Derivation of transcriptomics-based points of departure for twenty per- or polyfluoroalkyl substances (PFAS) using a larval fathead minnow (Pimephales promelas) reduced transcriptome assay.

Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization. Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization. Larval fathead minnows (Pimephales promelas; 5-6 days post-fertilization) were exposed to each of the 20 PFAS, in concentration response, for 24 h in 96-well plate format. For each assay, 30 mM and 9.5 mM stock solutions in DMSO were used to prepare a dilution series spanning from 30 mM to 0.0095 mM (1/2 log spacing between concentrations) in DMSO. Solutions in DMSO were then diluted 300-fold in sand filtered, UV-treated Lake Superior water (LSW) to generate a series of aqueous nominal concentrations ranging from 100 µM to 0.03 µM (1/2 log-spacing between concentrations), each, including control treatments, containing 0.33% DMSO. Larval fathead minnows (five days post fertilization) were transferred to wells of three 96-well (1 ml wells; NEST), one animal per well. The volume (variable from organism loading) in each well of the three plates was reduced to a uniform volume of 50 µl using a Biomek i5 liquid handler (Beckman), and then 550 µl of dosing solution was added to each well, bringing the total volume to 600 µl. On each of the three replicate plates five individual fish were exposed to each concentration of two PFAS test chemicals, eight individuals were exposed to control water, and the remaining eight wells were treated with reference chemicals (150 µg CuSO4/L). In total, 15 fish (distributed across 3 plates) were exposed to each PFAS treatment and 24 fish (distributed across 3 plates) were exposed to control water. Following the addition of the exposure solutions, plates were incubated at 25C for 24 h. At the end of each exposure period, each well was inspected under a dissecting microscope. Mortalities were enumerated wells containing dead animals noted. Any non-lethal phenotypic abnormalities such as edema, spinal malformations, or unusual behaviors were noted. A Biomek i5 liquid handler was used to transfer 100 µl of exposure water from one of the plates (Plate A) to be used for exposure verification (below) to a new 96 well plate. All wells from plates B and C were de-watered and frozen at -80 until homogenized. For homogenization, 75 µl of Biospyder homogenization buffer and a 2.3 mm stainless steel bead added. The fish were then homogenized in 96 well plate format using a bead mixer mill (Omni-Bead Ruptor; 30 Hz for 2 min). Homogenates corresponding to the same well position on each of two replicate plates (both from the same treatment) were pooled together (except in the case of mortality on one or both plates). This resulted in five replicate pools (independent biological replicates) per treatment, each representing the mean of two individuals. For controls there were eight replicate pools, each representing the mean of two individuals, however, the controls were shared for both PFAS tested on the same plate. PFAS concentrations in the exposure medium (collected from plate A) were measured at the end of each 24 h exposure.

传统毒性测试已无法跟上新化学品商业化投放的步伐。因此，针对鱼类与野生生物可能接触的诸多化学品，用于风险评估的毒性数据往往十分匮乏，甚至完全缺失。全氟和多氟烷基物质（Per- and polyfluoroalkyl substances, PFAS）正是这一困境的典型代表：绝大多数PFAS的生态危害仍未得到阐明。本研究采用高通量测试方法，针对20种性质各异的PFAS，探究了其暴露24小时后，受精后5~6天的黑头呆鱼（Pimephales promelas）幼体中引发协同基因表达响应的浓度。本研究采用简化转录组学策略，检测了1832个基因的全身表达水平，受试的20种PFAS的转录组起始点（transcriptomic point of departure, tPOD）中位数为10 µM。长链羧酸（12~13个碳氟链）、8个碳氟链的二元醇、N-烷基磺酰胺以及端基齐聚物磺酸均属于活性最强的PFAS之列，可在低于1 µM的浓度下引发基因表达响应。除少数例外外，黑头呆鱼幼体的tPOD与人类细胞系中基于全转录组响应得到的结果一致。不过，黑头呆鱼幼体的tPOD往往高于大型溞（Daphnia magna）暴露于同种PFAS时的tPOD。tPOD对应的浓度范围与已报道的鱼类其他亚致死效应浓度重合（已有10种PFAS的相关数据，涵盖不同物种、生命阶段与研究设计）。尽管如此，黑头呆鱼的tPOD仍比北美五大湖支流中检测到的水相PFAS浓度高出数个数量级，这表明即使对于生物富集潜力较强的PFAS，这些水域也具备充足的安全边际。总体而言，本研究结果广泛支持使用黑头呆鱼幼体转录组学测试方法，推导用于生态风险优先排序的筛选水平效应效能估算值。

本研究将受精后5~6天的黑头呆鱼幼体以浓度梯度暴露于20种PFAS中，于96孔板中完成24小时暴露。每种测试均使用30 mM与9.5 mM的二甲基亚砜（dimethyl sulfoxide, DMSO）储备液，配制浓度范围为30 mM至0.0095 mM（浓度间隔为1/2对数单位）的DMSO梯度溶液。随后将DMSO溶液以300倍稀释于砂滤、紫外灭菌的苏必利尔湖湖水（Lake Superior water, LSW）中，得到浓度范围为100 µM至0.03 µM的水相名义浓度梯度（浓度间隔为1/2对数单位），所有处理（含对照组）的DMSO终浓度均为0.33%。

将受精后5天的黑头呆鱼幼体转移至三块96孔板（每孔容积1 ml，品牌为NEST）的孔中，每孔投放1条幼体。使用Biomek i5液体处理系统（Beckman）将每孔体积（因幼体接种量存在差异）统一调整至50 µl，随后向每孔加入550 µl暴露液，使终体积达到600 µl。在三块重复板中，每种PFAS浓度均设置5条幼体暴露，对照组设置8条幼体，剩余8个孔加入参考化学品（150 µg CuSO4/L）。总计每种PFAS处理设置15条幼体（分布于三块板），对照组设置24条幼体（分布于三块板）。

加入暴露液后，将板置于25℃下孵育24小时。暴露结束后，在体视显微镜下观察每孔情况，统计死亡幼体数量并记录死体所在孔位，同时记录非致死表型异常情况，如水肿、脊柱畸形或异常行为。使用Biomek i5液体处理系统将其中一块板（板A）中的100 µl暴露水转移至新的96孔板，用于暴露浓度验证。将板B与板C的所有孔内液体抽干后，于-80℃冻存直至均质化。

均质化步骤：向每孔加入75 µl Biospyder均质缓冲液与1颗2.3 mm不锈钢珠，使用珠磨式均质仪（Omni-Bead Ruptor；30 Hz，均质2分钟）以96孔板格式完成幼体均质。将两块重复板（来自同一处理组）中相同孔位的匀浆合并（若其中一块或两块板存在死体则除外），最终每个处理组得到5个重复混合样本（独立生物学重复），每个样本代表2条幼体的混合匀浆。对照组得到8个重复混合样本，每个样本同样代表2条幼体的混合匀浆，但同一板内测试的两种PFAS共用对照组样本。

在每次24小时暴露结束后，测定暴露介质（取自板A）中的PFAS浓度。