Exposure to perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at ppb concentration induces chronic toxicity in Daphnia carinata
接触ppb浓度的全氟辛烷磺酸（PFOS）而非全氟辛酸（PFOA）会诱发水蚤的慢性毒性

Received 17 October 2020
2020 年 10 月 17 日收到

Received in revised form 10 December 2020
2020 年 12 月 10 日收到修订稿

Accepted 13 December 2020
2020 年 12 月 13 日接受

Available online 9 January 2021
2021 年 1 月 9 日在线提供

Editor: Henner Hollert 编辑：Henner Hollert

Acute and chronic toxicity
急性和慢性毒性

Comet assay 彗星试验

Daphnia carinata 水蚤

Perfluorooctanoic acid 全氟辛酸

Perfluorooctanesulfonate
全氟辛烷磺酸

(c) 2021 Elsevier B.V. All rights reserved.
(c) 2021 Elsevier B.V. 保留所有权利。保留所有权利。

Per- and polyfluoalkyl substances (PFAS) are a group of synthetic organic chemicals containing fully fluorinated carbon chains. These chemicals have been used in a variety of industrial and consumer applications for over six decades (Buck et al., 2011; Giesy et al., 2010; Kissa,
全氟和多氟烷基物质（PFAS）是一组含有全氟碳链的合成有机化学品。六十多年来，这些化学品一直被用于各种工业和消费用途（Buck 等人，2011 年；Giesy 等人，2010 年；Kissa、

2001). Among more than 5000 PFAS known to be used in commerce, perfluoroalkylsulfonates (PFSA) characterized by a terminal sulfonate group attached to the perfluoroalkyl chain, and perfluoroalkyl carboxylic acids (PFCA) with carboxylate group as the terminal group received wide attention in recent years (Lindstrom et al., 2011b). High-energy carbon-fluorine ( ) bond provides PFAS with extreme thermal, photolytic, hydrolytic stability and resistance to microbial degradation, and these compounds have been used as wetting, lubricating, staining, and corrosion resistance agents. Among the several PFAS, perfluorooctanesulfonate (PFOS) has been shown to biomagnify in the aquatic food web (Giesy and Kannan, 2001). PFAS have been detected in human milk, blood, and seminal plasma from various countries worldwide (Guruge et al., 2005; Kudo and Kawashima, 2003; Mulkiewicz et al., 2007; Liu et al., 2020a). Perfluorooctanoic acid (PFOA) was reported to be present in groundwater and surface water in areas near the point and non-point sources (Mak et al., 2009). PFAS exhibit differential fates and toxicities in the environment, which may be related to polar functional moieties (Giesy et al., 2010). The environmental persistence and widespread distribution of PFAS in terrestrial and aquatic environments (Table S1) have raised concern about their ecological toxicity (Berthiaume and Wallace, 2002; Shi et al., 2007). The persistency of PFAS has been shown to be related to the length of the alkyl chain, with shorter chain compounds having lesser toxicity (Giesy et al., 2010; Olsen et al., 2007).
2001).在已知用于商业用途的 5000 多种 PFAS 中，以全氟烷基链上连接的末端磺酸基为特征的全氟烷基磺酸盐（PFSA）和以羧酸基为末端基团的全氟烷基羧酸（PFCA）近年来受到广泛关注（Lindstrom 等人，2011b）。高能碳-氟（ ）键使全氟烷基羧酸具有极高的热稳定性、光解稳定性、水解稳定性和抗微生物降解性，这些化合物已被用作润湿剂、润滑剂、染色剂和抗腐蚀剂。在几种 PFAS 中，全氟辛烷磺酸（PFOS）已被证明会在水生食物网中产生生物放大作用（Giesy 和 Kannan，2001 年）。在全球多个国家的母乳、血液和精浆中都检测到了全氟辛烷磺酸（Guruge 等人，2005 年；Kudo 和 Kawashima，2003 年；Mulkiewicz 等人，2007 年；Liu 等人，2020a）。据报道，全氟辛酸（PFOA）存在于点源和非点源附近地区的地下水和地表水中（Mak 等人，2009 年）。全氟辛酸在环境中表现出不同的命运和毒性，这可能与极性功能分子有关（Giesy 等人，2010 年）。PFAS 在陆地和水生环境中的环境持久性和广泛分布（表 S1）引起了人们对其生态毒性的关注（Berthiaume 和 Wallace，2002 年；Shi 等人，2007 年）。研究表明，全氟辛烷磺酸的持久性与烷基链的长度有关，链短的化合物毒性较低（Giesy 等人，2010 年；Olsen 等人，2007 年）。

Though the industrial production of PFOS and its derivatives is restricted in many countries since 2000, several related analogues with varying chain lengths and telomeres that can degrade to yield PFOA and other perfluorocarboxylic acids are still being produced and utilized in commerce (Jensen and Leffers, 2008). Studies on the acute toxicity of PFAS to aquatic invertebrates, plants, and animals are limited (Giesy et al., 2010; Latała et al., 2009; Bartlett et al., 2020; Gaballah et al., 2020; Ghisi et al., 2019; Sivaram et al., 2020).
尽管自 2000 年以来，许多国家限制了全氟辛烷磺酸及其衍生物的工业生产，但目前仍在生产和使用几种具有不同链长和端粒的相关类似物，它们可以降解生成全氟辛酸和其他全氟羧酸（Jensen 和 Leffers，2008 年）。有关全氟辛烷磺酸对水生无脊椎动物、植物和动物急性毒性的研究十分有限（Giesy 等人，2010 年；Latała 等人，2009 年；Bartlett 等人，2020 年；Gaballah 等人，2020 年；Ghisi 等人，2019 年；Sivaram 等人，2020 年）。

Daphnia is an ecologically important organism that is widely distributed in freshwater aquatic ecosystems (Benzie, 2005). Daphnids have been commonly used in ecotoxicity testing of several chemicals across the globe (Chen et al., 2015; Liu et al., 2020b). Acute toxicity of PFOS and PFOA to freshwater invertebrates such as Daphnia magna, D. pulicaria, Unio complanatus, Moina macrocopa, and Chydorus sphaericus has been reported (Boudreau et al., 2002; Dang et al., 2012; Ji et al., 2008). Several authors have emphasized the need to use native species for ecotoxicity testing (Cáceres et al., 2007a, 2007b; Phyu et al., 2004; Harmon et al., 2003). An Australian cladoceran, namely, Daphnia carinata, has been used as a model invertebrate species for toxicity tests (Phyu et al., 2004; Cáceres et al., 2007a). D. carinata are similar in size to D. magna, which makes them an ideal candidate for chronic toxicity studies (Phyu et al., 2004). The use of native species in toxicity testing is ecologically relevant, which will reduce the variation in toxicity due to regional differences (Harmon et al., 2003). In the present study, we examined acute, chronic, and genotoxicity of PFOA and PFOS to D. carinata, a native Australian freshwater cladoceran.
水蚤是一种重要的生态生物，广泛分布于淡水水生生态系统中（Benzie，2005 年）。水蚤通常被用于全球多种化学品的生态毒性测试（Chen 等人，2015 年；Liu 等人，2020b）。据报道，全氟辛烷磺酸和全氟辛酸对大型水蚤、D. pulicaria、Unio complanatus、Moina macrocopa 和 Chydorus sphaericus 等淡水无脊椎动物具有急性毒性（Boudreau 等人，2002 年；Dang 等人，2012 年；Ji 等人，2008 年）。一些作者强调了使用本地物种进行生态毒性测试的必要性（Cáceres 等人，2007a，2007b；Phyu 等人，2004；Harmon 等人，2003）。澳大利亚的一种无脊椎动物，即卡氏水蚤（Daphnia carinata），已被用作毒性测试的无脊椎动物模型物种（Phyu 等人，2004 年；Cáceres 等人，2007a）。鲫鱼的大小与大型鲫鱼相似，因此是慢性毒性研究的理想候选物种（Phyu 等人，2004 年）。在毒性测试中使用本地物种与生态相关，这将减少因地区差异造成的毒性差异（Harmon 等人，2003 年）。在本研究中，我们检测了全氟辛烷磺酸和全氟辛烷磺酸对 D. carinata（一种原生的澳大利亚淡水蛤类）的急性、慢性和遗传毒性。

