Abstract ［Objectives］ This study was conducted to investigate the effect of tetrabromodiphenyl ether (BDE-47) on the activity of 11-ketotestosterone (11-KT) in tilapia liver, with a view to understanding the potential hazard of BDE-47 on fish and aquatic ecosystems from the perspective of sex steroid hormones.
　　［Methods］Adopting the semi-static water exposure method, 3 exposure concentrations of 5, 50, and 500 μg/L and 5 sampling time of 1, 3, 7, 15, and 30 d were set to investigate the effect of BDE-47 on 11-ketotestosterone in tilapia liver.
　　［Results］ The low concentration of BDE-47 (5 μg/L) had no effect on the 11-KT level of tilapia liver; and when exposed to high concentrations of BDE-47 (50 and 500 μg/L), 11-KT in the liver of tilapia was first suppressed and then returned to the normal level. Because the fish reproductive process is completed under the coordinated regulation of sex steroid hormones, significant changes of 11-KT in the liver of tilapia may cause its reproductive dysfunction to a certain extent.
　　［Conclusions］This study provides relevant toxicological data for promoting the formulation (revision) of relevant water quality standards and the formulation of limit standards, and facilitating the protection of aquatic living resources and aquatic ecosystems.
　　Key words 2, 2′, 4, 4′-Tetrabromodiphenyl ether; Tilapia; Liver; 11-Ketotestosterone
　　Received: January 13, 2021  Accepted: March 4, 2021
　　Supported by National Key R&D Program (2020YFD0900502); Special Project of National Characteristic Freshwater Fish Industry Technology System (CARS-46).
　　Shunlong MENG (1982-), male, P. R. China, researcher, PhD, devoted to research about environmental toxicology, fishery environmental protection, aquatic product quality and safety risk assessment.
　　*Corresponding author. E-mail: chenjz@ffrc.cn; xup@ffrc.cn.
　　Polybrominated diphenyl ethers (PBDEs) are brominated aromatic hydrocarbons, which are divided into 209 homologues based on the number of bromine atoms in their molecules. PBDEs began to be industrialized in the 1940s and 1950s. As excellent flame retardants, PBDEs are widely added to rubber, resin, polyurethane and other polymer materials to make fireproof materials. However, due to the strong stability of PBDEs and their release into the environment following the incineration of waste industrial products containing PBDEs, they have been widely detected in water, air, soil, sediments, animals and plants, and humans. Because PBDEs have the characteristics of environmental persistence, long-distance transmission, bioaccumulation, endocrine disrupting effects, etc., the environmental problems they cause have attracted more and more attention from the society, and they have been listed as new POPs and their production and use have been restricted. 　　The reproductive process of bony fishes is regulated by the hypothalamus-pituitary-gonad-liver axis［1］. The first step in the regulation process is the secretion of gonadotropin-releasing hormone from the hypothalamus. The second step is to transport the gonadotropin-releasing hormone to the anterior pituitary through the circulatory system to bind to the gonadotropin-releasing hormone receptor in the pituitary. The third step is that the conjugate instructs the anterior pituitary to secrete gonadotropins, including luteinizing hormone (LH) and follicle stimulating hormone (FSH). In the fourth step, the circulatory system transports FSH and LH to the gonads to bind to the follicle-stimulating hormone receptor and luteinizing hormone receptor of the gonads. The fifth step is that the conjugate guides the gonads to secrete steroid hormones, including E2, T and 11-KT. The sixth step is to transport steroid hormones to various parts to regulate the changes in fish reproductive organs and physiological processes［1］. Some pollutants at low concentrations will not cause fish death or growth abnormalities, but they will participate in the physiological and biochemical reactions at the molecular level in the fish body and interfere with normal physiological activities, such as too-high or too-low sex hormone levels［2］. Fish sex steroid hormones, such as T, 11-KT, E2, etc., play an important role in the regulation of fish gonadal development, yolk formation, egg cell maturation, sperm formation, etc. during the sexual maturation of fish［3］. The pathway of fish VTG synthesis is as follows: fish ovaries synthesize estrogen, and transport it to the liver to bind with ERs in the liver, forming a hormone-receptor complex, which transforms its conformation to bind to the promoter sequence on RNA, which leads to the expression of the VTG gene to initiate the synthesis of VTG polypeptide chain and finally synthesize VTG［4］. VTG is transported into the ovary to produce vitellin then through related enzymatic reactions［5］.
