Acute and Sublethal Effects of Ethylmercury Chloride on Chinese Rare Minnow (Gobiocypris Rarus): Accumulation, Elimination, and Histological Changes

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 bioaccumulation 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 environments 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 material 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. during the whole experimental process, compared with the

1 3 710 Bulletin of Environmental Contamination and Toxicology (2019) 102:708–713 control group. The fish were observed to swim slowly and Pesticides Database). The differing results perhaps originate have a diminished response. After 1 h of exposure, the mal- from the variations in fish species and size. adjustment (swimming and response) was alleviated but still The histological differences in gill and liver from the con- not lively compared with the control group. Fish in the expo- trol and exposure groups were observed. The control gill had sure group preferred to stay in the corner. Prior to death, fish a fresh red color as well as normal and clear configurations were not able to maintain body balance, breathing quickly of lamellae and epithelial cells (Fig. 1a). In the high concen- and sometimes suddenly leaped toward the surface. Red gore tration EtHg exposure groups (40, 50, and 80 µg Hg L−1), appeared at the belly and white floc attached on the fish. widespread necrosis was distinctly observed in epithelial, Further, the fins became whitened and the skeleton in the pillar, and mucous cells; concurrently, telangiectasis and fin was blurry. exfoliation of epithelial cells at the base of secondary lamel- The lethal concentration dose (LC 50) was calculated lae were also observed (Fig. 1b). Liver hepatocytes in the based on the death rate and linear regression. The 24, 48, 72, control group showed normal polygonal structures, with per- and 96 h LC50 of EtHg chloride were 24.8 (95% confidence fectly clear homogeneous cytoplasm and basophilic granules interval 23.4–26.2), 15.3 (14.6–16.1), 14.5 (13.9–15.1), and (Fig. 1c). The nucleus was roughly round and filled with −1 13.8 (13.3–14.4) µg L , respectively. The LC 50 of EtHg light-colored chromatin, and small and red nucleolus could chloride showed an obvious toxicity to Chinese rare minnow be easily found. However, hepatocytes from the exposure (4 months old). Previous studies have also suggested the group had a weakly basophilic cytoplasm, with a slightly high toxicity of EtHg to western mosquitofish (Gambusia gray color. Small edema, vacuoles, and pyknotic nucleus −1 affinis) (LC 50 77.0 µg L ) and moderate toxicity to chan- could be clearly observed. Furthermore, the nucleolus of −1 nel catfish (Ictalurus punctatus) (LC50 3033 µg L ) (PAN some cells disappeared (Fig. 1d). These results clearly

Fig. 1 Representative histological slice of liver (a, control and b, ectasis (T) and exfoliation of epithelial cells at the base of secondary exposure) and gill (c, control and d, exposure) of Chinese rare min- lamellae were found; c control liver; d exposed liver, small oedema now. a control gill; b, exposed gill, widespread necrosis of epithelial (O), vacuoles (V), necrosis (N) and pyknotic nucleus (PN) could be cells, pillar cells and mucous cells was observed distinctly; telangi- clearly seen; nucleolus of some cells disappeared

