Effects of electromagnetic radiation from offshore wind power on the physiology and behavior of two marine fishes

1、Introduction：

Offshore wind power is one of the most established/developed power generation methods in the field of renewable energy technology, and it has very positive significance in solving the energy crisis and adjusting the energy structure (Tsai et al., 2023). According to the “China Wind Power Development Roadmap 2050”, China's open offshore wind en- ergy resources in sea areas with 5 to 50 m underwater and 100 m above water are 500 million kW, with a total area of 3.94 million km2 (Ren et al., 2021). In fact, with the construction and operation of offshore wind power facilities, there have been concerns about the environ- mental consequences of transmitting power through an extensive

network of cables between devices and onward to onshore grid systems (Farr et al., 2021).
Offshore wind turbines, booster stations, and submarine cables are the main sources of electromagnetic radiation (ER) associated with offshore wind power. In addition, wind turbines and booster stations are located on the sea surface and electromagnetic radiation attenuates quickly from different media (Ai et al., 2022). Accordingly, the impact of ER from wind turbines and booster stations on marine life appears to be limited. Since the cable insulation shield shell is grounded and shields the electric field, the outside of the cable is mainly exposed to magnetic field radiation (Hutchison et al., 2018). Therefore, with the increasing scale of high-voltage, high-current generated by the long-distance laying

of submarine cables in large-scale marine wind farms, the expansion of the ER-affected area and the intensification of its impact are likely to affect marine ecological environment (Riefolo et al., 2016).
To date, most laboratory research on the physiological and ecolog- ical effects of ER has focused on terrestrial organisms. For example, ER of a certain intensity has been shown to reduce hemoglobin in rats (Cakir et al., 2009), pulsed electromagnetic fields can cause abnormalities in multiple indicators in rats, increase apoptosis and necrotic cells, and cause body damage (Emre et al., 2011). Juutilainen (2005) found that ELF had no obvious effect on the growth and development of mammals, but promoted bone development. Other studies have shown that when a pulsed magnetic field is applied, the ECG amplitude in rats increases several folds, and the amplitude rises as the current increases (Slabbekoorn et al., 2010). ELF has been observed to influence the memory ability of mice “window effect” (Duarte et al., 2021). It is worth noting that marine organisms share similarities with terrestrial mam- mals in terms of their nervous systems, sensory organs, and mechanisms for stabilizing their internal environments, so this raises the question: how will electromagnetic radiation affect the marine fishes?
There is no doubt that the introduction of large quantities of ER into the marine environment will impact aquatic organisms (Tricas and Gill, 2011). In fact, the effects of ER on the behavior of different marine or- ganisms vary considerably, especially among crustaceans. For example, the Antarctic sand flea (Gondogeneia antarctica) become disoriented after being exposed to extremely low-frequency electromagnetic fields of 20 nT and below for 1 min (Tomanova and Vacha, 2016), while the Euro- pean lobsters (Homarus gammarus) show no obvious change in their behavioral pattern when subjected to a magnetic field of 200 μT (Taormina et al., 2020). However, the edible zodiac crab (Cancer pagu- rus) shows obvious magnetotactic behavior (Scott et al., 2018). Simi- larly, when Hediste diversicolor was exposed to magnetic field interference, its burrowing behavior was significantly enhanced (Jakubowska et al., 2019). For fish, when the electric current generates a magnetic field, migratory fish and other marine life may be affected, as magnetic fields can influence their direction of movement (Nyqvist et al., 2020). Additionally, submarine cables may disrupt or alter the geomagnetic patterns used by migrating fish, thereby affecting physio- logical processes such as reproduction and development (Gill et al., 2014). However, fish as an important biological and economic compo- nent of the marine ecosystem (Dyck and Sumaila, 2010), their ability to perceive and adapt to electromagnetic radiation is still unclear. Furthermore, beyond the uncertain physiological mechanisms, toxic endpoints of ER are rarely considered.
The large yellow croaker (Larimichthys crocea) is the most widely farmed seawater fish species with the highest aquaculture output in China (Ding et al., 2020), whose annual production exceeds that of all other marine fish species cultured in single cages (Sun et al., 2017). In addition, it has extremely high nutritional and economic value and is very popular among consumers (Wang et al., 2017). The black sea bream (Acanthopagrus schlegelii) is a warm-water bottom-dwelling fish widely distributed in the northwestern Pacific Ocean. Due to its habitat char- acteristics, it also has a very high economic value and is a primary focus of aquaculture expansion in China (Ma et al., 2008). Thus, the aqua- culture habitats of both fish species are strongly affected by ER from offshore wind power facilities (Zheng et al., 2012). In summary, studying the impact of electromagnetic radiation on these two fish species is crucial for optimizing the economic benefits of aquaculture and maintaining their ecological stability. In this experiment, L. crocea andA. schlegelii were taken as experimental organisms to investigate the physiological and behavioral effects of different electromagnetic radia- tion intensities. Five electromagnetic radiation intensities, 0mT, 0.5mT, 1.0mT, 1.5mT, and 2.0mT were applied over a total exposure of 28 days. Behavioral observations and biochemical analyses were conducted to establish the relationship between biological behaviours and physio- logical mechanisms, revealing the mechanisms of toxicity of electro- magnetic radiation on these two fish species. This study can provide

valuable insights for assessing the toxicity and risk associated with offshore wind power facilities and their intensity to offshore economi- cally important fish.

