell division. MN arise from acentric chromosome fragments or whole chromosomes during anaphase and are not incorporated into main nuclei at telophase (Hintzsche et al.,

Fig. 5. Cytotoxicity endpoints (NAs: apoptotic (Ap), fragmented (Fr), 8-shaped nucleus and binucleated (BN) cells) induced in L. balthica gills cells after exposure to EMF of 1 m T for a period of 12 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20.

Fig. 7. Cytotoxicity endpoints (NAs: apoptotic (Ap), fragmented (Fr), 8-shaped nucleus and binucleated (BN) cells) induced in H. diversicolor coelomocytes after exposure to 1 m T EMF for a period of 12 days. Asterisks (*) denote significant differences from the control group. In each group, N = 20.

3.4. Genotoxicity and cytotoxicity responses in H. diversicolor

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In the current study, a 12 day-long experiment was performed with invertebrate species to assess long-term effects caused by ELF-EMF. Twelve-day exposure duration was sufficient to record the elevation of all the analyzed geno- and cytotoxicity endpoints. During the second experiment, which was designed to assess the EMF influence on genoand cytotoxicity endpoints in teleost fish during early development, the elevation of 6 out of the 8 endpoints was noted after 40 days of exposure. However, standard errors of the mean of the analyzed endpoints indicated a high deviation of the sample mean from the actual group mean. The study by Mansourian et al. (2016) revealed a nonlinear time response and indicated that the maximum EMF effect on apoptosis in vitro can be noted between 72 h and 5 days. The high standard error detected in our study may suggest time-dependent induction, elimination of the analyzed geno- and cytotoxicity endpoints as well as variability in the susceptibility of the fish. Moreover, fish susceptibility to EMF during the embryonic and larval development can vary. Further studies of EMF with a frequent sampling period are necessary to determine fluctuations, differential occurrence, life span of geno- and cytotoxicity endpoints and to compare the susceptibility of early life stages. At present the exact mechanism of EMF genotoxic and cytotoxic effect on organisms is unknown. Shen et al. (2016) emphazised that the EMF-provoked molecular irregularities are responsible for DNA damage. Several studies suggested that EMF prolongs the life of free radicals and induces oxidative DNA damage (Yokus et al., 2005; Lai and Singh (2004); Zmyslony et al., 2000). As reported by Simkò (2007), cell type specific redox status may lead to diverse EMF-induced effects. The present study indicated different genotoxicity and cytotoxicity endpoints and their frequencies in analyzed cells of selected aquatic organisms exposed to EMF. Disruption of redox homeostasis can initiate DNA damage; however, further effects of EMF depend on cell types and their homeostatic state (Simkò, 2007). Other underlying mechanisms may be an induction of electric currents in DNA (Porath et al., 2000; Wan et al., 2000) or altered structure and function of proteins, which are related to DNA metabolism (Ravera et al., 2006; Sahebjamei et al., 2007; Blank and Soo (2001)). However, multiple factors may lead to DNA damage. Most of the bioelectromagnetics research indicated oxidative stress as a main mechanism responsible for the genotoxicity and cytogenetic damage (Wolf et al., 2005; Yokus et al., 2005; Bułdak et al., 2012; Mahmoudinasab et al., 2016). The intracellular effects of EMF on our test organisms are probably also indirect and elucidation of the real nature of these effects needs future research.

