Bulletin of Environmental Contamination and Toxicology (2019) 102:708–713 https://doi.org/10.1007/s00128-018-2513-3 Acute and Sublethal Effects of Ethylmercury Chloride on Chinese Rare Minnow (Gobiocypris rarus): Accumulation, Elimination, and Histological Changes Dandan Cao1 · Bin He1,2 · Yongguang Yin1,2,3 Received: 15 October 2018 / Accepted: 30 November 2018 / Published online: 4 December 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Ethylmercury (EtHg) has been widely observed in the environment due to anthropogenic contamination and/or environmental ethylation of inorganic mercury. Herein, the acute and sublethal effect of EtHg chloride on Chinese rare minnow (Gobiocypris rarus) as a fish model was studied. EtHg chloride showed an obvious toxicity to 4-month-old Chinese rare minnow (LC50 24.8 µg L−1 (as Hg) at 24 h). Histological analysis revealed that acute EtHg exposure can induce necrosis, telangiectasis and exfoliation of epithelial cells in the gill, as well as edema, vacuoles, and pyknotic nuclei in hepatocytes. Sublethal dose exposure revealed a very high accumulation of EtHg in fish, which is subsequently metabolized to inorganic mercury and eliminated after depuration. A new mercury species, possibly diethylmercury, was also observed as the metabolite of EtHg in rare minnow. The present study provides useful information for assessing the risks of EtHg and understanding its bioac- cumulation in aquatic organisms. Keywords Ethylmercury · Chinese rare minnow · Histopathological change · Bioaccumulation · Elimination Mercury, a widespread pollutant that affects both human The inevitable release of EtHg during its production and use and ecosystem health, has drawn great concerns due to has led to contamination of river water (up to 30 µg L−1 in its increasing environmental concentration in recent years the effluent-contaminated river water) (Acosta et al. 2015), (Driscoll et al. 2013). In general, organic mercury com- soils (Hintelmann et al. 1995), sediments (Hintelmann et al. pounds are more toxic to organisms than the inorganic forms 1995), and even in river fish (Yamanaka and Ueda 1975). (Boening 2000). For example, methylmercury (MeHg), an More importantly, in environments absent of direct point organomercury specie, has high environmental prevalence, sources, EtHg has been observed in snow and surface water bioaccumulation, and toxicity (Boening 2000), for which (at 10 ng L−1 level) (Paudyn and Vanloon 1986), wetland it has been extensively studied in recent years. Similarly, soils and sediments (Cai et al. 1997; Siciliano et al. 2003; ethylmercury (EtHg) has also been widely observed in the Holmes and Lean 2006; Mao et al. 2010), canal sediments environment due to its use as a fungicide and preservative in (Cavoura et al. 2017), and forest soils (Kodamatani et al. agricultural and pharmaceutical products (Geier et al. 2007). 2018). Given the lack of a point source, EtHg in these envi- ronments is possibly derived from chemical (Hempel et al. 2000; Yin et al. 2012) or biological ethylation (Fortmann * Yongguang Yin [email protected] et al. 1978). EtHg has also been observed in reference mate- rial estuarine sediments (ERM CC580) (Kodamatani and 1 State Key Laboratory of Environmental Chemistry Tomiyasu 2013), although its origin, from anthropogenic and Ecotoxicology, Research Center for Eco-Environmental contamination or naturally ethylation, is still not konwn. Sciences, Chinese Academy of Sciences, Beijing 100085, China Despite EtHg being widely present in the environment and consumer products, e.g., cosmetics, pharmaceuticals, 2 University of Chinese Academy of Sciences, Beijing 100049, China and vaccines, its toxicity and bioaccumulation has not been studied in as much detail as those of MeHg. Herein, the acute 3 Laboratory of Environmental Nanotechnology and Health, Research Center for Eco-Environmental Sciences, Chinese (96 h) and sublethal (15 days exposure and 6 days elimina- Academy of Sciences, Beijing 100085, China tion) effects of EtHg chloride on a fish model, Chinese rare Vol:.(1234567890)1 3 Bulletin of Environmental Contamination and Toxicology (2019) 102:708–713 709 minnow (Gobiocypris rarus), were studied to asses its toxic- On days 15 (after EtHg exposure) and 21 (after elimination ity and bioaccumulation on/in aquatic organisms. Analysis experiment), five fish were taken from each group, dissected of EtHg and total mercury (THg) in fish tissue and histologi- for later histopathological analysis of liver and gill. cal slices of liver and gill was performed following acute and Each fish was homogenized to 5 mL and stored at − 20°C sublethal exposure. Due the fast degradation of thimerosal until analysis. Homogenized fish tissue (2 mL) was pro- (ethylmercurithiosalicylate) to EtHg in