D. carinata, obtained from the Department of Environment and conservation (NSW), Australia and maintained in our laboratory was used in this study (Cáceres et al., 2007a). Twenty-four-hour old (neonates) cladocerans were obtained from a continuous culture in glass bottles with natural spring water in deionized water. The cultures were maintained with a light and dark photoperiod at (Cáceres et al., 2007a). The light intensity was maintained at at the surface of the water. The cladoceran medium was renewed thrice a week, and the animals were fed with green alga Raphidocelis subcapitata (at cells day ). The alga was grown in Bold's basal medium (Megharaj et al., 2000) and maintained under the same condition as the experiment. In order to test the sensitivity of the daphnid culture maintained in our laboratory, acute toxicity tests were performed at periodic intervals (once every three months) as per OECD guidelines (Test No.211, OECD, 2012), with the reference toxicant potassium dichromate .
本研究使用了从澳大利亚新南威尔士州环境与保护部（Department of Environment and conservation，NSW）获得并在我们实验室中饲养的 D. carinata（Cáceres 等人，2007a）。在 玻璃瓶中用 天然泉水和去离子水进行连续培养，获得 24 小时大（新生）的梭鱼。培养物的光周期为 和 ，光照强度保持在 （Cáceres 等人，2007a）。水面的光照强度保持在 。培养基每周更新三次，并向动物投喂绿色藻类 Raphidocelis subcapitata（在 细胞 天 ）。这种藻类在 Bold 的基础培养基（Megharaj 等人，2000 年）中生长，并在与实验相同的条件下维持。为了测试本实验室所培养的水蚤的敏感性，根据经合组织指南（测试编号 211，经合组织，2012 年），以参考毒物重铬酸钾 为参照，定期（每三个月一次）进行急性毒性测试。

Perfluorooctanoic acid (95%) and perfluorooctanesulfonic acid potassium salt used in this study were obtained from SigmaAldrich Australia. The stock solution of PFOA was prepared in deionized water ( ), and the PFOS stock solution ( ) was prepared in dimethylformamide (DMF) and diluted in distilled water to achieve . From this, the treatment concentrations were prepared using cladoceran water in polypropylene containers.
本研究中使用的全氟辛酸（95%）和全氟辛烷磺酸钾 均来自澳大利亚 SigmaAldrich 公司。PFOA 的储备溶液用去离子水配制 ( ) ，PFOS 的储备溶液 ( ) 用二甲基甲酰胺（DMF）配制，并用蒸馏水稀释 ，得到 。在此基础上，使用聚丙烯容器中的蛤蜊水制备处理浓度。

The acute toxicity test was performed as per OECD guidelines (OECD, 2000) with slight modifications (Cáceres et al., 2007a). Toxicity tests were conducted in the cladoceran growth medium. Six to twelve hours old neonatal cladocerans were used for testing. Before the acute toxicity test, an initial range-finding test was conducted with test concentrations of . Based on the range finding test the following concentrations were selected for the acute toxicity: and for PFOA and and for PFOS. The sensitivity of . carinata was checked by exposing the fleas to a known toxicant up to under the same experimental conditions. The survival test was conducted at a temperature of with a photoperiod of and dark cycle under light intensity. Cladoceran growth medium without the test chemicals served as controls. Toxicity tests were conducted in triplicate with ten organisms per replication. Cladocerans exhibiting immobility within after the gentle stirring of the test container were considered immobilised, which was the endpoint for determining acute toxicity. Immobility (mortality) after 24 and were recorded from each treatment and control.
急性毒性试验是根据经合组织指南（经合组织，2000 年）略加修改后进行的（Cáceres 等人，2007a）。毒性测试在桡足类生长培养基中进行。测试使用的是六到十二小时大的新生衣藻。在进行急性毒性试验之前，先进行了初步的范围测定试验，试验浓度为 。急性毒性测试选择了以下浓度的全氟辛烷磺酸（PFOA）： 和 ；全氟辛烷磺酸（PFOS）： 和 。在相同的实验条件下，将跳蚤暴露于已知毒物 至 ，以检测 . carinata 的敏感性。存活试验在 的温度下进行，光照强度为 ，光周期为 和 暗周期。不含受试化学品的桡足类生长培养基作为对照。毒性试验一式三份，每份十个生物。轻轻搅拌试验容器后，在 内表现出不动的桡足类被视为固定，这是确定急性毒性的终点。记录了每种处理和对照组在 24 小时和 之后的不动性（死亡率）。

The chronic toxicity test was performed according to an OECD standard protocol (OECD, 2012). In order to conduct the chronic toxicity at environmentally relevant concentrations, the neonates (6-12 h old) were exposed to the test chemicals (from stock solutions prepared in cladoceran water) at a concentration ranging from 0 to for 21 days. One neonate was placed in each container ( polypropylene containers containing test solution), and each treatment had ten replicates. Test containers were monitored every to record mortality and for the renewal of test solutions. The animals were fed daily with . subcapitata at a concentration of cells . The incubation conditions were similar to that reported for the acute toxicity assay. The endpoints for chronic effects were days to first brood, average offspring in each brood, and total live offspring. The survival of adults in each treatment was also documented during the chronic exposure period.
慢性毒性试验根据经合组织标准协议（经合组织，2012 年）进行。为了在环境相关浓度下进行慢性毒性试验，将新生儿（6-12 小时大）暴露于浓度为 0 至 的试验化学品（来自 在蛤蜊水中制备的储备溶液）中，持续 21 天。每个容器（ ，内装 试验溶液的聚丙烯容器）中放置一个新生儿，每个处理有 10 个重复。每隔 对试验容器进行监测，以记录死亡率和更新试验溶液。每天用 . subcapitata 喂养动物，浓度为 cells 。培养条件与急性毒性试验报告的条件相似。慢性效应的终点是第一窝产仔天数、每窝产仔的平均后代数以及活后代总数。在慢性接触期间，还记录了各处理中成虫的存活率。

