Bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in zebrafish

GU Shunxi; TANG Bin; CAI Fengshan; YAN Xiao; YAO Lijun; LI Jialu; YAO Lili; XIE Chun; ZHENG Jing

doi:10.7524/j.issn.0254-6108.2022101804

2023 Volume 42 Issue 4

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GU Shunxi, TANG Bin, CAI Fengshan, YAN Xiao, YAO Lijun, LI Jialu, YAO Lili, XIE Chun, ZHENG Jing. Bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in zebrafish[J]. Environmental Chemistry, 2023, 42(4): 1085-1096. doi: 10.7524/j.issn.0254-6108.2022101804

Citation:

GU Shunxi, TANG Bin, CAI Fengshan, YAN Xiao, YAO Lijun, LI Jialu, YAO Lili, XIE Chun, ZHENG Jing. Bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in zebrafish[J]. Environmental Chemistry, 2023, 42(4): 1085-1096. doi: 10.7524/j.issn.0254-6108.2022101804

Bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in zebrafish

1.
School of Public Health, the key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, 550025, China
2.
State Environmental Protection Key Laboratory of Environmental Pollution Health Risk Assessment, Research Group of Emerging Contaminants, South China Institute of Environmental Sciences, Ministry of Ecology and Environment, Guangzhou, 510655, China
3.
State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, China
4.
University of Chinese Academy of Sciences, Beijing, 100049, China

Corresponding author: ZHENG Jing, zhengjing@scies.org
Received Date: 18/10/2022
Accepted Date: 10/11/2022
Available Online: 27/04/2023

Fund Project: the National Natural Science Foundation of China（42007341）.

Abstract

In the present study, bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in tissues of zebrafish (Danio rerio) were investigated after administered DBDPE to zebrafish via the diet. The experiment consists of a 28-day of uptake period and a 77-day of depuration period. Bioaccumulation constants, including the assimilation efficiency (α), depuration rate constants (k_d), half-life (t_1/2), and biomagnification factor (BMF), for DBDPE in tissues of zebrafish were calculated. Biotransformation products of DBDPE were screened in serum of zebrafish using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and was confirmed by the MS2 spectrum. Similar absorption curves were found for DBDPE in muscle, liver, brain, and ovary of zebrafish, with α in a range of 10.1% ± 1.34% to 18.3% ± 3.78%. Depuration of DBDPE in zebrafish tissues followed a pseudo first-order kinetics, with k_d values ranged from (3.66 ± 0.16) × 10⁻²·d⁻¹ to (4.69 ± 0.06) × 10⁻²·d⁻¹, and t_1/2 values ranged from (14.8 ± 0.19) d to (19.0 ± 0.83) d, respectively. The BMF of DBDPE in zebrafish tissues were (0.72 ± 0.04) d to (1.12 ± 0.23) d, indicating a potential bioaccumulation capacity of DBDPE in fish, which was also significantly correlated to the functions and fat content of individual tissues. Four debromination, three hydroxylation, one methoxy, and two methylsulfonyl products of DBDPE were identified in serum of zebrafish. The results of this study demonstrated that DBDPE had obvious biotransformation processes in zebrafish, which affected its bioaccumulation potential in fish.
- DBDPE,
- zebrafish,
- bioconcentration,
- biotransformation.

References

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沈阳化工大学材料科学与工程学院沈阳 110142

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近年来，随着十溴联苯醚（decabromodiphenyl ether，Deca-BDE）的逐步禁用，十溴二苯乙烷（decabromodiphenyl ethane，DBDPE；化学结构见图1）作为其主要替代产品，生产量和使用量呈逐年升高的趋势，现已成为使用量最大的新型溴代阻燃剂（novel brominated flame retardants，NBFRs）^[1-2]. DBDPE作为一种添加型NBFRs，不与产品产生化学键结合，因而在生产、加工和使用等过程中极易释放到周围环境中^[3]. 此外，DBDPE还具有潜在的内分泌干扰效应^[4]、生殖发育毒性^[5]以及肝脏毒性^[6]等，进而对生物和人体造成健康危害. 我国作为DBDPE生产和使用大国，其环境污染问题以及由此带来的潜在健康风险不容忽视^[7-8]. 研究表明，在广州市污水处理厂中DBDPE是主要的卤代阻燃剂类（HFRs）污染物之一，且呈现逐年递增的趋势^[9]. 在我国东江下游水体中DBDPE也是检出的主要污染物，占HFRs总量的64%^[10]. 因此，DBDPE潜在的环境健康风险问题需引起高度重视.