2. Materials and methods：
   2.1. Generation of magnetic field
   The magnetic field was generated using a Helmholtz coil. Based on the electromagnetic field distribution characteristics of the Helmholtz coil, the experimental fish tank was positioned along the central axis of the Helmholtz coil. The entire fish tank was placed within a uniform magnetic field area encompassing two-thirds of the radius from the center. The coil was connected to an external power supply (WANPTEK, GPS605D, China) to generate a magnetic field, with its strength adjustable by varying the current and voltage.
   2.2. Experimental animals
   L. croceas (10.9 ± 1.6 cm body length, 17.3 ± 5.2 g wet weight, mean ± SD) and A. schlegelii (5.1 ± 1.7 cm body length, 3.5 ± 1.4 g wet weight, mean ± SD) were collected from Xiangshan County (29o 28,39.288"N, 121o 52,9.012"E), Zhejiang Province. The temporary rearing conditions were recorded as follows: sand-filtered seawater temperature 22.0 ± 0.5 oC, pH 7.99 ± 0.1, salinity 21 ± 0.5 PSU, and dissolved oxygen 6.1 ± 0.5 mg/L. A total of 600 fish from each species were kept in two separate breeding barrels (1500 L) respectively, and acclimated for two weeks under conditions simulating their natural habitat. The fish were fed twice a day.
   2.3. Experimental treatments
   After a two-week acclimation period, 300 healthy L. crocea and A. schlegelii were selected. They were exposed to different strengths of ER treatments. No individual deaths occurred during the 21-days exposure period and 7 days recovery period. Different fish respond differently to different paradigms of ER (Nyqvist et al., 2020). Such as, Yuan et al. (2016) found that the survival rate and behavior of A. schlegelii were affected to a certain extent in a magnetic field with a strength of 1.0mT. Similarly, other studies have reported that fish exhibit escape behavior under a static magnetic field of 3.7mT under ER, etc. (Bochert and Zettler, 2004; Hermans et al., 2024). Therefore, considering that our experimental subjects were in the larval stage, each fish species was divided into five different groups based on ER intensity (each group had 3 replicate pools, with 20 fish per pool): 0mT group (control group), 0.5mT group, 1.0mT group, 1.5mT group, and 2.0mT group.
   2.4. Oxidative stress, immune and nutritional biomarkers measurement
   Total protein (TP) was detected through the Coomassie Brilliant Blue (G-250) method (Bradford, 1976). Following the kit instructions, 0.1 g of the intestine was combined with 0.9 mL of normal saline. The mixture was homogenized using a Teflon Potter-Elvehjem homogenizer (CEBO- 64, China) at 4 oC. Subsequently, the samples were centrifuged at 2500 rpm for 10 min to obtain the supernatant. Absorbance measurements were conducted using an automatic microplate analyzer (Flexstation®3, Molecular Devices, California, USA).
   The activity of Superoxide Dismutase (SOD) was determined by measuring the reduction of nitrite. Catalase (CAT) activity was deter- mined by measuring the initial rate of absorbance decrease at 405 nm using the ammonium molybdate method, as described by Goth (1991). Glutathione (GSH) levels were assessed following the procedure out- lined by Gheita and Kenawy (2014). Malondialdehyde (MDA) levels were quantified using the thiobarbituric acid (TBA) assay.
   Acid phosphatase (ACP) activity (product code: A060-2-1) and
3. Alkaline phosphatase (AKP) activity were measured by the micro- enzyme labeling method. Lysozyme (LZM) activity was measured using the turbidimetric method.
4. Glucose (GLU) levels were detected using the glucose oxidase method, while lactic acid (LD) levels were measured by colorimetry. Cortisol levels were assessed through an enzyme-linked reaction. All the above measurements were performed using commercial kits (A045-2-2 for TP, A001-3 for SOD, A007-1-1 for CAT, A006-2-1 for GSH, A003-1-1 for MDA, A060-2-1 forACP, A059-2 forAKP, A050-1-1 for LZM, A154-1- 1 for GLU, H094-1-1 for Cortisol, Nanjing Jiancheng Bioengineering Institute, China), absorbance measurements were obtained using an automatic microplate analyzer (Flexstation®3, Molecular Devices, Cal- ifornia, USA).
5. 2.5. Lateral line cell viability and reactive oxygen species assay
6. Cell viability (product code: C0038) was determined using kits from Beyotime biotech (Shanghai, China), which is based on WST-8 and widely used for the rapid and highly sensitive detection of cell prolif- eration and cytotoxicity (absorbance was measured at 450 nm). Reactive
7. oxygen species (ROS) (product code: E004-1-1) levels were assessed using kits from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China) via a chemical fluorescence method (fluorescence measured at an excitation wavelength of 488 nm). The excitation light and absorbance were measured using an automatic microplate analyzer (Flexstation®3, Molecular Devices, California, USA).
8. 2.6. Behavioral biomarkers measurement
9. The frequency of operculum and fin movements was calculated by counting the number of operculum and fin movements per minute. The swimming velocity was measured by placing the fish in a 55 cm × 45 cm × 30 cm tank, recording their movement with a camera (KNR0508, KOH-I-NOOR, Nanjing, China) for 5 min (repeated three times for each group), and analyzing the recordings using idTracker version 2.1 (Instituto Cajal, Spain). The feeding frequency was calculated by measuring the feed consumption in the feeding area after feeding and recording the fish's residence time in the area. Feeding frequency = feed weight (g)/residence time (min).

2.7. Statistical analysis
All data were assessed for normal distribution using Levene's test (SPSS 26.0). The two-way ANOVA was used to analyze the effects of electromagnetic intensity and sampling time points. Student's t-test was applied to examine significant differences between parameters in different treatments. Results were presented as mean ± standard devi- ation, with P < 0.05 indicating significant differences between groups. Finally, the data were subjected to PCA and visualized using Origin Pro 2021 and GraphPad Prism 10.0.