DNA damage was evaluated using alkaline single cell gel electrophoresis or comet assay, as described in Prasath et al. (2016). Six-hour old neonates were exposed to each test chemicals , ) for at a concentration similar to the chronic toxicity test to replicate as environmentally relevant as possible, and also high concentrations were included in the assay to generate observable and distinguishable results. Tests were conducted in triplicate with ten
按照 Prasath 等人（2016 年）的描述，使用碱性单细胞凝胶电泳或彗星试验评估 DNA 损伤。将六小时大的新生儿暴露于每种受试化学品 , ) ，浓度与慢性毒性试验相似，以尽可能复制与环境相关的情况，同时在试验中加入高浓度，以产生可观察和可区分的结果。试验一式三份，每份十个
organisms per replicate. After the test duration, juvenile fleas were collected and transferred to beakers using a glass tube; subsequently, the fleas were laid onto a blotting paper. When excess water was adsorbed, the organisms were rapidly picked using a needle, carefully avoiding damage (when gently touched with a needle, the organisms adhered to the needle tip), and transferred into a microcentrifuge tube containing of phosphate-buffered saline (PBS), containing EDTA and dimethyl sulfoxide (DMSO), and disintegrated mechanically by grinding. Hydrogen peroxide treatment was used as a positive control (Diamantino et al., 2000; Yang et al., 2019). The suspension was centrifuged at for (Thermo Scientific Heraeus Fresco 21) to separate the debris and cells. Fifty microlitres of the cells were mixed with of low melting agarose and mixed thoroughly by pipetting. Comet assay slides were coated with of cells-agarose suspension and allowed to solidify at for . The alkaline comet assay was performed according to the manufacturer's instructions (Trevigen comet assay protocol, 8405 Helgerman Ct.). About 50 cells per slide ( 3 slides per treatment) were analyzed using a fluorescence microscope (Olympus BX41) at magnification. DNA damage was expressed as the percent DNA (Lent et al., 2012) in tail using an image analysis computerized method by CometScore software (TriTek Corp., Sumerduck, VA, USA).
每个重复的生物数量。试验时间结束后，收集幼蚤并用玻璃管转移到烧杯中，然后将幼蚤放在吸墨纸上。当吸附了多余的水分后，用针迅速挑取生物体，小心避免损伤（当用针轻轻触碰时，生物体会粘附在针尖上），并将其转移到装有 磷酸盐缓冲盐水（PBS）的 微离心管中，PBS 中含有 EDTA 和 二甲基亚砜（DMSO），并通过研磨进行机械分解。过氧化氢 处理作为阳性对照（Diamantino 等，2000；Yang 等，2019）。悬浮液在 下离心 （Thermo Scientific Heraeus Fresco 21）以分离碎片和细胞。将 50 微升细胞与 的低熔琼脂糖混合，并用移液器充分混合。用 细胞-琼脂糖悬浮液涂布彗星测定载玻片，并让其在 固化， 。碱性彗星试验按照制造商的说明进行（Trevigen 彗星试验方案，8405 Helgerman Ct.）。使用荧光显微镜（Olympus BX41）在 倍率下分析每张玻片上的约 50 个细胞（每个处理 3 张玻片）。使用 CometScore 软件（TriTek Corp., Sumerduck, VA, USA）的图像分析计算机方法，以尾部 DNA 的百分比（Lent 等人，2012 年）表示 DNA 损伤。

The test chemicals PFOA and PFOS were added to cladoceran water (at a final concentration of each), and the samples were incubated under prevailing experimental conditions. Untreated water samples served as controls. All the experiments were conducted in triplicate. After 48 and , the samples were analyzed for PFAS by high-performance liquid chromatography and mass spectroscopy (Das et al., 2015).
将测试化学品全氟辛烷磺酸和全氟辛烷磺酸添加到桡足类水体中（最终浓度分别为 ），并在现行实验条件下对样本进行培养。未经处理的水样作为对照。所有实验均一式三份。在 48 小时和 之后，采用高效液相色谱法和质谱法对样品进行 PFAS 分析（Das 等人，2015 年）。

The cladoceran growth medium was filtered using Millipore® filters prior to the analysis of heavy metals, dissolved organic carbon (DOC), and major ions such as nitrate, phosphate, sulphate, fluoride, chloride, and bromide. The heavy metal analysis was carried out in an inductively coupled plasma mass spectrophotometer (Agilent 7500 series) (Detection limit - ). The major ions were determined by ion chromatography (ICS 2000 series, Dionex Ion Chromatography System, Hong Kong) with AS19 column (Instrument detection limit: ), and the dissolved organic carbon (DOC) was analyzed using total organic carbon analyzer ( 1010 OI Analytical, PO Box 9010 College Station, Texas, 77842-9010, USA). The concentrations of PFOS and PFOA used in the study were measured using HPLC-MS (Agilent 1100 series) (Das et al., 2015). The spiked samples were injected onto a C-18 column and eluted with a short gradient comprised of methanol and an aqueous ammonium acetate buffer. The eluent was then introduced into the ESI source, and the negative ions were selected and detected by MS, operating in the selected ion monitoring (SIM) mode. Quantitation was performed using the Chemstation Software through the extraction of specific ions: for PFOA and for PFOS.
在分析重金属、溶解有机碳（DOC）和主要离子（如硝酸盐、磷酸盐、硫酸盐、氟化物、氯化物和溴化物）之前，使用 Millipore® 过滤器过滤桡足类生长培养基。重金属分析在电感耦合等离子体质谱仪（Agilent 7500 系列）中进行（检测限 - ）。主要离子采用离子色谱法（ICS 2000 系列，Dionex 离子色谱系统，香港）和 AS19 色谱柱进行测定（仪器检测限： ），溶解有机碳（DOC）采用总有机碳分析仪（1010 OI Analytical, PO Box 9010 College Station, Texas, 77842-9010, USA）进行分析。研究中使用的全氟辛烷磺酸和全氟辛酸的浓度是通过高效液相色谱-质谱仪（Agilent 1100 系列）测定的（Das 等人，2015 年）。将加标样品注入 C-18 色谱柱，用甲醇和乙酸铵水缓冲液组成的短梯度洗脱。然后将洗脱液引入 ESI 源，在选择离子监测 (SIM) 模式下通过质谱选择和检测负离子。使用 Chemstation 软件通过提取特定离子进行定量：全氟辛烷磺酸为 ，全氟辛烷磺酸为 。