　　The sex differentiation of fish is easily affected by exogenous hormones, and even some fish have sexual reversal due to the stimulation of exogenous hormones. Therefore, environmental exogenous hormones will affect the normal reproduction process of fish and have a negative impact on the ecosystem. In this study, the effect of tetrabromodiphenyl ether (BDE-47) on the activity of 11-ketotestosterone (11-KT) in the liver of tilapia was investigated, hoping to understand the potential hazards of BDE-47 to fish and aquatic ecosystems from the perspective of sex steroids, and obtain relevant toxicological data for promoting the formulation (revision) of relevant water quality standards and the formulation of limit standards and facilitating the protection of aquatic living resources and water ecosystem. 　　Materials and Methods
　　Materials
　　The test fish used in this experiment was Oreochromis niloticus. The fish were taken from the test site of the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences. After 4 weeks of domestication, the fish with strong activity were selected to start the experiment. The average body length and weight were measured before the start of the experiment. The results showed that the average body weight was (38.30±3.98) g (n=40), and the average body length was (13.57±0.50) cm (n=40).
　　Experimental water
　　The experimental water was ordinary tap water after three days of aeration. During the experiment, the water temperature was 24.3-25.5 ℃, the dissolved oxygen was 6.3-7.0 mg/L, and the pH value was 6.5-7.5. The experimental water met the fishery water quality standard (GB11607-89).
　　Main reagents and instruments
　　Main reagents: Tetrabromodiphenyl ether (Wuhan Kaimeike Chemical Technology Co., Ltd.); dimethyl sulfoxide (DMSO) (Jiangsu Qiangsheng Functional Chemical Co., Ltd.); enzyme-linked immunoassay kit (Shanghai Lengton Biotechnology Co., Ltd.).
　　Main instruments: Glass homogenizer; constant temperature water bath; low temperature refrigerated centrifuge (Sigma 26K); multi-function microplate reader (Spectra Max M5); constant temperature box; pipettes of various specifications; distilled water; test tubes; beakers; absorbent paper; measuring cylinder.
　　Preparation of reagent mother liquor: A certain amount of BDE-47 (50 mg) was accurately measured, dissolve in DMSO, and diluted to constant volume in a 25 ml brown volumetric flask. The prepared BDE-47 mother liquor had a concentration of 2×103 mg/L and stored in the dark at 4 ℃ for later use.
　　Experimental design
　　This experiment was set with 3 concentration gradients: 5, 50, and 500 μg/L, and a blank control and a solvent control were also set (the solvent concentration was the same as the solvent concentration in the highest concentration of poisoning solution). The poisoned groups and the control were all set with three parallels. 300 L aquaria were equipped with 200 L of the above-mentioned solutions in various concentrations, respectively, and 40 healthy tilapias were randomly added in each parallel group. The tilapia were fed once in the morning and once in the evening every day during the temporary rearing and experiment with a total feeding amount of 3% of the tilapia body weight, and oxygenation was performed with air compressor. The renewing static water quality contact test method was used. During the experiment, half of the contaminated water was discharged from each water tank every day, and the drug and water was added to make the total poisoning solution volume and drug concentration unchanged. The experimental period was 30 d［7］. 　　In order to obtain the actual content of BDE-47 in each test group during the experiment, we referred to the method provided by Sha Jingjing et al. to sample and determine the experimental water. After analysis, the BDE-47 concentrations after 24 h of exposure were as follows: 0, 0, 4.86, 57.28, and 343.92 μg/L, respectively. The results showed that the actual concentration in the high-dose concentration groups were lower than the concentrations set in the experimental design. Relevant data show that due to its high lipophilicity, PBDEs have very low water solubility in actual water bodies and are easy to adsorb on container walls and in bottom sludge. Therefore, it was normal for the measured concentrations of the water samples to be slightly lower than the experimental design concentrations during the experiment.
　　Sample collection and testing
　　The tilapia samples of each concentration group were collected on the 1st, 3rd, 7th, 15th, and 30th d after exposure. Feeding was stopped the day before sampling. At the time of sampling, 2 test fish were randomly selected from each group of 3 parallels, and measured for their body lengths and weights, and then tilapia were quickly put to death to take 1 g of fish liver. Three parts of liver were preserved, and the remaining 3 parts of liver entered the next step of the experiment.  The liver tissue was rinsed with normal saline at 4 ℃ to remove blood clots on it, wiped to remove water and put into a small beaker. First, a pipette was used to transfer 2/3 of the pre-cooled normal saline ［the total volume of normal saline (ml) was 9 times the tissue weight (mg)］ into the beaker, and the liver tissue was cut with scissors while keeping the temperature low. The liver fragments were poured from the beaker into a homogenizer, and then the beaker was flushed with the remaining 1/3 of normal saline, which was then poured into the homogenizer. The liver fragments were fully ground to a homogenate (the homogenization process needed to be kept at low temperature). Centrifugation was performed at 3 000 r/min and 4 ℃ for 10 min in a low-temperature centrifuge. The supernatant was added in a centrifuge tube and stored in a refrigerator at 4 ℃.
