Sterechinus neumayeri fertilisation and larval development toxicity tests

This dataset contains results of toxicity tests with early life stages of the sea urchin Sterechinus neumayeri as part of the AAS Project 3054 'Ecological risks from oil products used in Antarctica: characterising hydrocarbon behaviour and assessing toxicity on sensitive early life stages of Antarctic marine invertebrates.'

Dataset consists of excel spreadsheets with separate spreadsheets for each test. Test details are outlined on worksheets 'Test conditions' and results of test in worksheet 'Counts'.

This metadata record contains the results of toxicity tests conducted to characterise the response of Antarctic nearshore marine invertebrates to hydrocarbon contaminants in fuels commonly used in Antarctica as part of AAS Project 3054. This dataset contains results of toxicity tests conducted at Davis Station in 2010/11 summer season to test the sensitivity of fertilisation and early life stages of the sea urchin Sterechinus neumayeri to fuels in seawater. The three fuel types used were: Special Antarctic Blend diesel (SAB), Marine Gas Oil diesel (MGO) and an intermediate grade (180) of marine bunker Fuel Oil (IFO). Test treatments were obtained by experimentally mixing fuel and seawater in temperature controlled cabinets at -1 degrees C to prepare a mixture of fuel hydrocarbons in filtered seawater (FSW) termed the water accommodated fraction (WAF). WAF was produced by adding fuel to seawater in Pyrex glass bottles using a ratio of 1:25 fuel : FSW. This mixture was stirred at slow speed with minimal vortex for 18 h on a magnetic stirrer then settled for 6 h before the water portion was drawn from beneath the fuel.

Mature S. neumayeri were collected from the outlet of Ellis Fjord, East Antarctica (68.62°S, 77.99°E) in December and early January 2010/11. Sea urchins were collected from shallow nearshore waters less than 1m deep, placed in 20 L buckets of seawater and transported to Davis station. They were held for 1–2 d in a flow-through aquarium at -1 plus or minus 1°C, with macroalgae from the collection site as a food source, before being used for testing. Seawater for experiments was collected ~20 m from the shoreline north of Davis station (68°34’ S, 77°57’ E). Collected seawater was filtered to 0.45 µm (FSW) and stored in 30 L polyethylene containers at 0°C.

Fertilisation and early embryo toxicity tests.
Effects of WAFs on fertilisation and on development to the 2 cell stage were determined in static tests in which both eggs and sperm were pre-exposed to SAB, MGO and IFO 180 WAFs, fertilised within treatments and developed to the 2 cell stage (G1, G2, G3). Gamete exposure and fertilisation was done in a temperature controlled room at 0°C.

Test vessels were 22 mL borosilicate glass vials with foil lined lids holding 20 mL of test solution. There were 10 vials for each treatment; 5 replicates for fertilisation and 5 replicates for the 2 cell endpoint. To pre-expose eggs, 5 mL of prepared egg solution was added to vials that contained 5 mL of 2, 20 and 100% WAFs and FSW controls, to give final treatment concentrations of 1, 10 and 50% WAF dilutions and FSW controls. Vials were sealed, swirled gently to mix and left standing for 20 min. To pre-expose sperm, pooled sperm were activated by dilution in FSW to the density required for a sperm to egg ratio of 800:1. One µL of sperm solution was added to vials containing 5 mL of FSW and gently mixed. Five mL of this solution was then added to vials containing 5 mL of 2%, 20% and 100% WAFs (final treatments of 1, 10 and 50% WAF dilutions) and FSW controls. The vials were sealed, swirled gently to mix and left for 15 mins. After the gamete exposure period was complete, for each treatment the contents of the sperm vials were added to the egg vials with a final target concentration of ~10 eggs per mL. Vials were sealed and placed into temperature-controlled cabinets set at -1 plus or minus 1°C. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.3 plus or minus 0.5°C.

Tests were terminated at 4 h for the fertilisation endpoint, and at 11 h for the 2 cell endpoint by the addition of 1 mL of 2.5% (v/v) buffered glutaraldehyde. Samples were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. Fertilisation was assessed according to the presence or absence of a fertilisation membrane in the first 100 eggs counted, to obtain the percentage of eggs fertilised in each replicate. The 2 cell endpoint was assessed in the first 100 embryos counted, as the percentage of embryos in each replicate with normal first cleavage.

Embryonic and larval toxicity tests. Effects of fuel WAFs on embryonic and larval development were tested with 1, 10, and 100% WAFs of SAB, MGO and IFO 180 and FSW control, with 5 replicates per treatment. Eggs and sperm were collected and density of solutions adjusted as described above to obtain the optimal sperm to egg ratio of 800:1.

