Flame retardants in the livers of the Eurasian otter collected from Scotland between 2013 and 2015 (PBMS)

Otters found dead were examined by Cardiff University Otter Project to determine sex and age. Age-class (adult, sub-adult or juvenile) was estimated from a combination of morphometric data and indicators of reproductive activity. Nutritional and reproductive status, lesions, growths and concretions were noted. Tissue samples, including the liver, were taken as part of the post-mortem examination. Between 2013 and 2015, livers were collected from 16 otters found dead in Scotland for analysis of PBDE, tri-BDE through nona-BDEs, residues. Age was estimated using weight and signs of sexual maturity. Females weighing less than 2.1 kg and males weighing less than 3 kg were recorded as juvenile. Females above this weight but with no sign of reproductive activity (nipples not showing, immature uterus) and males with a baculum less than 60 mm in length were recorded as sub-adult. Females above this weight and with signs of having reproduced at least once and males above this weight and with a baculum length greater than or equal to 60 mm were recorded as adult. Liver samples were analysed at the centralized analytical laboratories at the Centre for Ecology & Hydrology, Lancaster. Concentrations of 32 BDEs (6 tri-BDEs, 6 tetra-BDEs, 6 penta-BDEs, 4 hexa-BDEs, 2 hepta-BDEs, 5 octa-BDEs and 3 nona-BDEs) were quantified. A sub- sample of each liver (~1 g) was thawed, weighed accurately, ground with sand and dried with anhydrous sodium sulphate. Each sample was spiked with labeled recovery standards (13C PBDEs and 13C BFRs) and Soxhlet extracted in DCM for 16 h. A small portion of the extract was evaporated to zero volume and the lipid content was determined gravimetrically. The remaining extract was cleaned using automated size exclusion chromatography followed by deactivated alumina column. All BDEs were analysed by GCMS. Each extract was spiked with labelled internal standards and 100μl of sample was injected into a GC-MS with programmable temperature vaporization (PTV) inlet. The PTV injector was kept at 55°C for 0.45 min, and heated to 325°C at a rate of 700°C min-1 and kept at 325°C for 5 min. Then the temperature was reduced to 315°C min-1 at a rate of 10°C min-1. The GC-MS had a 25 m HT8 column (0.22 mm internal diameter and 0.25μm film thickness, SGE Milton Keynes, UK) and the carrier gas was helium (2.0 ml min-1). The temperature programme was: isothermal at 80°C for 2.4 min, 25°C min-1 to 200°C, 5°C min-1 to 315°C and was held at 315°C for 9.8 min. Residues were quantified as recovery corrected concentrations using an internal standard correction method and calibration curves based on PBDE standards (Greyhound Ltd, Birkenhead, UK and LGC Ltd, Teddington, UK).. Average recoveries for 13C-PBDE recovery standards for BDE 28, BDE 47, BDE 126, BDE 153 and BDE 197 ranged between 94% and 98%. Liver concentrations of flame retardants are reported as ng/g wet weight (wet wt). When all summed PBDE concentrations were calculated, individual congener concentrations below the limit of detection (non-detected) were assigned a zero value.

卡迪夫大学水獭项目（Cardiff University Otter Project）对发现的死亡水獭开展解剖检查，以确定其性别与年龄。研究人员通过形态测量数据与繁殖活动指征的结合，对水獭的年龄分级（成年、亚成年或幼年）进行估算，同时记录其营养与繁殖状况、病变、增生及钙化凝结物。作为尸检的一部分，研究人员采集了包括肝脏在内的组织样本。2013年至2015年间，研究人员从苏格兰境内发现的16只死亡水獭体内采集肝脏样本，用于分析多溴二苯醚（PBDE）、从三溴二苯醚（tri-BDE）到九溴二苯醚（nona-BDEs）的各类同系物残留。研究人员通过体重与性成熟迹象来估算水獭年龄：体重低于2.1千克的雌性个体与体重低于3千克的雄性个体被归类为幼年个体；体重高于上述阈值但无繁殖活动迹象（乳头未发育、子宫未成熟）的雌性个体，以及阴茎骨（baculum）长度小于60毫米的雄性个体被归类为亚成年个体；体重高于该阈值且存在至少一次繁殖经历迹象的雌性个体，以及体重高于该阈值且阴茎骨长度大于或等于60毫米的雄性个体则被归类为成年个体。肝脏样本在兰卡斯特生态与水文中心（Centre for Ecology & Hydrology, Lancaster）的集中分析实验室完成检测。研究人员对32种溴二苯醚同系物（BDEs）的浓度进行定量分析，其中包含6种三溴二苯醚、6种四溴二苯醚、6种五溴二苯醚、4种六溴二苯醚、2种七溴二苯醚、5种八溴二苯醚及3种九溴二苯醚。每份肝脏样本取约1克的子样本，经解冻、精准称重后用砂粒研磨，再以无水硫酸钠干燥。每份样本均加入碳13标记的多溴二苯醚（13C PBDEs）与碳13标记的溴系阻燃剂（13C BFRs）作为回收率标准品，随后用二氯甲烷（DCM）进行16小时索氏提取。取少量提取液蒸发至干，通过重量法测定样本的脂质含量。剩余提取液先经自动化尺寸排阻色谱净化，再通过脱活氧化铝柱进一步纯化。所有溴二苯醚同系物均采用气相色谱-质谱联用仪（GC-MS）进行分析。每份提取液均加入标记内标，随后取100微升样本注入带有程序升温汽化（PTV）进样口的气相色谱-质谱联用仪中。程序升温汽化进样口先在55℃下维持0.45分钟，随后以700℃每分钟的速率升温至325℃并保持5分钟，再以10℃每分钟的速率降温至315℃并维持该温度。该气相色谱-质谱联用仪配备25米长的HT8色谱柱（内径0.22毫米，膜厚0.25微米，产自英国米尔顿凯恩斯的SGE公司），载气为氦气，流速为2.0毫升每分钟。色谱柱升温程序设置为：80℃下恒温保持2.4分钟，以25℃每分钟的速率升温至200℃，再以5℃每分钟的速率升温至315℃并保持9.8分钟。残留量采用内标校正法与基于多溴二苯醚标准品（英国伯肯黑德的Greyhound有限公司与英国特丁顿的LGC有限公司提供）绘制的校准曲线，换算为经回收率校正的浓度值进行定量。针对BDE 28、BDE 47、BDE 126、BDE 153及BDE 197的碳13标记多溴二苯醚回收率标准品，其平均回收率介于94%至98%之间。阻燃剂在肝脏中的浓度以纳克每克湿重（ng/g wet wt，湿重）为单位报告。在计算总多溴二苯醚浓度时，所有低于检测限（未检出）的同系物浓度均赋值为零。