The concentration of test chemicals that caused 50% mortality in the daphnids in each treatment with confidence limits were calculated using Probit analysis in Minitab 17.0 (Minitab, In. Pennsylvania, US) statistical software. Significant differences between the treatment groups and the control were determined by Tukey's test using SPSS Statistics 22.0 software. No observed effect concentrations (NOEC) were determined based on the results of the Tukey's analyses. Species sensitivity distribution (SSD) curve was plotted for PFOA and PFOS concentrations causing effects ( ) in water-flea from published literature (Table S3) and also from the present study. The SSD generator spreadsheet (Version 1) from the USEPA was downloaded from http://www.epa.gov/caddis/da_software_ssdmacro. , was used to generate SSD plot. The geometric means of values were used to generate the SSD when the Daphnid species with more than one published literature was available.
使用 Minitab 17.0（Minitab，In. Pennsylvania，US）统计软件中的 Probit 分析法，计算出各处理中造成水蚤 50% 死亡的试验化学品浓度以及 置信限。处理组与对照组之间的显著差异 ，使用 SPSS 统计 22.0 软件进行 Tukey's 检验。根据 Tukey's 分析结果确定无观测效应浓度（NOEC）。根据已发表的文献（表 S3）和本研究的结果，绘制了 PFOA 和 PFOS 浓度对水蚤造成 影响 ( ) 的物种敏感性分布（SSD）曲线。从 http://www.epa.gov/caddis/da_software_ssdmacro 下载了美国环保局的 SSD 生成器电子表格（第 1 版）。 用该电子表格生成 SSD 图。如果水蚤物种有多个已发表的文献，则使用 值的几何平均数来生成 SSD。

The physico-chemical characteristics of the cladoceran growth medium is presented in Table S-2. The cladoceran water contained less than of dissolved organic carbon. The was 6.96 , and the electrical conductivity was . Sodium, calcium, magnesium, potassium, nitrate, phosphate, sulphate, and chloride were detected at concentrations of . Heavy metals like copper, arsenic, lead, zinc, chromium, cadmium, cobalt, and nickel were less than the instrument's reporting limit.
表 S-2 列出了衣藻生长介质的物理化学特征。衣藻水的溶解有机碳含量低于 。 为 6.96，电导率为 。钠、钙、镁、钾、硝酸盐、磷酸盐、硫酸盐和氯化物的检测浓度为 。铜、砷、铅、锌、铬、镉、钴和镍等重金属的含量低于仪器的报告限值。

Potassium dichromate was used as a positive control in the acute toxicity experiment. The values for potassium dichromate in D. carinata at 24 and were 0.28 and , respectively. No mortality occurred in the negative control (cladoceran medium) in the acute toxicity assay. Among the test chemicals, the for PFOS was lower than PFOA at both 24 and (Table 1). The NOEC for both PFOS and PFOA decreased with the increase in test duration. The mortality of . carinata increased with the increase in PFOS/PFOA concentration, as represented in the dose-response relationship analysis (Fig. S1). Table S3 summarizes acute toxicity data reported for PFOS and PFOA in Daphnids. The species sensitivity distribution (SSD) curve for PFOS and PFOA comprised of LC50 data from 12 different freshwater aquatic organisms, including candidates from the genera Daphnia. SSD curve showed that . carinata is very sensitive to PFOS but less sensitive than Hyalella azteca to PFOA (Fig. 1).
重铬酸钾在急性毒性实验中用作阳性对照。在 24 小时和 时，重铬酸钾在 D. carinata 中的 值分别为 0.28 和 。在急性毒性实验中，阴性对照组（桡足类培养基）没有出现死亡现象。在测试化学品中，全氟辛烷磺酸在 24 和 时的 均低于全氟辛酸（表 1）。随着试验时间的延长，全氟辛烷磺酸和全氟辛酸的无观测效应浓度均有所下降。 . carinata 的死亡率随着全氟辛烷磺酸/全氟辛酸浓度的增加而增加，如剂量-反应关系分析所示（图 S1）。表 S3 总结了所报告的全氟辛烷磺酸和全氟辛酸对水蚤的急性毒性数据。全氟辛烷磺酸和全氟辛酸的物种敏感性分布（SSD）曲线由 12 种不同淡水水生生物的半数致死浓度数据组成，其中包括水蚤属的候选生物。SSD 曲线显示 . carinata 对全氟辛烷磺酸非常敏感，但对全氟辛酸的敏感性低于 Hyalella azteca（图 1）。

Chronic toxicity experiment was conducted for 21 days, and the effects of PFOS and PFOA on D. carinata survival, reproduction, and population parameters were evaluated. Parameters such as mortality, days to first brood, average offspring per brood, and total living offspring of D. carinata were assessed, and the results are shown in Table 2. Higher concentrations of PFOS and PFOA significantly extended the time to the first brood, and the average offspring in each brood decreased. When compared to the acute toxicity results, chronic toxicity was more sensitive in D. carinata at lower concentrations. The results also revealed that PFOS and PFOA at caused up to and mortality, respectively, in D. carinata. Also, at higher concentrations of PFOS
进行了为期 21 天的慢性毒性实验，评估了全氟辛烷磺酸和全氟辛酸对鲤科鱼类存活、繁殖和种群参数的影响。评估了鲤科鱼类的死亡率、首次产卵天数、每窝平均后代数和存活后代总数等参数，结果见表 2。较高浓度的全氟辛烷磺酸和全氟辛酸明显延长了第一窝育雏的时间，每窝平均后代数减少。与急性毒性结果相比，较低浓度的全氟辛烷磺酸和全氟辛酸对鲤科鱼类的慢性毒性更为敏感。研究结果还显示，全氟辛烷磺酸和全氟辛酸浓度为 时，会分别导致鲤鱼死亡，最高可达 和 。此外，在较高浓度的全氟辛烷磺酸

Table 1 表 1

Acute toxicity of PFOA and PFOS to Daphnia carinata. Values in the parentheses represents confidence interval.
全氟辛烷磺酸和全氟辛烷磺酸对水蚤的急性毒性。括号中的数值代表 置信区间。

(a)

(b)

Fig. 1. Species sensitivity distribution (SSD) based on of water flea species exposed to (a) PFOA and (b) PFOS. See Table S3 in the supplementary materials for the sources of values obtained for the generation of SSD.
图 1.基于 的暴露于 (a) 全氟辛烷磺酸和 (b) 全氟辛烷磺酸的水蚤物种敏感性分布 (SSD)。关于生成 SSD 所获得的 值的来源，请参见补充材料中的表 S3。

(1 and ), reproduction was completely inhibited, resulting in no offspring. Also, there was a delay in the first brood with an increase in the concentration of PFOS and PFOA. Besides, the offspring number in each brood was reduced due to the decrease in the total living offspring.
(1 和 )，繁殖受到完全抑制，没有后代。此外，随着全氟辛烷磺酸和全氟辛酸浓度的增加，第一窝的繁殖也会推迟。此外，由于存活后代总数减少，每窝的后代数量也减少了。

In contrast to the acute toxicity, comet assay showed that low concentrations of PFOA and PFOS could cause significant effects on D. carinata. The percent DNA in the tail was found to be significantly higher under PFOS concentrations of 1.0 and . The genotoxicity in D. carinata exposed to PFOA was significant at (Fig. 2). In accordance with acute and chronic toxicity experiments, PFOS was found to be more genotoxic than PFOA.
与急性毒性不同的是，彗星试验表明，低浓度的全氟辛烷磺酸和全氟辛烷磺酸会对鲤科鱼造成显著影响。在全氟辛烷磺酸浓度为 1.0 和 时，尾部 DNA 百分比明显升高。 时，鲤鱼暴露于全氟辛烷磺酸的遗传毒性显著（图 2）。根据急性和慢性毒性实验，发现全氟辛烷磺酸的遗传毒性高于全氟辛酸。