　　The determination index was 11-ketotestosterone (11-KT) in tilapia liver. The determination operation steps were based on the operation method of the kit.
　　Statistical analysis
　　Considering that the experimental duration was 30 d, which is a relatively long time for tilapia with a relatively short life cycle, the enzyme activity and hormone level of the tilapia body might also change to some extent due to its own growth during the experiment. Therefore, in order to more scientifically evaluate the effects of BDE-47 on the liver enzyme activity and hormone levels of tilapia, the results of the experiment were expressed as relative values, namely: the measurement result of the treatment group at the same measurement time/the result of the blank measurement group×100%. The data results were expressed as mean±standard deviation (mean±sd) (n=3). The experimental data were analyzed for variance with SPSS 17.0 statistical software, and Tukeys multiple comparisons were used to analyze the differential effects of concentration and time. 　　Results and Analysis
　　During the entire experimental period, there was no death of tilapia. The effect of BDE-47 on the 11-KT content of tilapia liver is shown in Fig. 1. During the entire experimental period, there was no significant difference in enzyme activity between the BDE-47 solvent control group and the blank control group (P>0.05). On the 1st d of exposure, the contents of various groups were basically the same, and there were no significant differences (P>0.05). BDE-47 had no effect on the content of 11-KT in the liver of tilapia in a short time. On the 3rd d after exposure, the contents of the 50 and 500 μg/L groups decreased, so the BDE-47 inhibited the 11-KT content of tilapia liver, and the inhibitory effects reached 60%, leading to significant differences (P<0.05). On the 7th d of exposure, there were no significant changes in the 11-KT content between various concentration groups. There was no significant difference between the 5 and 50 μg/L groups (P>0.05), and only the 500 μg/L group showed inhibition, which reached 20%. On the 15thd after exposure, the 11-KT contents in various concentration groups did not change significantly, and the differences were not significant (P>0.05). On the 30thd after exposure, the contents of 11-KT in various concentration groups were basically the same, and there was only a difference between the 5 and 500 μg/L groups. In general, at concentrations of 5 and 50 μg/L, the time effects of BDE-47 on the 11-KT content of tilapia liver
　　Agricultural Biotechnology2021
　　had the same changing trends, generally first decreasing and then recovering to the level the same as the control group. At the concentration of 500 μg/L, the content decreased in the early stage, recovered to the level equal to that of the control group in the middle of the experimental period, and decreased again in the later stage.
　　Conclusions and Discussion
　　Fish have two main androgens, namely T and 11-KT, the main function of which is to maintain the activity of spermatogenic cells and testicular sustentacular cells, and to regulate the spermatogenesis process of male fish［8-10］. The T of fish is synthesized by the tunica vaginalis layer on the follicular membrane and converted to 11-KT under certain conditions. The contents of the two hormones in the fish have a strong correlation. Research data shows that the concentration of 11-KT in the blood is usually higher than that of T, and it is the main androgen in fish. Because T and 11-KT are easily affected by exogenous environmental hormones, they are often used as biomarkers for the evaluation of environmental hormones［11-13］. 　　Studies on tilapia show that under natural conditions, when tilapia grows to sexual maturity, with the development of testes and sperm formation in the fish, the content of 11-KT in its plasma gradually rises during the process of excretion［14］. The significant reduction of 11-KT content in male tilapia fish may cause reproductive disorders such as decreased testis weight, decreased sperm count, and decreased sperm motility. Therefore, in the liver of tilapia under the stress of BDE-47 in this study, a significant decrease (P<0.05) in the content of 11-KT was determined, indicating to a certain extent that BDE-47 at a concentration higher than 50 μg/L produced a environmental hormone interference effect and might have caused damage to the reproductive system of tilapia. This study showed that when tilapia was exposed to two concentrations of 50 and 500 μg/L, the 11-KT content was significantly reduced on the 3rd d of the experimental period (P<0.05), and then recovered. The 11-KT content was inhibited at the 3rd d, but then recovered with the prolonged exposure time. It was inferred that when tilapia was exposed to the two concentrations of 50 and 500 μg/L, the 11-KT content in the liver was stressed for a short time. After a period of time, the tilapia body developed a compensation mechanism, and the 11-KT content in the liver returned to the normal level.