DBDPE亲脂性较高（辛醇-分配系数，lg K_ow = 11.1）^[11]，具有在水生生物体内富集和放大的潜力. 目前关于DBDPE生物富集特征的相关研究普遍集中于野外食物链调查，且研究结果并不一致. 针对东江流域鲮鱼、罗非鱼和鲢鱼等3种鱼类的研究结果表明，DBDPE可以在鱼体内显著富集^[12]. 关于我国南海海域水生食物网的研究发现，DBDPE具有明显的生物放大作用^[13]. 然而在太湖水域水生食物链中DBDPE的研究结果显示，DBDPE出现营养级稀释现象^[14]. 除污染物自身的理化性质外，污染物在生物体内的吸收和代谢转化等也是影响其生物富集特征的重要因素. 然而，目前仅有少量研究报道了DBDPE的生物转化过程. 将DBDPE暴露斑马鱼胚胎后，在胚胎内共检出7种可能的脱溴代谢产物^[15]. 以大鼠为模式生物，经口暴露DBDPE后也检出7种代谢产物^[16]. 通过开展沉积物-水-泥螺暴露实验结果发现，DBDPE在泥螺体内的生物转化主要为脱溴过程^[17]. 以上研究结果表明，DBDPE生物富集特征仍然存在争议，在生物体内的转化过程具有潜在的物种差异性. 目前对DBDPE在鱼体内的生物富集以及生物转化尚缺乏深入认识，且有关生物转化过程对DBDPE生物富集的影响仍有待进一步研究.

综上所述，本研究以斑马鱼作为受试生物，开展室内DBDPE饮食暴露实验，探讨DBDPE在肌肉、肝脏、性腺和脑等不同组织内的生物富集特征；并对暴露鱼体血清中DBDPE潜在的生物转化产物进行筛查和鉴定，推导其可能的生物转化路径，进而评估生物转化对DBDPE生物富集特征的影响. 主要目的是探讨DBDPE在鱼类中的组织特异性分布特征，以及DBDPE在斑马鱼体内的生物转化特征. 相关研究结果有望为深入认识DBDPE的环境行为和归趋、准确评估其生态环境健康风险提供科学依据，具有重要的意义.

1. 材料与方法（Materials and methods）

1.1. 实验材料与试剂

仪器：气相色谱-质谱联用仪（GC-MS，7890N-5977A；Agilent，美国）,色谱柱DB-5HT（15 m×0.25 mm×0.1 µm；Agilent，美国），超高效液相色谱-四极杆-飞行时间质谱联用仪（UPLC-QTOF-MS，1290-G6545；Agilent，美国），RRHD Eclipse Plus95-C18色谱柱（3 mm×150 mm，1.8 µm；Agilent，美国），离心机（Sigma，德国），氮吹仪（Organomation，美国），冷冻干燥机（北京博医康实验仪器有限公司，中国），数控超声波清洗器（上海安谱实验科技公司，中国），涡旋振荡器（Troemner，美国），电子天平（梅特勒托利多公司，瑞士）.

耗材：CNWBOND HC-C18 SPE 填料（40—63 μm），CNWBOND PSA QuEChERS专用超洁净填料（40—63 μm），10 mL和15 mL玻璃旋盖离心管，1 mL 无菌连针注射器，亲水PTFE针式滤器（0.22 μm）（上海安谱实验科技有限公司，中国），鱼饲料（茂名市粤诚饲料有限公司，中国），无水硫酸钠（阿拉丁生化科技公司，中国），Supelco Florisil® ENVI固相萃取（SPE）柱（500 mg, 3 mL；Merck，德国），巴斯德吸管（Witeg Labortechnik Gmb，德国）.