2017). The majority of previously performed genotoxicity studies were dealing with medical applications of MF or radiofrequency EMF, thus undisputable conclusions about the EMF effect on aquatic biota cannot be drawn from their results. Very few in vivo genotoxicity and cytotoxicity studies of EMF have been carried out and such evidence is limited to magnetic induction values, which generally occur in the environment. Considering that EMF potential to induce NAs in aquatic animals was not investigated previously, it is difficult to compare and discuss the results of the current study. The formation of MN in vivo or in vitro after exposure to EMF was previously investigated in newborn rat astrocytes (Miyakoshi et al., 2005), in rat primary astrocytes and male C57BL/6 J mice (Herrala et al., 2018), in rat tracheal cell lines (Lagroye and Poncy, 1997) and in human blood cells (Stronati et al., 2004). However, in those studies the genotoxic effect of selected intensity EMFs was not detected. Low-frequency EMF emits nonionizing radiation. However, the energy is considered not strong enough to break intermolecular chemical bonds, therefore effects of EMF at the molecular level are rather of indirect nature (Lagroye and Poncy, 1997; Vijayalaxmi and Obe, 2005). Although EMF has the ability to penetrate into deep tissues and affect cell functions, the produced effects highly depend on field strength, frequency and exposure period (Pilla, 2013; Carpenter and Ayrapetyan (1994)). Ruiz-Gómez and Martínez-Morillo (2009) in review on DNA strand breaks induced by magnetic field exposure concluded that MF could be a co-inductor of DNA damage but not a genotoxic agent. Therefore, the intracellular effects of EMF can occur indirectly. Notwithstanding, other studies reported the in vivo sensitivity of mammals to the genotoxicity of EMF. Significant oxidative DNA damage and lipid peroxidation in rats exposed to ELF-EMF for 50 and 100 days (50 Hz, 0.97 m T) were found (Yokus et al., 2005). Higher frequency of MN formation was detected in Wistar rat (bone marrow cells) exposed to 50 Hz, 1 m T EMF (Erdal et al., 2007). Tkalec et al. (2013) showed that radiofrequency EMF (RF-EMF 900 MHz) induces DNA damage in the earthworm Eisenia fetida coelomocytes. In this study, exposure to EMF induced a significant elevation of NB in analyzed tissue cells of all the three aquatic species. Several experimental studies showed that genotoxic action of EMF may be mediated by an interaction with DNA-repair mechanisms (Robison et al., 2002; Ruediger, 2009). Other nuclear abnormalities such as nuclear buds, nuclear buds on filament and cells with blebbed nuclei are associated with MN (Fenech et al., 2016; Harabawy and Mosleh, 2014). Exposure of L. balthica to EMF resulted in a significant increase of all NAs associated with MN. These nuclear abnormalities are biomarkers of gene amplification and elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells (Fenech et al., 2011, 2016). An increased frequency of nuclear buds was found in human liver and neural cells lines (C3A and SH-SY5Y) exposed to ELF magnetic field (10 and 50 μT) (Maes et al., 2016). In the present study, significant induction of certain cytotoxicity endpoints was detected in O. mykiss and L. balthica cells. Cells with 8shaped nuclei showed the largest increase of all the analyzed cytotoxicity endpoints in both species. Frequencies of apoptotic and binucleated cells were significantly elevated only in L. balthica gills. Bi-nucleated and 8-shaped nucleus cells are formed during abnormal cell division and are the result of blocked cytokinesis (Cavas et al., 2005; Baršienė et al., 2014). Apoptosis is a type of programmed cell death and a mechanism-based biomarker of pollutants and environmental stressors (Sokolova, 2009; Kiss, 2010). In contrast, several studies did not detect changes in cytotoxicity response after EMF exposure (Mahmoudinasab et al., 2016; Ross et al., 2018). No cytotoxic effect and morphological changes in MCF-7 cells were noted after exposure to EMF at 0.25 m T and 0.50 m T (Mahmoudinasab et al., 2016). Ross and co-authors (2018) reported that 5 Hz, 0.4 m T EMF does not induce any cytotoxic effects in MSCs/pericytes after 2 weeks of exposure. However, the meta-analysis of the published data performed by Mansourian and co-authors (2016) demonstrated that ELF-EMF have a significant impact on apoptosis level in vitro.

5. Conclusions The present study indicated a significant genotoxic and cytotoxic activity of EMF in aquatic organisms in vivo. It provided the first evidence that EMF of the value typically generated by submarine cables, significantly and negatively affected important benthic species and early life stages of a typical representative of salmonid fish. Therefore, it might be concluded that the cables that are present in the natural aquatic environment may pose threat to those organisms. Micronuclei together with other nuclear abnormalities are markers of cytogenetic damage such as chromosomal loss or disruption of the mitotic apparatus. Therefore, affected integrity of genetic information can lead to cell death, genomic instabilities, which can cause a variety of diseases and disorders, including cancer development. Moreover, our results revealed the usefulness of selected geno- and cytotoxicity endpoints for the investigation of EMF-inflicted cytogenetic damage on aquatic organisms. Based on the study results, L. balthica could be proposed as one of the most suitable bio-indicators for the assessment of EMF-induced geno- and cytotoxicity. The evaluation of EMF geno- and cytotoxicity in teleost fish (O. mykiss) during early development stages suggests the existence of time-dependent induction and elimination of the analyzed endpoints. Moreover, the potential to acclimate or individual sensitivity to EMF exposure may appear. However, further studies are required to 114

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reveal the unique mechanisms involved in EMF induced genotoxicity and cytotoxicity in different aquatic organisms. The findings presented in this paper are also important because currently there are no standards set for the introduction of magnetic fields into the aquatic environment but technical solutions that may reduce the introduction of EMF are available. They include, for example, laying cables in close proximity to each other or using direct current cables of high voltage (HVDC), which introduce a static magnetic field. Therefore, effects of static and alternating fields on genotoxic and cytotoxic responses as well as other biomarkers of environmental stress in marine and freshwater species require urgent attention and more extensive research.

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