PFOA and PFOS were found to be stable in cladoceran medium during incubation under the experimental conditions, with both PFOA and PFOS completely recovered at the end of the incubation period.
在 的实验条件下培养期间，发现全氟辛烷磺酸和全氟辛烷磺酸在桡足类培养基中很稳定，在培养期结束时，全氟辛烷磺酸和全氟辛烷磺酸完全恢复。

Table 2 表 2

Chronic toxicity (21 d) of PFOA and PFOS to D. carinata.
全氟辛烷磺酸和全氟辛烷磺酸对 D. carinata 的慢性毒性（21 天）。

Note: nd refers "not determined": this is because no offspring were found in the 1 and of PFOS treatment group during the experiment. "ind": individual. Different letters in the same column indicate significant differences between treatments (analysis of variance with Tukey's test, ).
注：nd 指 "未确定"：这是因为全氟辛烷磺酸浓度为 1 和 的处理组在实验过程中未发现后代。"ind"：个体。同一栏中不同字母表示不同处理之间存在显著差异（采用 Tukey's 检验的方差分析， ）。

Results of the acute toxicity assay showed that PFOS was more toxic than PFOA to D. carinata. This result is similar to that reported previously by Lu et al. (2015), which showed that the toxicity of perfluorononanoic acid (PFNA), a perfluorinated carboxylic acid was lower than the PFOS to D. magna ( PFNA - 80.9 and PFOS ). Similar to our study, the toxicity of PFOS was reported to be higher than that of PFOA in both D. magna and Moina macrocopa with the values of 37.4 and , respectively (Ji et al., 2008). However, the NOEC (without losing the ability to be mobile) for PFOA exposed . carinata in our study was found to be several-fold lower than that for D. magna, and M. macrocopa (Ji et al., 2008), which indicates D. carinata used in this study was highly sensitive to PFOA and PFOS. Boudreau et al. (2003) reported value for PFOS to D. magna and D. pulicaria as 130 and , respectively. Similarly, at , PFOS was found to be more toxic ( ) than PFOA ( ) to D. magna (Li, 2009).
急性毒性检测结果表明，全氟辛烷磺酸对鲤科鱼类的毒性高于全氟辛酸。这一结果与 Lu 等人（2015 年）之前的报道相似，该报道显示全氟羧酸（PFNA）对鲤鱼的毒性低于全氟辛烷磺酸（ PFNA - 80.9 和 PFOS ）。与我们的研究类似，据报道，全氟辛烷磺酸对大型鲤科鱼类和大鲤科鱼类的毒性高于全氟辛酸，其 值分别为 37.4 和 （Ji 等人，2008 年）。然而，在我们的研究中，发现暴露于 PFOA 的 . carinata 的无观测效应浓度（在不失去移动能力的情况下）比 D. magna 和 M. macrocopa 低数倍（Ji 等人，2008 年），这表明本研究中使用的 D. carinata 对 PFOA 和 PFOS 高度敏感。Boudreau 等人（2003 年）在 上报告了 PFOS 对 D. magna 和 D. pulicaria 的影响值，分别为 130 和 。同样，在 ，发现全氟辛烷磺酸（ ）比全氟辛酸（ ）对大型蚤的毒性更大（Li，2009 年）。

The temperate freshwater fish, fathead minnow (Pimephales promelas) exhibited higher toxicity to PFOS ( ) than PFOA ( ) (Beach et al., 2006; Hekster et al., 2003). A rotifer, Brachionus calyciflorus, also showed higher sensitivity to PFOS ( ) than PFOA ( of ) (Zhang et al., 2013). Thus, the higher toxicity of PFOS than that of PFOA could be related to specific bioaccumulation potential of the former than the latter. Also, a direct relationship between fluorinated carbon chain length and the toxicity of PFAS with long-chain PFAS exhibiting higher toxicity than short chain PFAS to D. magna (Barmentlo et al., 2015) and fish has been reported (Lee et al., 2020). Furthermore, the variation in the toxicity of PFOA and PFOS among various aquatic species (Table S3) could be due to factors such as species specificity
温带淡水鱼黑头鲦（Pimephales promelas）对全氟辛烷磺酸（ ）的毒性高于全氟辛酸（ ）（Beach等人，2006年；Hekster等人，2003年）。轮虫 Brachionus calyciflorus 对全氟辛烷磺酸 ( ) 的敏感性也高于全氟辛酸 ( of ) （Zhang 等人，2013 年）。因此，全氟辛烷磺酸的毒性高于全氟辛酸，可能与前者的生物累积潜力高于后者有关。此外，据报道，氟化碳链长度与全氟辛烷磺酸的毒性之间存在直接关系，长链全氟辛烷磺酸对大型鲤科鱼类（Barmentlo 等人，2015 年）和鱼类的毒性高于短链全氟辛烷磺酸（Lee 等人，2020 年）。此外，PFOA 和 PFOS 在不同水生物种中的毒性差异（表 S3）可能是由于物种特异性等因素造成的。

Fig. 2. DNA damage in D. carinata exposed to PFOA and PFOS. The results were expressed as per cent DNA in tail by comet assay (number , mean standard error of mean). Tukey pairwise comparisons showing treatment sharing same letter are not statistically significant, . (variations in protein content, temperature, ), habitat, diet, exposure concentration, exposure duration, and laboratory conditions. The bioaccumulation of PFAS with longer fluorinated carbon chains ( C10) was found to be higher in European Perch (Perca fluviatilis), and the concentration of PFAS was closely related to the trophic ecology when compared to the size or age of the fish (Åkerblom et al., 2017). Ahrens et al. (2016) reported that the diet of fish influences the accumulation of short/long-chain PFAS since piscivorous fish accumulated more long-chain PFAS than the herbivorous and omnivorous fish which accumulated more of short-chain PFAS.
图 2.暴露于全氟辛烷磺酸和全氟辛烷磺酸的 D. carinata 的 DNA 损伤。结果用彗星试验尾部 DNA 百分比表示（数量 ，平均值 平均值的标准误差）。Tukey 配对比较显示字母相同的处理无统计学意义， 。（蛋白质含量、温度、 ）、栖息地、饮食、接触浓度、接触时间和实验室条件的变化。研究发现，欧洲鲈鱼（Perca fluviatilis）体内氟化碳链较长的全氟辛烷磺酸（ C10）的生物累积率较高，与鱼的大小或年龄相比，全氟辛烷磺酸的浓度与营养生态密切相关（Åkerblom 等人，2017 年）。Ahrens等人（2016年）报告称，鱼类的食性会影响短链/长链全氟辛烷磺酸的积累，因为食鱼积累的长链全氟辛烷磺酸多于食草性和杂食性鱼类，后者积累的短链全氟辛烷磺酸较多。