1 3 Bulletin of Environmental Contamination and Toxicology (2019) 102:708–713 711 demonstrate the damage and histological changes induced 0.1 µg L−1 and 1 µg L−1 exposure groups, respectively. by EtHg on the gill and liver of Chinese rare minnow. After depuration, the concentration of both THg and EtHg The concentrations of MeHg, EtHg, and THg in the whole decreased accordingly, demonstrating the fast elimination fish were determined by HPLC-AFS and AFS. In the control of both EtHg and inorganic mercury from organisms. Sim- group, the concentrations of EtHg were below the limit of ilar to acute exposure, the fraction of EtHg in THg firstly detection (10 µg kg−1), respectively. MeHg was observed in increased to over 70% after sublethal exposure (Fig. 3c), both the exposure and control groups (< 50 µg L−1), with indicating that, in the early stage of exposure, the accumu- no apparent difference being observed between them, indi- lation of EtHg is much faster than its biodegradation. For cating that MeHg is not a metabolite of EtHg and is rather the 0.1 µg L−1 exposure group, the fraction of EtHg in THg derived from artemia as fish food. Under the acute exposure increased even after depuration. However, for the 1 µg L−1 scenario, both THg and EtHg were detected in fish tissues exposure group, after the first increase, the fraction of (Fig. 2), demonstrating the bioaccumulation of mercury in EtHg in THg leveled off after day 9, then decreasing sig- fish. For all the acute exposure samples, EtHg accounted nificantly after depuration (day 15), indicating inhibited for approximately 32.1% ± 4.2% of the THg under differ- EtHg accumulation and/or enhanced EtHg degradation at ent exposure concentrations. Despite EtHg being partially the 1 µg L−1 exposure level compared to that at 0.1 µg L−1. degraded to inorganic mercury in water during the exposure The C–Hg bond of EtHg is comparably less stable than procedure, HPLC-AFS results showed that the percentage of that of MeHg and therefore EtHg is prone to decomposi- degraded EtHg was no more than 20%. Moreover, the bio- tion to inorganic mercury. In vivo and in vitro studies have concentration of inorganic mercury is generally lower than shown that the degradation EtHg is much higher than that that of organic mercury (Seixas et al. 2014). Therefore, the of MeHg (Suda and Takahashi 1992); indeed, the half- high fraction of inorganic mercury in fish indicates the bio- life of EtHg in monkey is of approximately 1 week (Bur- degradation of EtHg to inorganic mercury in the fish body. bacher et al. 2005). Whilst the fast biodegradation of EtHg The sublethal effects of EtHg on Chinese rare minnow reduces its neurotoxic potential faster than that of MeHg were subsequently investigated. Under sublethal exposure, (Magos et al. 1985), its degradation into inorganic mer- fish behavior did not change significantly. Besides hyper- cury potentially increases the risk of renotoxicity (Magos plasia of epithelial cells being observed in gill of fish in et al. 1985). the 1.0 µg L−1 exposure group, no other obvious histologi- Interestingly, a new mercury species peak was observed cal changes were found in gill and liver. The concentration in the fish in the later stage of exposure (Fig. 4), with a of THg and EtHg in fish in the sublethal exposure and longer retention time, and therefore higher hydrophobicity, depuration procedure was observed to increase with the than that of EtHg. Diethylmercury has been proposed as increase in EtHg concentration and exposure time (Fig. 3a, the possible biotransformation product of thimerosal in a b). The bioconcentration factors for EtHg at 15 days of previous study (Drum 2009). Then we synthesized dieth- exposure were 21,821 L kg−1 and 4423 L kg−1 for the ylmercury from aqueous derivitization of inorganic diva- lent mercury by sodium tetraethylborate (Ma et al. 2014). The retention time of the unknown mercury compound was shown to be consistent with that of diethylmercury, indicating its likely nature. Considering the more toxic nature of diethylmercury than that of EtHg, the in vivo formation of diethylmercury should be confirmed by other characteriza- tion methods in the future study. In summary, the acute and sublethal toxic effects of EtHg were observed in a fish model. EtHg chloride showed an obvious toxicity to 4-month-old Chinese rare minnow. The high concentration of EtHg and inorganic mercury in exposed fish demonstrated the high accumulation and degradation of EtHg in fish. Further, fast elimination of EtHg and inorganic mercury were also observed in the depuration experiment.

Fig. 2 The accumulation of EtHg and THg in Chinese rare minnow after acute exposure. (note: the fishes were sampled (n = 4) at 24 h for 50 µg L−1 and 80 µg L−1 exposure group, and at 96 h for 5 µg L−1 and 10 µg L−1 exposure group, respectively)

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Fig. 3 Concentration of THg (a) and EtHg (b) and percentage of EtHg/THg (c) in sublethal exposed fish (n = 4 for each data)

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