实验所用试剂均为色谱纯，包括正己烷（HEX），丙酮（ACE），二氯甲烷（DCM），甲醇（MeOH），乙酸乙酯（EtAC）和异辛烷（上海安谱实验科技有限公司，中国）；优级纯甲酸（阿拉丁生化科技公司，中国）；标准品包括DBDPE，OH-BDE85，BDE128和BDE181（> 99.0%，AccuStandard，美国）.

受试生物：性成熟雌性斑马鱼（Brachydanio rerio，AB种野生型；购自中国科学院水生生物研究所）.

1.2. 斑马鱼暴露实验以及生物样品收集

1.2.1. 斑马鱼饲养

将性成熟雌性斑马鱼（体长（4.01±0.16） cm，体重（0.65±0.21） g）养殖于15 L水族玻璃缸内（n = 40）；养殖水为经过爆气和活性炭过滤的自来水（pH = 7.4±0.2；水温（28±0.5） ℃；光暗比为14 h:10 h），每天食物投放量以全部鱼体重2%计；待斑马鱼适应14 d后开始暴露实验，并于实验前5 d停止喂食.

1.2.2. 暴露与取样

斑马鱼暴露实验参考经合组织（Organization for Economic Co-operation and Development; OECD）推荐的化学物质鱼体生物富集测试指南^[18-20]，简述如下：将斑马鱼（n = 200）随机分配于5个15 L玻璃水族箱中（每缸40条）作为暴露组；另取50条作为实验对照组. 暴露剂量参考我国华南地区河流污泥样品中DBDPE平均浓度（1995 ng·g⁻¹ dw）^[8]，将染毒鱼食设计剂量为2000 ng·g⁻¹，以模拟DBDPE在实际环境中的生物富集潜力；对照组用未染毒的相同鱼食进行喂养.

暴露期间，以前述饲养环境，每天用加标鱼食喂养斑马鱼. 连续暴露28 d后，将其转至干净的水族箱继续开展为期77 d净化实验，净化期采用未染毒的鱼食喂养. 实验过程中，每2 天更换1次实验水体. 分别于暴露期第7、14、21、28 d，以及清除期7、21、35、49、63、77 d进行取样；每次取样，从每个暴露组水族箱中随机选取4尾斑马鱼，共计20尾. 测量每条鱼的体长和体重；断尾取血，解剖分离鱼体性腺、肝脏、脑和肌肉等组织，并记录其重量. 每次取样的血液合并为一个样品，离心（4000 r·min⁻¹，10 min）后获得血清；肌肉、肝脏和脑等则分别将5尾鱼对应组织合并为一个样品（n = 4）. 除血清外，其他生物样品在合并、称重后冷冻干燥. 每次取样时，随机从对照组中取5尾斑马鱼，各组织处理方式与暴露组相同.

1.3. 实验分析

1.3.1. 样品前处理

鱼食和斑马鱼组织样品前处理参考文献报道的方法^{[15, 17, 21]}，并做适当优化，简述如下：准确称取鱼食或鱼体组织样品置于干净的玻璃离心管中，冷冻干燥后加入适量无水硫酸钠、3 mL HEX:ACE混合溶剂（1:1，V/V）、内标溶液20 μL（BDE128，1000 ng·mL⁻¹），静置过夜. 样品经超声萃取（100 kHz，15 min，35 ℃）、离心（4000 r·min⁻¹，10 min）后，取上清液转移至干净的10 mL玻璃离心管；重复上述超声萃取步骤2次，合并萃取液. 取1 mL萃取液用于测定脂肪含量，剩余萃取液（8 mL）氮吹浓缩至近干，HEX复溶至1 mL. 浓缩液采用Florisil 固相萃取（SPE）柱（500 mg, 3 mL）分离纯化；SPE柱依次用6 mL EtAC和6 mL HEX预淋洗；上样后，用10 mL HEX：DCM混合溶剂（1:1，V/V）进行洗脱. 洗脱液氮吹浓缩至近干，加入180 μL异辛烷和20 μL回收率指示物（BDE181，1000 ng·mL⁻¹），涡旋混合均匀后转至进样瓶中，置于-20 ℃冷冻保存，待上机测试.

