Stable isotope and elemental analyses of an ex-situ experiment testing the effects of mining-generated sediment plumes and de-oxygenation on the juveniles of the hydrothermal vent mussel Bathymodiolus azoricus

We report the results of an aquaria-based experiment testing the effects of low oxygen and suspended particles generated during a potential mining activities accident on the juveniles of the vent mussel Bathymodiolus azoricus. Mussels were collected from the Lucky Strike hydrothermal vent field (Azores, NE Atlantic) at 1700 m water depth in 2021. Mussels were maintained in 1 l aquaria and exposed to four experimental treatments for a period of two weeks at the DeepSeaLab aquaria facilities (Okeanos-University of the Azores): (1) control conditions (no added sediments and normal seawater oxygen ); (2) low oxygen (difference of 30 µmol from the normal oxygen concentration); (3) suspended polymetallic sulphide (PMS) particles; (4) Low oxygen + suspended polymetallic sulphide (PMS) particles. PMS particles were obtained by grinding PMS inactive chimney rocks collected at the hydrothermal vent field Lucky Strike. Particles types were in concentration of 420 mg/l on day 8 and day 11. The putative effects of low oxygen, PMS particles, and the cumulative effect(s) were evaluated through measurements of bulk mussel tissue samples by stable isotopes and elemental analyses, as follows: Continuous flow isotope mass spectrometry (CF-IRMS) was performed on a SerconHydra 20–22 (Sercon, UK) and Isoprime IRMS stable isotope ratio mass spectrometer, coupled to a EuroEA (EuroVector, Italy) elemental analyser for online sample preparation by Dumas-combustion. Delta Calculation was performed according to δ = [(Rsample – Rstandard)/Rstandard]*1000, where R is the ratio between the heavier isotope and the lighter one. δ15 NAir values are referred to air, δ13 CVPDB values are referred to PDB (Pee Dee Belemnite), and δ18O are referred to -VSMOW. The reference materials used were IAEA C3, IAEA CH7; IAEA 600; IAEA N1 and N2; IAEA 601 and IAEA 602. EuroEA (EuroVector, Italy) elemental analyser was used for online sample preparation by Dumas-combustion. For the elemental analyses the Certified Reference Materials were Sulfanilamide for C, N, H, S and Atropine for O.

本研究报道了一项基于水族箱的受控实验结果，该实验旨在探究潜在采矿活动事故中产生的低氧环境与悬浮颗粒物，对深海贻贝（Bathymodiolus azoricus）幼体的影响。

实验所用贻贝于2021年采自东北大西洋亚速尔群岛的幸运罢工热液喷口场（Lucky Strike hydrothermal vent field），采样水深为1700米。实验贻贝被饲养于1升规格的水族箱内，并于亚速尔大学Okeanos平台的深海实验室水族设施中，接受为期两周的四种实验处理：
（1）对照组：无外源沉积物添加，海水溶解氧浓度维持正常水平；
（2）低氧组：溶解氧浓度较正常水平低30 μmol；
（3）悬浮多金属硫化物（PMS）颗粒物组；
（4）低氧+悬浮多金属硫化物（PMS）颗粒物复合处理组。

实验所用PMS颗粒物通过研磨采自幸运罢工热液喷口场的非活性热液烟囱岩制备获得。实验第8天与第11天，水体中悬浮颗粒物浓度均设置为420 mg/L。

本研究通过稳定同位素分析与元素分析方法，对混合贻贝组织样品进行检测，以评估低氧胁迫、PMS颗粒物的单独效应及其复合效应，具体检测流程如下：
检测采用连续流同位素比值质谱法（CF-IRMS），所用设备为英国Sercon公司的SerconHydra 20–22型与Isoprime IRMS稳定同位素比值质谱仪，该设备与意大利EuroVector公司的EuroEA元素分析仪联用，通过杜马斯燃烧法完成在线样品前处理。

同位素比值的δ值计算遵循公式：δ = [(R样本 – R标准品)/R标准品]×1000，其中R为重同位素与轻同位素的丰度比值。其中δ¹⁵N_Air值以大气为参考基准，δ¹³C_VPDB值以PDB（佩德箭石，Pee Dee Belemnite）为参考基准，δ¹⁸O值以-VSMOW为参考基准。

本次实验所用的标准参考物质包括IAEA C3、IAEA CH7、IAEA 600、IAEA N1、IAEA N2、IAEA 601以及IAEA 602。前述EuroEA元素分析仪（意大利EuroVector公司）同样通过杜马斯燃烧法完成在线样品前处理。

元素分析所用的有证标准物质为：碳（C）、氮（N）、氢（H）、硫（S）采用磺胺作为标准物质，氧（O）采用阿托品作为标准物质。