The species sensitivity distribution method was used to predict the hazardous concentrations affecting a certain percentage of species in a community (Newman et al., 2000). In ecological risk assessment, for measuring the toxic effects of chemicals on individual organisms, the concentration-effect data derived from single-species toxicity are used. Usually, a reliable SSD can only be obtained with at least 10-15 single species toxicity estimates (Wheeler et al., 2002). In addition, SSD modelling can be adopted for deriving environmental quality standards when the toxicity data are insufficient (Valsecchi et al., 2017). When comparing the acute toxicity data for various taxonomic groups (algae, fish, rotifers, crustaceans), there is still a paucity of knowledge on the chronic/reproductive toxicity in lower trophic aquatic organisms. In addition, studies on the effects of PFAS at environmentally relevant concentrations are still limited (Sinclair et al., 2020). Therefore, we used toxicological endpoints from acute toxicity testing for Daphnia and other freshwater organisms from published literature for SSD modelling (Table S3). Among the freshwater organisms studied, D. carinata was found to be sensitive to PFOS, whereas for PFOA Hyalella azteca was found to be more sensitive (Lee et al., 2007) than D. carinata from this study. However, among the marine species, marine turbot (Psetta maxima) was reported to be the most sensitive to PFOA ( ) and PFOS ( ) (Mhadhbi et al., 2012). SSD analysis was previously used to report the threshold values for endocrinedisrupting chemicals, especially in terrestrial ecosystems using soil algae, soil nematodes, annelid worms, springtails and plants (monocots) exposed to bisphenol A (Kwak et al., 2018), invertebrates Eisenia andrei, Enchytraeus albidus and Folsomia candida and the plants Triticum aestivum (monocotyledonous) and Brassica rapa (dicotyledonous) exposed to triclosan (Amorim et al., 2010), and plants, earthworm, collembola, soil nematode, soil algae exposed to methyl and propyl parabens, (Kim et al., 2018; Kim et al., 2020).
物种敏感性分布法用于预测影响群落中一定比例物种的有害浓度（纽曼等人，2000 年）。在生态风险评估中，为了测量化学品对生物个体的毒性影响，会使用从单一物种毒性中得出的浓度-效应数据。通常，只有至少 10-15 个单物种毒性估计值才能获得可靠的 SSD（Wheeler 等人，2002 年）。此外，当毒性数据不足时，也可采用 SSD 模型来推导环境质量标准（Valsecchi 等人，2017 年）。在比较不同分类群（藻类、鱼类、轮虫、甲壳类）的急性毒性数据时，对低营养级水生生物的慢性/生殖毒性仍然缺乏了解。此外，有关全氟辛烷磺酸在环境相关浓度下的影响的研究仍然有限（Sinclair 等人，2020 年）。因此，我们在 SSD 建模中使用了已发表文献中有关水蚤和其他淡水生物急性毒性测试的毒理学终点（表 S3）。在所研究的淡水生物中，发现水蚤对全氟辛烷磺酸比较敏感，而本研究发现 Hyalella azteca 比水蚤对全氟辛烷磺酸更敏感（Lee 等人，2007 年）。然而，据报道，在海洋物种中，大菱鲆（Psetta maxima）对全氟辛烷磺酸（ ）和全氟辛烷磺酸（ ）最为敏感（Mhadhbi 等人，2012 年）。SSD 分析以前曾被用于报告干扰内分泌的化学品的阈值，特别是在陆地生态系统中使用暴露于双酚 A 的土壤藻类、土壤线虫、环节动物蠕虫、春蜱和植物（单子叶植物）（Kwak et al、2018年），暴露于三氯生的无脊椎动物Eisenia andrei、Enchytraeus albidus和Folsomia candida以及植物Triticum aestivum（单子叶植物）和Brassica rapa（双子叶植物）（Amorim等人，2010年），以及暴露于对羟基苯甲酸甲酯和对羟基苯甲酸丙酯的植物、蚯蚓、鞘翅目线虫、土壤线虫、土壤藻类（Kim等人，2018年；Kim等人，2020年）。

Chronic toxicity studies on PFOA and PFOS in freshwater species are very limited. Chronic toxicity data forms an important part of an integrated environmental monitoring and assessment strategy (Yi et al., 2010). To our knowledge, thus far, there have been no published data on the toxicity of PFAS to D. carinata. From the chronic exposure study, it is evident that . carinata is sensitive to higher concentrations ( 1.0 and ) of PFOS and PFOA, with a significant reduction in growth and reproductive parameters. We assessed reproductive performance as the time to first brood and average offspring per brood in D. carinata exposed to PFOA and PFOS. We found that PFOS elicited
关于全氟辛烷磺酸和全氟辛烷磺酸在淡水物种中的慢性毒性研究非常有限。慢性毒性数据是综合环境监测和评估战略的重要组成部分（Yi 等人，2010 年）。据我们所知，迄今为止还没有关于全氟辛烷磺酸对鲤科鱼类毒性的公开数据。从慢性暴露研究中可以看出， . carinata 对较高浓度（1.0 和 ）的全氟辛烷磺酸和全氟辛酸敏感，生长和生殖参数显著降低。我们评估了暴露于全氟辛烷磺酸和全氟辛烷磺酸的鲤科鱼类的繁殖性能，即首次育雏时间和每窝平均后代数。我们发现全氟辛烷磺酸会导致
greater reproductive toxicity than PFOA. Chronic exposure of D. magna to PFOS at was reported to cause mortality (Boudreau et al., 2003). In the present study, PFOS concentration as low as affected the survival of D. carinata. Similarly, exposure of D. magna to PFOA at and PFOS at 10 and significantly reduced the hatching (Seyoum et al., 2020). From these observations, it is evident that survival and reproductive toxicities exerted by PFOS and PFOA could cause risk to lower trophic organisms in the aquatic ecosystems.
与全氟辛烷磺酸相比，全氟辛烷磺酸的生殖毒性更大。据报道，D. magna 长期接触 的全氟辛烷磺酸会导致 死亡（Boudreau 等人，2003 年）。在本研究中，全氟辛烷磺酸浓度低至 会影响鲤鱼的存活。同样，将 D. magna 暴露于 的 PFOA 以及 10 和 的 PFOS 会显著降低孵化率（Seyoum 等人，2020 年）。从这些观察结果可以看出，全氟辛烷磺酸和全氟辛酸的生存和生殖毒性可能会对水生生态系统中的低营养级生物造成危害。