斑马鱼血清样品分析参考Chen等^[22]的处理方法，并对其进行优化：准确称重合并的血清（—100 μL）样品于2 mL离心管，加入100 μL MeOH和20 μL甲酸，以及20 μL内标溶液（OH-BDE85，1000 ng·mL⁻¹），混匀后静置15 min；加入200 μL MeOH:ACE （1:1，V/V），涡旋混匀（1800 r·min⁻¹，2 min）；离心（8000 r·min⁻¹，10 min）后取上清转移至干净的离心管中；重复萃取步骤3次，合并上清液；萃取液加入5 mg PSA和5 mg C18除脂；涡旋（1800 r·min⁻¹，2 min）、离心（8000 r·min⁻¹，10 min）后取上清；过亲水PTFE针式滤器（0.22 μm），并氮吹至近干，加入100 μL MeOH复溶，涡旋混合均匀后转移至进样2 mL瓶中,置于−20 ℃冷冻保存，用于DBDPE脱溴产物筛查分析.

1.3.2. 仪器分析方法

DBDPE定量参考Tang等^[21]报道的仪器方法，分析仪器为Agilent GC-MS（7890N-5977A）；离子源为负化学电离源（ECNI），采用选择离子检测模式（SIM）. 载气为高纯氦气，甲烷为反应气体；色谱柱采用DB-5 HT毛细管柱（15 m×0.25 mm，0.10 µm；Agilent）. DBDPE扫描离子为m/z 79和81. 升温程序：初始温度为110 ℃，保持5 min；以20 ℃·min⁻¹升至200 ℃，保持4.5 min；以10 ℃·min⁻¹升至310 ℃，保持5 min. 采用不分流进样，进样体积为1 μL. 传输线、进样口和离子源温度分别为280 ℃、290 ℃、250 ℃. 此外，采用全扫描（SCAN）模式下分析斑马鱼组织及血清中DBDPE的脱溴代谢产物.

参考Wang^[20]等报道的方法，使用安捷伦超高效液相色谱-四极杆-飞行时间质谱（UPLC-QTOF-MS；Agilent 1290-6465）对斑马鱼血清中DBDPE的代谢产物进行筛查分析. 采用RRHD Eclipse Plus95-C18色谱柱（3 mm×150 mm，1.8 µm）分离，柱温40 ℃；进样体积4 µL；流动相A为乙腈，B为含0.05%（V/V）甲酸的超纯水，洗脱梯度见表1，流速为0.3 L·min⁻¹. 采用电喷雾离子源正负（ESI+和ESI-）两种模式采集数据；扫描模式的参数设置：采集模式为自动（Auto MS/MS）和靶向（Targeted MS/MS）两种相结合；扫描质荷比（m/z）50—1200之间；毛细管电压：3500V（ESI-）/4000V（ESI+）；碰撞能设置：5、10以及20 eV；采集速率：MS为2.5 spectra·s⁻¹，MS/MS为4 spectra·s⁻¹；气体流速：9 L·min⁻¹，气体温度：325 °C；鞘气流速：11 L·min⁻¹，鞘气温度：350 °C；锥孔电压：65 V.