Two semi-static tests (EL1, EL2) were done to test effects of WAFs on embryos and larvae when first exposed as zygotes (eggs fertilised in FSW then exposed to treatments before the first cleavage). To fertilise eggs, sperm were activated by their addition to 10 mL of FSW, and 1 µL of this sperm solution was added to beakers containing 700 mL of egg solution and gently mixed. After two hours, the mixture was stirred with a glass rod to maintain a homogeneous suspension while aliquots were transferred into 100 mL glass vials filled with 80 mL of test treatment, to a final density of ~10 zygotes per mL.

Three tests (GL1, GL2, GLP) were done to test effects of WAFs on larval development with exposure commencing as gametes. One mL aliquots of egg mixture were added to vials containing 80 mL of test solution (to a density of ~10 eggs per mL) and left for 20 min. Sperm were activated in 10 mL of FSW and 0.1 mL aliquots added to the vials to fertilise eggs within treatments at a sperm to egg ratio of 800:1. Two exposure regimes were used; continuous semi-static WAF renewal (GL1 and GL2) and a single static pulse of WAF exposure up to the 4 d unhatched blastula stage, followed by post exposure recovery in FSW up to the 21 d pluteus stage (GLP).

Vials were left uncovered and placed in a temperature controlled cabinet at -1 plus or minus 1°C with an 18 h light, 6 h dark photoperiod. Tests were under semi-static conditions, with test solutions renewed every 4 d. Water quality data was collected at each water change.

Treatment renewals were done by removing and replacing approximately 90% of test solution. Disposable syringes with silicon tubing attached to the nozzle, and with the end of the tubing covered with plankton mesh, were used to withdraw test solution while preventing embryos/larvae from being removed. The vials were then refilled to the 80 mL mark with fresh test solutions. Treatment renewals for tests EL1, EL2 and GL1, GL2 were with freshly made WAFs every 4 d. For the single pulse WAF exposure test (GLP) on the first treatment renewal at 4 d, treatment solutions were removed as described above, and replaced with FSW. All subsequent 4 d renewals for test GLP were with FSW.

To maintain the volume and salinity of test treatments a small volume of purified and deionised (Milli-Q) water at -1°C was stirred into the vials to the 80 mL mark every 2 d between water changes. Water quality measurements were made at the start of tests and pre and post treatment renewals. Mean water quality parameter measurements were pH 8.08 plus or minus 0.10, salinity 36.6 plus or minus 0.9‰ and dissolved oxygen 11.1 plus or minus 0.61 mg/L. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.0 plus or minus 1.0°C.

In tests where exposure commenced as zygotes, endpoints were the embryonic 4-8 cell (20 h) and unhatched blastula (48 h) stages, and the larval blastula (6–7 d) and gastrula (14–15 d) stages. In tests with exposure commencing as gametes, endpoints were the larval blastula, gastrula and early 4-arm pluteus (21–24 d) stages. At each endpoint a sample was taken from each replicate by drawing an aliquot with a glass pipette and transferring it to a vial, to which 1 mL of 2.5% (v/v) buffered glutaraldehyde was added. Embryo and larvae were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. The first 30 individuals in each sample at the 4-8 cell and unhatched blastula endpoints, and the first 100 individuals at the blastula, gastrula and pluteus endpoints, were assessed for normality. Test EL1 ended at the blastula stage and tests EL2 and GL2 at the gastrula stage as there were insufficient numbers of larvae remaining to continue the test beyond these stages. All remaining larvae were counted at the final endpoint.

Chemical analysis of water accommodated fractions
Total hydrocarbon content (THC) in WAFs were derived from replicate tests conducted under the same conditions but without test organisms. In these tests at 0°C, the concentrations of freshly made WAFs of each of the three fuels, and the depletion of hydrocarbons from 100%, 50%, 10% and 1% WAFs at multiple time points over 7 d were measured. Extracts were analysed for THC with GC-FID. Total hydrocarbon content was reported as the sum of hydrocarbons (µg/L) in the range less than n-C9 to C28 (Dataset AAS_3054_THC_WAF).

For fertilisation, and 2 cell embryonic development assays that were done in sealed vials, measured values in freshly decanted 50% and 10% WAF dilutions were used as the exposure concentrations.

For the embryonic and larval toxicity tests that were done in open vials, the exposure concentrations of THC in WAFs were modelled from the measured concentrations in WAF depletion tests. Exposure concentrations used to model sensitivity estimates were derived by calculating the time weighted mean THC between pairs of successive measurements in the 100% WAFs and dilutions to give overall exposure concentrations for each time point. These modelled concentrations integrated the loss of hydrocarbons over time, and renewal of test solutions at 4 d intervals.