organisms as well as cessed for organomercury speciation according to Yin et al. their similar metabolisms (Fortmann et al. 1978), the present (2007). Briefly, 2 mL of 25% KOH (inCH 3OH, m/v%) were study also aids in the understanding of toxicity and biodis- added to 2 mL of homogenized fish tissue in a 50 mL centri- tribution of thimerosal. fuge tube and shaken mechanically overnight. Then, 6 mL of CH2Cl2 were added and 1.5 mL of concentrated HCl was added dropwise, followed by shaking for 15 min to extract Materials and Methods organic mercury into the CH 2Cl2 phase. After centrifuga- tion at 3000 rpm for 15 min, 4–5 mL of the CH2Cl2 phase EtHg (98%, Merck-Schuchardt) was dissolved in methanol were transferred into a 10 mL glass tube and 1 mL of a as solvent to obtain a stock solution of 2000 mg L−1 (as 10 mmol L−1 solution of sodium thiosulfate were added. Hg) and kept at 4°C. The working solutions were freshly The glass tube was shaken for 45 min and centrifuged at prepared by the dilution of the stock solution in de-ionized 3500 rpm for 15 min. The water phase was then collected water to suitable concentrations just before use. All the other and injected into a high performance liquid chromatogra- reagents used were of analytical grade or above. It should be phy atomic fluorescence spectrometry (HPLC-AFS) system noted that all the concentrations of EtHg in solution or tissue for EtHg analysis (Yin et al. 2007). The optimized instru- were calculated based on Hg. mental parameters were given in the previous study (Yin A batch of 4-month-old fingerling Chinese rare minnow et al. 2007). The HPLC-AFS system and sample preparation 3.00 ± 0.36 cm in length and 0.27 ± 0.06 g in weight were procedure were validated by the determination of MeHg in pre-raised in a flow-through system continuously supplied a certified reference material (DORM-4) and EtHg-spiked with dechlorinated and aerated tap water with the follow- fish muscle (85.4% ± 3.5% recovery with 1000 µg kg−1 EtHg ing characteristics: pH 7.9 ± 0.2; oxygen concentration, spiking). −1 −1 5–7 mg L ; hardness of CaCO3, 200 mg L ; conductivity, Homogenized fish tissue (2 mL) was added to a PTFE −1 650 µS cm ; water temperature, 22.5°C–25.5°C. All fish digestion container. Concentrated HNO 3 (2 mL) was then were fed twice daily with artemia and kept under a 12 h added and the solution was left to predigest at 60°C over- light/12 h dark cycle. night. After cooling, 0.5 mL of H 2O2 were added, and the A series of EtHg solutions (5, 10, 20, 30, 40, 50, and PTFE digestion container was placed in stainless steel bombs 80 µg Hg L−1, blank control, and solvent control) were and maintained at 160°C in an oven for 8 h. After cooling administered to rare minnow (20 fish per group). The cul- to room temperature, the digestion solution was transferred ture density was < 1 g fishL −1 (fresh weight). The water in to a 25 mL PET bottle and diluted to 20 mL with de-ionized the exposure and control groups was refreshed every 12 h to water. The THg in the resulting solution was determined by maintain a relatively stable concentration of EtHg and good AFS (BRAIC Analytical Instrumental Company, Beijing). water quality. The fish were not fed in the acute exposure The liver and gill of fish from the control and exposure procedure and dead fish were removed as soon as possible. groups were submitted to cytological analysis. The tissue Fish were collected at the end of exposure, dried with filter specimens were fixed in 10% formalin, washed and dehy- paper, then it was dissected to obtain the liver and gill for drated with ethanol, and then embedded in paraffin blocks. later histopathological analysis. Serial 4–5 µm sections were cut from the block samples Four groups (0.1 and 1 µg Hg L−1, blank control, and and stained with hematoxylin and eosin, then observed and solvent control) were set in sublethal exposure with 40 fish photographed under a light microscope (Olympus BX41 TF, per group for 15 days, and subsequently transferred to EtHg- Japan). free water and raised for a further 6 days to assess elimina- Difference of Hg concentrations in the tissues were per- tion. The culture density was < 1 g of fish L−1. Due to the formed by one-way ANOVA by SPSS 17.0 for windows. sublethal exposure design, no fish death was observed in the whole exposure procedure. The EtHg exposure solution (or water) in each group was refreshed every 12 h to maintain Results and Discussion the relatively stable exposure concentration of EtHg and good water quality. All experimental fish were fed twice Following the exposure to high concentrations of EtHg daily. Four fish were sampled from each group to determine (acute exposure), life behavior showed significant changes EtHg and THg at 3, 6, 9, 12, 15, 18, and 21 days of exposure.