　　The low concentration of BDE-47 (5 μg/L) had no effect on the 11-KT level of tilapia liver; and when exposed to high concentrations of BDE-47 (50 and 500 μg/L), 11-KT in the liver of tilapia was first suppressed and then returned to the normal level.
　　BDE-47 with a concentration higher than 50 μg/L caused abnormal 11-KT levels in the liver of tilapia, and the endocrine system of tilapia was disturbed, causing a certain degree of disorder in the anabolic process of sex steroid hormones. Because the fish reproductive process is completed under the coordinated regulation of sex steroid hormones, significant changes of 11-KT in the liver of tilapia may cause its reproductive dysfunction to a certain extent.
　　References
　　［1］ HOU J. Effects of microcystin-LR on zebrafish reproduction and growth and its mechanism［D］. Wuhan: Huazhong Agricultural University, 2017. (in Chinese)
　　［2］ LI WM, YIN DQ, HU SQ, et al. Effects of two chloric-nitroanilines on serum sex steroids in carp (Carassius auratus)［J］. Journal of Nanjing University: Natural Science, 2001, 37(6): 707-712. (in Chinese) 　　［3］ MENG SL. Effects of the environmental estrogen methomyl on the hypothalamic-pituitary-gonad axis and antioxidant defense system of male tilapia［D］. Nanjing: Nanjing Agricultural University, 2014. (in Chinese)
　　［4］ ZHANG J. Study on the effects of A/O, A-2/O and typical advanced oxidation technologies on reducing wastewater toxicity［D］. Dalian: Dalian University of Technology, 2014. (in Chinese)
　　［5］ CHEN YP, QIAN XY, PAN N, et al. Effect of printing-dying sewage on vitellogenin and metallothionein level of Carassius auratus［J］. Journal of Hydroecology, 2011, 32(6): 95-99. (in Chinese)
　　［6］ LIU T, MENG SL, QIU LP, et al. The effect of methomyl on caspase activity in male tilapia testis［J］. Environmental Science & Technology, 2016, 39(12): 37-41. (in Chinese)
　　［7］ WEI H, ZHANG GF. The harm of environmental endocrine disruptors (EDCs) to aquaculture［J］. China Fisheries, 2006, (3): 72-74. (in Chinese)
　　［8］ LI GL, LIU XC, LIN HR. Aromatase inhibitor letrozole induces sex inversion in the protogynous red spotted grouper (Epinephelus akaara)［J］. Acta Physiologica Sinica, 2005, 57(4): 473-479. (in Chinese)
　　［9］ HU HX, CHANG BB, WANG WEI, et al. The function and research progress of 11-ketotestosterone in sturgeon gonadal development ［J］. China Fisheries, 2020, (5): 104-106. (in Chinese)
　　［10］ LAI XJ, CHEN SX, LAI GY, et al. Chinese herbs Epimedium brevicornum Maxim. and Cuscuta chinensis Lam. induced ovary development in Japanese eel Anguilla japonica［J］. Journal of Fishery Sciences of China, 2019(2): 314-321. (in Chinese)
　　［11］ ZHUANG TF, HAN J, LIU LY, et al. Reproductive toxicity caused by co-exposure of zebrafish to progestin megestrol acetate and estrogen 17α-ethinylestradiol［J］. Environmental Chemistry, 2017, 36(7):1440-1450. (in Chinese)
　　［12］ LIU SZ, LYU XY, GAO GB, et al. Effect of 17 alpha-methyl testosterone (MT) on hormone levels of Gobiocypris rarus［J］. Animal Husbandry and Feed Science, 2016, 37(6):1-3. (in Chinese)
　　［13］ HUA JH, HAN J, GUO YY, et al. Reproductive toxicity in male zebrafish after long-term exposure to low concentrations of progestin levonorgestrel［J］. Asian Journal of Ecotoxicolog, 2019, 14(2):176-186. (in Chinese)
　　［14］ LIU XL, WANG Q, JIA LZ, et al. Alterations in testicular histology and the mRNAs of enzymes responsible for sex steroid synthesis in the zebrafish Danio rerio exposed to nonyphenol［J］. Acta Scientiae Circumstantiae, 2011, 31(11): 2523-2529. (in Chinese)