Chronic exposure of D. magna to ammonium salt of PFOA resulted in a delay in the time to first brood, reduction in the number of broods per parent organism, and also reduced the number of neonates per surviving parent (Colombo et al., 2008). Although the concentrations of PFOA and PFOS in freshwater ranges between up to tens of hundreds of (Castiglioni et al., 2015; Hansen et al., 2002; Rostkowski et al., 2006; Sinclair et al., 2006; So et al., 2007; Wilson et al., 2007). Elevated concentrations of PFAS have been reported, for example, of PFOA at manufacturing and disposal sites (Xiao et al., 2015), 42,000 PFOA and PFOS in the groundwater underlying landfill (Oliaei et al., 2006), 84,000 PFOA and 30,000 PFOS in the leachate from landfill (Oliaei et al., 2013) and PFOA and PFOS in the pond used to store wastewater arising from AFFF at a military base (Arias et al., 2015). The major sources of groundwater contamination of PFAS are manufacturing facilities (Davis et al., 2007; Hoffman et al., 2011), fire-training sites (Cheryl et al., 2003), agricultural fields applied with PFAS-contaminated biosolids (Lindstrom et al., 2011a), and industrial disposal sites (Xiao et al., 2015). Therefore, aquatic environments nearby those point sources of PFAS can have an impact on the health and survival of lower trophic aquatic organisms such as water fleas.
magna 长期接触全氟辛烷磺酸铵盐会导致第一次产卵时间延迟、每个亲本的产卵数量减少，并且每个存活亲本的新生儿数量也会减少（Colombo 等人，2008 年）。尽管全氟辛烷磺酸和全氟辛烷磺酸在淡水中的浓度介于 至数十百 之间（Castiglioni 等人，2015 年；Hansen 等人，2002 年；Rostkowski 等人，2006 年；Sinclair 等人，2006 年；So 等人，2007 年；Wilson 等人，2007 年）。据报道，PFAS 的浓度已经升高，例如， ，生产和处置场地的 PFOA（Xiao 等人，2015 年），垃圾填埋场地下水中的 42,000 PFOA 和 PFOS（Oliaei 等人，2006 年），84,000 PFOA 和 PFOS、2006年）、垃圾填埋场渗滤液中的84,000 PFOA和30,000 PFOS（Oliaei等人，2013年）以及军事基地用于储存AFFF废水的池塘中的 PFOA和 PFOS（Arias等人，2015年）。全氟辛烷磺酸污染地下水的主要来源是生产设施（Davis 等人，2007 年；Hoffman 等人，2011 年）、消防训练场地（Cheryl 等人，2003 年）、施用了受全氟辛烷磺酸污染的生物固体的农田（Lindstrom 等人，2011a）以及工业弃置场（Xiao 等人，2015 年）。因此，这些全氟辛烷磺酸点源附近的水生环境会对水蚤等低营养级水生生物的健康和生存产生影响。

Generally, genotoxicity tests, in combination with comet assay, are widely used in aquatic environmental monitoring (Frenzilli et al., 2009; Kim and Hyun, 2006). In vitro test systems of aquatic species using fishcell lines are commonly used in comet assays (Cotelle and Ferard, 1999; Nehls and Segner, 2005). In this study, however, D. carinata was exposed to PFOA and PFOS in vivo, and the DNA damage was assessed in subsequently isolated nucleoids. Both PFOA and PFOS caused genotoxic effects to D. carinata. DNA strand breaks increased with an increase in the concentration of PFOS and PFOA. Among the two chemicals, PFOS caused greater DNA damage to D. carinata than PFOA. Compared with the wellknown genotoxic compounds (Kirkland et al., 2016; Kirkland et al., 2008; Zounková et al., 2007), PFOS showed relatively higher genotoxicity. Similarly, PFOS has been known to cause genetic injuries in rodents and chickens (Guruge et al., 2009; Rosen et al., 2008; Yeung et al., 2009). Studies on gene expression conducted in zebrafish embryos have revealed that exposure to PFOS resulted in both developmental toxicity and DNA damage mediated apoptosis (Shi et al., 2008). In green mussels (Perna viridis), both PFOS and PFOA were reported to elicit DNA damage such as DNA strand breaks, fragmentation, chromosomal breaks, and apoptosis (Liu et al., 2014).
一般来说，遗传毒性试验与彗星试验相结合，广泛用于水生环境监测（Frenzilli 等人，2009 年；Kim 和 Hyun，2006 年）。彗星试验通常使用鱼细胞系的水生物种体外测试系统（Cotelle 和 Ferard，1999 年；Nehls 和 Segner，2005 年）。然而，在本研究中，D. carinata 在体内暴露于全氟辛烷磺酸和全氟辛烷磺酸，并在随后分离的核苷酸中对 DNA 损伤进行了评估。全氟辛烷磺酸和全氟辛烷磺酸都对鲤科鱼类产生了基因毒性影响。DNA链断裂随着全氟辛烷磺酸和全氟辛酸浓度的增加而增加。在这两种化学物质中，全氟辛烷磺酸对鲤科鱼类 DNA 造成的损伤比全氟辛酸更大。与众所周知的基因毒性化合物（Kirkland 等人，2016 年；Kirkland 等人，2008 年；Zounková 等人，2007 年）相比，全氟辛烷磺酸的基因毒性相对更高。同样，已知全氟辛烷磺酸会对啮齿动物和鸡造成遗传损伤（Guruge 等人，2009 年；Rosen 等人，2008 年；Yeung 等人，2009 年）。在斑马鱼胚胎中进行的基因表达研究表明，接触全氟辛烷磺酸会导致发育毒性和 DNA 损伤介导的细胞凋亡（Shi 等人，2008 年）。据报告，在绿贻贝（Perna viridis）中，全氟辛烷磺酸和全氟辛酸都会引起DNA损伤，如DNA链断裂、碎片、染色体断裂和细胞凋亡（Liu等人，2014年）。

