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Exposure to perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at ppb concentration induces chronic toxicity in Daphnia carinata

Panneerselvan Logeshwaran , Anithadevi Kenday Sivaram , Aravind Surapaneni ,
Panneerselvan Logeshwaran , Anithadevi Kenday Sivaram , Aravind Surapaneni
Kurunthachalam Kannan , Ravi Naidu , Mallavarapu Megharaj
Kurunthachalam Kannan , Ravi Naidu , Mallavarapu Megharaj
Global Centre for Environmental Remediation, Faculty of Science, The University of Newcastle, Callaghan, NSW 2308, Australia
澳大利亚新南威尔士州 2308 卡拉汉纽卡斯尔大学理学院全球环境修复中心
Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, The University of Newcastle, Callaghan, NSW 2308, Australia
纽卡斯尔大学环境污染评估与补救合作研究中心,澳大利亚新南威尔士州卡拉汉 2308 号
c South East Water, Frankston, Victoria 3199, Australia ARC Training Centre for the Transformation of Australia's Biosolids Resource, Bundoora, Victoria, Australia
澳大利亚维多利亚州本多拉 ARC 澳大利亚生物固体资源改造培训中心
e Department of Pediatrics and Department of Environmental Medicine, New York University School of Medicine, New York, NY 10016, USA
e 美国纽约大学医学院儿科系和环境医学系,纽约州纽约市,邮编 10016


  • Eco- and genotoxicity of PFOA and PFOS were studied in Daphnia carinata.
  • PFOS causes developmental and reproductive toxicity at environmental concentrations.
  • PFOA and PFOS did not show acute toxicity at environmental concentrations.
  • PFOS was more toxic than PFOA.
  • Comet assay revealed that PFOS could cause DNA damage.
    彗星试验显示,全氟辛烷磺酸可导致 DNA 损伤。


Article history: 文章历史:

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

Keywords: 关键词:

Acute and chronic toxicity
Comet assay 彗星试验
Daphnia carinata 水蚤
Perfluorooctanoic acid 全氟辛酸


Abstract 摘要

A B S T R A C T Widespread environmental contamination of per- and polyfluoroalkyl substances (PFAS) is well established. Nevertheless, few studies have reported on the aquatic toxicity of PFAS, especially in indicator species such as Daphnia. In this study, the toxicity of two major PFAS, namely perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS), was investigated on water flea (Daphnia carinata) using a battery of comprehensive toxicity tests, including a acute and a 21-day chronic assays. The survival, growth, and reproduction of D. carinata were monitored over a 21-day life cycle. PFOS exhibited higher toxicity than PFOA. The values (confidence interval) based on acute toxicity for PFOA and PFOS were 78.2 (54.9-105) and 8.8 (6.4-11.6) , respectively. Chronic exposure to PFOS for 21 days displayed mortality and reproductive defects in D. carinata at a concentration as low as . Genotoxicity assessment using comet assay revealed that exposure for to PFOS at 1 and significantly damaged the organism's genetic makeup. The results of this study have great implications for risk assessment of PFOS and PFOA in aquatic ecosystems, given the potential of PFOS to pose a risk to Daphnia even at lower concentrations ( ).
A B S T R A C T 全氟烷基和多氟烷基物质(PFAS)对环境的广泛污染已得到公认。然而,很少有研究报道 PFAS 的水生毒性,尤其是对水蚤等指示物种的毒性。本研究采用一系列综合毒性试验,包括 急性试验和 21 天慢性试验,研究了两种主要 PFAS(即全氟辛酸(PFOA)和全氟辛烷磺酸(PFOS))对水蚤(水蚤)的毒性。在 21 天的生命周期内对水蚤的存活、生长和繁殖进行了监测。全氟辛烷磺酸的毒性高于全氟辛酸。全氟辛烷磺酸和全氟辛烷磺酸急性毒性的 值(置信区间)分别为 78.2 (54.9-105) 和 8.8 (6.4-11.6) 。长期接触全氟辛烷磺酸 21 天后,当浓度低至 时,鲤鱼会出现死亡和生殖缺陷。使用彗星试验进行的遗传毒性评估显示, ,1 和 的全氟辛烷磺酸会严重破坏生物体的遗传结构。鉴于全氟辛烷磺酸即使在较低浓度下也可能对水蚤构成风险,这项研究的结果对全氟辛烷磺酸和全氟辛酸在水生生态系统中的风险评估具有重要意义 ( )。

(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。

1. Introduction 1.导言

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。

2. Materials and methods

2.1. Test organism and culture conditions

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 ,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 )。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。

2.2. Test compounds 2.2.试验化合物

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。

2.3. Acute immobilization tests on D. carinata
2.3.D. carinata 的急性固定试验

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): ;全氟辛烷磺酸(PFOS): 。在相同的实验条件下,将跳蚤暴露于已知毒物 ,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 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)。

2.4. Chronic toxicity assay

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。

2.5. Comet assay 2.5.彗星试验

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。

2.6. Stability of PFAS

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)。

2.7. Analytical methods 2.7.分析方法

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

2.8. Statistical analysis

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。

3. Results 3.成果

3.1. Physico-chemical properties of test water

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。

3.2. Acute toxicity of test chemicals

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)。

3.3. Chronic toxicity 3.3.慢性毒性

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 。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。
Compounds Cladoceran water Cladoceran 水
(Potassium dichromate) (重铬酸钾)