1.3.3. 质量控制与数据分析

使用未染毒生长发育正常的雌性斑马鱼肌肉作为基质，采用与样品相同流程，分析基质（n = 3）、基质加标（n = 3）、空白（n = 3）以及空白加标（n = 3）等4组平行样品. DBDPE的基质加标、空白加标回收率分别为109%±14.8%和84.2%±5.60%，相对标准偏差（RSD）均小于20%. 实验过程中，每批16个生物样品，设置2个程序空白样品同时分析，以检测实验过程中可能产生的背景污染. 程序空白样品中有少量DBDPE检出，并在报道浓度时做相应的空白校正. 定量限（LOQ）定义为程序空白样品中DBDPE浓度均值加上3倍标准偏差，为2.90 ng·g⁻¹ lw（脂重）. 所有样品中，内标（BDE 128）的回收率为87.5%±8.92%. 血清样品实验分析的过程中，设置两个程序空白样品，以检测实验过程中是否产生背景污染，程序空白样品中未检测出DBDPE，对照组样品基质对照，以检测是否为斑马鱼自身基质产生的影响.

1.3.4. 数据处理

参考（OECD）推荐的化学物质鱼体生物富集测试指南^{[18-19, 23-24]}，计算DBDPE在斑马鱼体内的生物富集常数.

净化阶段鱼体组织中DBDPE的浓度变化采用准一级动力学模型拟合：

式中，C₀和C_t分别为清除开始（t = 0）及清除时间t时，鱼体组织中DBDPE的浓度；k_d为清除速率常数（depuration rate constant）. 为获得多个净化时间点的k_d，将DBDPE浓度的对数值与净化时间进行线性拟合，k_d为回归趋线的斜率值，即：

DBDPE在鱼体组织中的生物半衰期（half-life，t_1/2）通过以下公式计算：

暴露阶段DBDPE的同化效率（assimilation efficiency，α）采用以下方程进行拟合：

式中，F为喂食比例（食物/g 斑马鱼/d，脂肪校正）；C_food为斑马鱼组织中DBDPE的浓度.

生物放大因子（biological magnification factor，BMF）计算公式为：

采用SPSS21.0对暴露组与对照组的体长、体重、不同组织浓度的差异是否具有显著性差异进行单因素方差分析（one-way ANOVA），显著性标准设置为P < 0.05，绘图采用Origin 2017.

1.3.5. DBDPE脱溴产物的鉴定

DBDPE代谢产物鉴定如下：（1）根据相关的文献报道^{[16-17, 25-26]}以及Biotransformer 3.0^[27]软件预测的代谢产物，并运用ChemDraw Ultra 20.0软件绘制化合物结构，构建PCDL数据库构建；（2）将MS数据导入Profinder进行批量处理，快速确定可能的产物精确分子量及出峰时间；（3）通过Auto MS/MS及Targeted MS/MS模式获取所有可能的产物的二级质谱图，将MS/MS数据导入Qualitative Analysis运用“Find by Auto-MS/MS”或者“Find by Targeted-MS/MS”查找化合物，并将结果以cef格式导出;（4）最后，将cef文件导入MSC分析，调用前期构建的PCDL数据库或者选择一些在线的化合物数据库（PubChem或ChemSpider）进行匹配，根据子离子的碎片信息推测产物的二级结构.

3. 结论（Conclusion）

本研究结果表明DBDPE在斑马鱼体内具有潜在的生物富集性；且组织功能及其脂肪含量对DBDPE的富集潜力具有重要作用. 此外，在斑马鱼体组织中筛查到DBDPE的脱溴代谢产物；而在血清中，除脱溴产物外，还筛查鉴定出DBDPE的羟基化代谢产物以及其他产物；表明DBDPE在斑马鱼体内除脱溴反应外，还存在氧化羟基化和甲氧基化等代谢反应，代谢转化也是影响DBDPE生物富集潜力的重要因素之一；同时，DBDPE代谢产物对环境的影响也值得引起关注. 本文系统探讨了DBDPE在斑马鱼体内的生物富集和转化过程，为准确评估其生态环境风险提供科学依据.

Figure (8) Table (2) Reference (34)

Bioaccumulation and biotransformation of decabromodiphenyl ethane (DBDPE) in zebrafish