DNA damage in common carp exposed to PFOS has also been documented (Hagenaars et al., 2008; Hoff et al., 2003). Perfluorinated chemicals could also cause DNA damage in human liver HepG2 cells (Eriksen et al., 2010). In human HepG2 cells, PFOA at caused DNA strand breaks and at caused micronuclei in a dosedependent manner (Yao and Zhong, 2005). Interestingly, PFOA but not PFOS caused DNA damage in Paramecium caudatum (Kawamoto et al., 2010). In Earthworm (Eisenia fetida), both PFOS and PFOA ( ) were reported to cause DNA damage in the coelomocytes (Zheng et al., 2016). As suggested in previous studies, the chain length of the perfluorinated chemical is the major contributing factor than the functional group that determines chemical's interaction with the genetic material (Hagenaars et al., 2008; Nobels et al., 2010). Similarly, our results showed that PFOS has higher genotoxicity than PFOA. However, there exist discrepancies in the genotoxic potential of PFAS. Thus, studies on human HepG2 cells, freshwater tilapia fish cells, and Paramecium caudatum showed that PFOA was capable of causing DNA damage (Freire et al., 2008; Kawamoto et al., 2010; Liu et al., 2007). However, in carps, no significant induction in DNA fragmentation was recorded even at a higher concentration of PFOS (Kim et al., 2010). In our study, it is evident that PFOS could cause significant DNA damage to . carinata at a concentration of .
接触全氟辛烷磺酸的鲤鱼的DNA损伤也有文献记载（Hagenaars等人，2008年；Hoff等人，2003年）。全氟化学品也会对人类肝脏 HepG2 细胞造成 DNA 损伤（Eriksen 等人，2010 年）。在人类 HepG2 细胞中，全氟辛酸浓度为 时会导致 DNA 链断裂，浓度为 时会导致微核，其发生与剂量有关（Yao 和 Zhong，2005 年）。有趣的是，全氟辛烷磺酸（PFOA）而非全氟辛烷磺酸（PFOS）会对钝顶水螅（Paramecium caudatum）造成 DNA 损伤（Kawamoto 等人，2010 年）。据报道，在蚯蚓（Eisenia fetida）体内，全氟辛烷磺酸和全氟辛酸（ ）都会导致其腹腔细胞的 DNA 损伤（Zheng 等人，2016 年）。正如之前的研究表明的那样，全氟化学品与遗传物质相互作用的主要因素是全氟化学品的链长，而不是官能团（Hagenaars 等人，2008 年；Nobels 等人，2010 年）。同样，我们的研究结果表明，全氟辛烷磺酸的遗传毒性高于全氟辛酸。然而，全氟辛烷磺酸的基因毒性潜力存在差异。因此，对人类 HepG2 细胞、淡水罗非鱼细胞和尾状副蚤进行的研究表明，全氟辛烷磺酸能够造成 DNA 损伤（Freire 等人，2008 年；Kawamoto 等人，2010 年；Liu 等人，2007 年）。然而，在鲤鱼中，即使使用较高浓度的全氟辛烷磺酸（ PFOS），也不会明显诱发 DNA 断裂（Kim 等人，2010 年）。在我们的研究中，全氟辛烷磺酸浓度为 时，显然会对 . carinata 造成严重的 DNA 损伤。

Both PFOA and PFOS are recalcitrant and persistent chemicals that are difficult to break down by either chemical or biological processes. Previous studies have demonstrated that there was no significant reduction from the initial concentration of PFOA/PFOS in the test water during the 35 day experiment period (Hanson et al., 2005; Sanderson et al., 2004). In this study, the stability of the test chemicals was found to remain unaffected at the end of 48 and of acute and genotoxicity experiments, respectively. Based on the results of our study, along with other published studies, it is suggested that the levels of PFOA and PFOS concentration in freshwater may have no acute harmful impact on the aquatic environment. However, chronic sublethal effects of PFOA and PFOS to freshwater animals have been investigated by a few studies. At sublethal concentrations, PFAS could cause endocrine disruption, and other chronic ecotoxicological effects that may be of great concern for aquatic fauna, which warrants further investigation (Oakes et al., 2005; Liu et al., 2007; Ulhaq et al., 2013). Further evidence on the long-term ecological effects of PFOA and PFOS on aquatic fauna will deliver significant data to assess the ecological risks of PFOA and PFOS effectively.
全氟辛烷磺酸和全氟辛烷磺酸都是难降解的持久性化学物质，很难通过化学或生物过程分解。先前的研究表明，在 35 天的实验期间，测试水中的全氟辛烷磺酸/全氟辛烷磺酸浓度与初始浓度相比没有明显降低（Hanson 等人，2005 年；Sanderson 等人，2004 年）。本研究发现，在急性和遗传毒性实验分别结束 48 天和 后，测试化学品的稳定性未受影响。根据我们的研究结果以及其他已发表的研究，淡水中的全氟辛烷磺酸和全氟辛烷磺酸浓度水平可能不会对水生环境造成急性有害影响。不过，也有少数研究调查了 PFOA 和 PFOS 对淡水动物的慢性亚致死效应。在亚致死浓度下，全氟辛烷磺酸可能会导致内分泌紊乱和其他慢性生态毒理效应，这可能会引起水生动物的极大关注，因此需要进一步调查（Oakes 等人，2005 年；Liu 等人，2007 年；Ulhaq 等人，2013 年）。关于全氟辛烷磺酸和全氟辛烷磺酸对水生动物的长期生态影响的进一步证据将为有效评估全氟辛烷磺酸和全氟辛烷磺酸的生态风险提供重要数据。

Our study demonstrated the acute, chronic, and genotoxic effects of two PFOS and PFOA to D. carinata. PFOS was found to be more toxic than PFOA. The chronic exposure to PFOS for 21 days induced significant inhibition of growth and reproduction and caused mortality. Genotoxicity assessment through comet assay showed that both the chemicals were capable of inducing DNA damage at high concentrations. Significant adverse effects were only detected at concentrations higher than under short term exposure ( ) to PFOA and PFOS; the effects could have prolonged on the health of organisms due to the irreversible nature of DNA damage resulting in permanent health effects. The sublethal effects of PFOA and PFOS on D. carinata need to be explored by using molecular markers. The present investigation emphasizes the need for using indigenous aquatic invertebrates in the assessment and monitoring of PFAS in natural ecosystems.
我们的研究证明了两种全氟辛烷磺酸和全氟辛酸对鲤科鱼类的急性、慢性和遗传毒性影响。研究发现，全氟辛烷磺酸的毒性高于全氟辛酸。长期接触全氟辛烷磺酸 21 天后，鲤鱼的生长和繁殖受到明显抑制，并导致死亡。通过彗星试验进行的遗传毒性评估表明，这两种化学物质在高浓度下都能导致 DNA 损伤。只有在短期接触（ ）全氟辛烷磺酸和全氟辛烷磺酸的浓度高于 时，才会检测到明显的不利影响；由于 DNA 损伤的不可逆性质，这些影响可能会对生物体的健康产生永久性影响。全氟辛烷磺酸和全氟辛烷磺酸对鲤科鱼类的亚致死效应需要利用分子标记进行研究。本调查强调了在评估和监测自然生态系统中的全氟辛烷磺酸时使用本地水生无脊椎动物的必要性。

Panneerselvan Logeshwaran: Conceptualization, Investigation, Methodology, Data curation, Validation, Writing - review & editing. Anithadevi Kenday Sivaram: Investigation, Methodology, Writing review & editing. Aravind Surapaneni: Writing - review & editing. Kurunthachalam Kannan: Writing - review & editing. Ravi Naidu: Writing - review & editing. Mallavarapu Megharaj: Conceptualization, Supervision, Writing - review & editing.
Panneerselvan Logeshwaran：构思、调查、方法、数据整理、验证、写作--审阅与编辑。Anithadevi Kenday Sivaram：调查、方法论、写作--审阅与编辑。Aravind Surapaneni：写作-审核与编辑Kurunthachalam Kannan：写作-审核与编辑拉维-奈杜写作 - 审核与编辑Mallavarapu Megharaj：构思、监督、写作--审阅和编辑。

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有任何可能会影响本文所报告工作的已知经济利益或个人关系。

The authors would like to acknowledge the Cooperative Research Centre for Contamination Assessment and Remediation of the Environment and the University of Newcastle for support and facilities.
作者感谢环境污染评估与补救合作研究中心和纽卡斯尔大学提供的支持和设施。

Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.144577.
本文的补充数据可在线查阅：https://doi. org/10.1016/j.scitotenv.2020.144577。

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