本数据集为AAS 3054项目"Ecological risks from oil products used in Antarctica: characterising hydrocarbon behaviour and assessing toxicity on sensitive early life stages of Antarctic marine invertebrates."（即《南极使用石油产品的生态风险：表征烃类行为并评估南极海洋无脊椎动物敏感早期生命阶段的毒性》）的一部分，包含以斯氏棘海胆（Sterechinus neumayeri）早期生命阶段为受试对象的毒性试验结果。

本数据集以Excel电子表格形式存储，每项试验对应单独的工作表。试验细节记载于"试验条件"工作表，试验结果则收录于"计数"工作表。

本元数据记录收录了AAS 3054项目中，为表征南极近岸海洋无脊椎动物对南极常用燃料中烃类污染物的响应而开展的毒性试验结果。本数据集包含2010/2011夏季在戴维斯站开展的毒性试验结果，旨在评估斯氏棘海胆（Sterechinus neumayeri）的受精过程与早期生命阶段对海水中燃料的敏感性。本次试验使用的燃料类型包括：南极专用混合柴油（Special Antarctic Blend diesel, SAB）、船用柴油（Marine Gas Oil diesel, MGO）以及IFO 180型中间级船用燃料油。

试验处理液通过以下方式制备：在-1℃的控温柜中，将燃料与过滤海水（filtered seawater, FSW）混合，得到燃料烃类在过滤海水中的混合物，即水可溶组分（water accommodated fraction, WAF）。具体制备流程为：按燃料与FSW的质量体积比1:25，将燃料加入派热克斯玻璃瓶中的海水中，以低速搅拌（尽量避免产生涡流）18小时，随后静置6小时，再从燃料层下方抽取水相部分。

2010年12月至2011年1月初，科研人员从南极东部埃利斯峡湾出口处（南纬68.62°，东经77.99°）采集成熟的斯氏棘海胆。海胆采自水深不足1米的近岸浅水区，放置于装有海水的20升桶中转运至戴维斯站。试验前，海胆在水温为-1±1℃的流水式水族箱中暂养1~2天，以采集地的大型藻类作为食物。试验用海水采自戴维斯站北侧海岸线附近约20米处（南纬68°34′，东经77°57′），经0.45μm过滤得到过滤海水（FSW），随后储存于0℃的30升聚乙烯容器中。

受精与早期胚胎毒性试验
WAF对受精过程及发育至2细胞阶段的影响通过静态试验测定：将卵子与精子分别预暴露于SAB、MGO及IFO 180型WAF中，在对应处理液中完成受精，并使其发育至2细胞阶段（G1、G2、G3组）。配子预暴露与受精过程均在0℃的控温室内完成。

试验容器为22mL硼硅酸盐玻璃小瓶，配有铝箔衬里的瓶盖，每瓶装入20mL试验溶液。每组处理设置10个重复小瓶：5个用于受精终点检测，5个用于2细胞阶段终点检测。卵子预暴露处理：向装有5mL 2%、20%、100% WAF及FSW对照液的小瓶中加入5mL配制好的卵子溶液，使最终处理液的WAF稀释浓度分别为1%、10%、50%，并设置FSW对照组。密封小瓶后轻轻摇匀，静置20分钟。精子预暴露处理：将混合的精子用FSW稀释至所需浓度，使精卵比达到800:1。取1μL该精子溶液加入装有5mL FSW的小瓶中，轻轻混匀。随后取5mL该混合液加入到装有5mL 2%、20%、100% WAF液及FSW对照液的小瓶中（最终WAF稀释浓度为1%、10%、50%，并设置FSW对照组）。密封小瓶后轻轻摇匀，静置15分钟。预暴露结束后，将每个处理组的精子混合液加入对应的卵子小瓶中，使最终卵子密度约为10个/mL。密封小瓶后放置于-1±1℃的控温柜中。使用马克西姆纽扣式数据记录仪（Maxim ibutton）每10分钟记录一次温度，平均水温为-1.3±0.5℃。

受精终点试验在4小时时终止，2细胞阶段终点试验在11小时时终止，终止方式为加入1mL 2.5%（体积比）缓冲戊二醛固定样品。使用塞奇威克-拉夫特计数板，在10倍放大倍数的光学显微镜下观察样品。受精率通过计数前100个卵子的受精膜有无情况进行评估，以得到每个重复组的受精百分比。2细胞阶段终点则通过计数前100个胚胎，统计每个重复组中正常完成第一次卵裂的胚胎占比。

胚胎与幼虫毒性试验
本次试验评估了三种燃料的WAF对胚胎与幼虫发育的影响，测试浓度为1%、10%、100% WAF，同时设置FSW对照组，每组处理设置5个重复。卵子与精子的采集及溶液浓度调整方式如前文所述，以确保精卵比达到最优的800:1。

开展两项半静态试验（EL1、EL2），以评估WAF对以受精卵为暴露起点的胚胎与幼虫的影响（即：在过滤海水中完成受精的卵子，在第一次卵裂前暴露于试验处理液中）。受精操作如下：将精子加入10mL FSW中激活，取1μL该精子溶液加入装有700mL卵子溶液的烧杯中，轻轻混匀。2小时后，用玻璃棒搅拌混合液以维持均匀悬浮状态，随后将等分试样转移至装有80mL试验处理液的100mL玻璃小瓶中，使最终受精卵密度约为10个/mL。

开展三项试验（GL1、GL2、GLP），以评估WAF对以配子为暴露起点的幼虫发育的影响。具体操作：取1mL卵子混合液加入装有80mL试验溶液的小瓶中，使最终卵子密度约为10个/mL，静置20分钟。将精子在10mL FSW中激活，取0.1mL等分试样加入小瓶中，以800:1的精卵比完成对应处理液中的受精。本次试验采用两种暴露方案：① 持续半静态更新WAF（GL1、GL2组）；② 单次静态脉冲暴露至4日龄未孵化囊胚阶段，随后将幼虫转移至FSW中恢复培养至21日龄长腕幼虫阶段（GLP组）。

小瓶开盖后放置于-1±1℃的控温柜中，光照周期为18小时光照、6小时黑暗。试验采用半静态条件，每4天更换一次试验溶液，每次换液时记录水质数据。

换液操作通过移除并更换约90%的试验溶液完成。使用连接硅树脂管的一次性注射器，且软管末端覆盖浮游生物网，以在抽取试验溶液的同时避免胚胎/幼虫被带出。随后向小瓶中加入新鲜试验溶液至80mL刻度线。EL1、EL2、GL1及GL2组的换液使用新鲜制备的WAF，每4天更换一次。对于单次脉冲暴露试验（GLP组），在第4天首次换液时，按上述方式移除原处理液，更换为FSW；后续所有每4天一次的换液均使用FSW。

为维持试验处理液的体积与盐度，在两次换液间隔的每2天，向小瓶中加入少量-1℃的密理博（Milli-Q）纯化去离子水，直至液面至80mL刻度线。在试验开始时、换液前后均进行水质测量。平均水质参数为：pH 8.08±0.10，盐度36.6±0.9‰，溶解氧11.1±0.61mg/L。使用马克西姆纽扣式数据记录仪每10分钟记录一次温度，平均水温为-1.0±1.0℃。

以受精卵为暴露起点的试验，其观测终点为胚胎的4-8细胞阶段（20小时）、未孵化囊胚阶段（48小时），以及幼虫的囊胚阶段（6~7日）与原肠胚阶段（14~15日）。以配子为暴露起点的试验，其观测终点为幼虫的囊胚阶段、原肠胚阶段与21~24日龄四腕长腕幼虫阶段。每个终点阶段时，从每个重复组中用玻璃移液管抽取等分试样，转移至小瓶中并加入1mL 2.5%（体积比）缓冲戊二醛固定。使用塞奇威克-拉夫特计数板，在10倍放大倍数的光学显微镜下观察胚胎与幼虫。对于4-8细胞和未孵化囊胚阶段的样本，计数前30个个体以评估发育正常率；对于囊胚、原肠胚及长腕幼虫阶段的样本，计数前100个个体以评估发育正常率。由于剩余幼虫数量不足，试验EL1在囊胚阶段终止，试验EL2与GL2在原肠胚阶段终止。所有试验在最终终点时统计全部剩余幼虫的数量。

水可溶组分的化学分析
WAF中的总烃含量（Total hydrocarbon content, THC）通过在相同条件下开展的无受试生物的平行试验获得。在0℃条件下的此类试验中，测定了三种燃料新鲜制备WAF的浓度，以及100%、50%、10%、1% WAF在7天内多个时间点的烃类消耗情况。使用气相色谱-火焰离子化检测器（GC-FID）对萃取液中的THC进行分析。总烃含量以n-C9至C28范围内的烃类总含量（μg/L）进行报告（数据集编号：AAS_3054_THC_WAF）。

对于密封小瓶中开展的受精与2细胞胚胎发育试验，使用新鲜倾出的50%与10% WAF稀释液的实测值作为暴露浓度。

对于开放式小瓶中开展的胚胎与幼虫毒性试验，WAF中的THC暴露浓度通过WAF消耗试验的实测值进行建模估算。用于敏感性评估建模的暴露浓度，通过计算100% WAF及其稀释液在连续两次测量之间的时间加权平均THC浓度得到，以得到每个时间点的总暴露浓度。该建模浓度整合了烃类随时间的消耗以及每4天一次的试验溶液更新过程。