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Perfluorinated alkyl substances impede growth, reproduction, lipid metabolism and lifespan in Daphnia magna

Asmerom Seyoum, Ajay Pradhan, Jana Jass, Per-Erik Olsson *
Asmerom Seyoum、Ajay Pradhan、Jana Jass、Per-Erik Olsson *
The Life Science Center-Biology, School of Science and Technology, Örebro University, SE-701 82 Örebro, Sweden
瑞典厄勒布鲁大学科技学院生命科学中心-生物学,SE-701 82 Örebro


  • Per- and polyfluorinated alkyl substances altered lipid metabolism in Daphnia magna.
  • Daphnia magna reproduction is impaired by both PFOS and PFOA.
  • The longevity of Daphnia magna is reduced by PFOS but not PFOA
  • Daphnia magna is a good animal model to investigate pollutants mechanism of action.


Article history: 文章历史:

Received 17 December 2019
2019 年 12 月 17 日收到
Received in revised form 18 May 2020
2020 年 5 月 18 日收到修订稿
Accepted 22 May 2020
接受 2020 年 5 月 22 日
Available online 25 May 2020
可于 2020 年 5 月 25 日在线查阅
Editor: Daniel Wunderlin

Keywords: 关键词:

Perfluorooctane sulfonate (PFOS)
Perfluorooctanoic acid (PFOA)
Fatty acid 脂肪酸
PFAS toxicity 全氟辛烷磺酸的毒性
Gene expression 基因表达
Fecundity 繁殖力


Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds with unique stability accompanied with hydrophobic and lipophobic properties. Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are of high concern due to their wide application in consumer and industrial products, extreme persistence, abundant occurrence in the environment and their toxic effect to humans and animals. However, knowledge on the molecular mechanisms of toxicity and the effects on reproduction output remain scarce. In this study, we analyzed the effects of PFOS and PFOA on Daphnia magna. Acute toxicity, development, reproduction, lipid metabolism (lipid-accumulation) and lifespan was investigated, as well as the expression of genes related to these endpoints. Exposure of PFOS and PFOA at 1, 10 and did not cause acute lethality. Hatching was reduced following exposure to both compounds, and lifespan was decreased following exposure to PFOS. Body length of Daphnia magna was reduced significantly by PFOS following 7 days exposure. Lipid staining revealed that all PFAS exposures increased lipid accumulation. qRT-PCR analysis of genes involved in lipid metabolism suggests that the increase in lipid content could be due to inhibition of genes involved on absorption and catabolism of fatty acids. Exposure to both PFOA and PFOS reduced the fecundity significantly. Downregulation of genes involved in development and reproductive process, including vtg2, vasa, EcRA, EcRB, usp, jhe, HR3, ftz-F1, E74 and E75 were observed. The alterations in developmental and reproductive genes as well as the disturbed lipid metabolism provides mechanistic insight into the possible causes for decreased fecundity and lifespan observed following exposure to both PFOS and PFOA.
全氟和多氟烷基物质(PFASs)是一种合成的有机氟化合物,具有独特的稳定性以及疏水和疏脂特性。全氟辛烷磺酸(PFOS)和全氟辛酸(PFOA)广泛应用于消费品和工业产品,具有极强的持久性,在环境中大量存在,对人类和动物具有毒性作用,因此备受关注。然而,有关其毒性的分子机制以及对生殖产出的影响的知识仍然匮乏。本研究分析了 PFOS 和 PFOA 对大型蚤的影响。研究了急性毒性、发育、繁殖、脂质代谢(脂质积累)和寿命,以及与这些终点相关的基因表达。接触 1、10 和 的全氟辛烷磺酸和全氟辛酸不会导致急性致死。暴露于这两种化合物后,孵化率降低,而暴露于 全氟辛烷磺酸后,寿命缩短。接触 全氟辛烷磺酸 7 天后,大型蚤的体长明显缩短。对参与脂质代谢的基因进行的 qRT-PCR 分析表明,脂质含量的增加可能是由于参与脂肪酸吸收和分解代谢的基因受到了抑制。暴露于全氟辛烷磺酸和全氟辛烷磺酸会显著降低繁殖力。观察到参与发育和生殖过程的基因下调,包括 vtg2、vasa、EcRA、EcRB、usp、jhe、HR3、ftz-F1、E74 和 E75。发育和生殖基因的改变以及脂质代谢的紊乱从机理上揭示了暴露于全氟辛烷磺酸和全氟辛酸后生育能力和寿命下降的可能原因。
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
© 2020 作者。出版商:Elsevier B.V.本文为 CC BY-NC-ND 许可下的开放存取文章 ( http://creativecommons.org/licenses/by-nc-nd/4.0/)。

1. Introduction 1.导言

Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds where the hydrogen atoms on the carbon

backbone are replaced by fluorine atoms with a charged functional group of either a carboxylate, alcohol, or sulfonate terminal group (Buck et al., 2011; Conder et al., 2008). Due to the strong electron withdrawing effect of fluorine, the bond is the strongest existing covalent bond ( ), which attributes to the chemical and thermal stability of PFASs (O'Hagan, 2008; Parsons et al., 2008). This unique stability together with their hydrophobic and lipophobic nature, contributes to their use in a wide variety of industrial and commercial products, such as lubricants, fire retardants, polymer additives, pesticides, protective coatings for clothing and other surfactants (Key et al., 1997; Kissa, 2001). The high-energy chemical bond in PFAS makes them resistant to hydrolysis, photolysis, biodegradation, and metabolism. These properties lead to PFAS persistence in the environment and allows bioaccumulation and biomagnification in the food web (Hekster et al., 2003; Schultz et al., 2003). Due to the reported toxicity, both the production and use of Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) is now restricted in Europe (EC_1907/2006, 2006) and North America (USEPA, 2017).
骨架被带有羧酸盐、醇或磺酸盐末端带电官能团的氟原子取代(Buck 等人,2011 年;Conder 等人,2008 年)。由于氟具有很强的退电子效应, 键是现有最强的共价键( ),这也是全氟辛烷磺酸具有化学和热稳定性的原因(O'Hagan,2008 年;Parsons 等人,2008 年)。这种独特的稳定性加上其疏水性和疏脂性,使其被广泛应用于各种工业和商业产品中,如润滑剂、阻燃剂、聚合物添加剂、杀虫剂、服装防护涂层和其他表面活性剂(Key 等人,1997 年;Kissa,2001 年)。全氟辛烷磺酸中的高能化学键使其不易水解、光解、生物降解和新陈代谢。这些特性导致 PFAS 在环境中持久存在,并在食物网中产生生物累积和生物放大作用(Hekster 等人,2003 年;Schultz 等人,2003 年)。由于所报告的毒性,全氟辛烷磺酸(PFOS)和全氟辛酸(PFOA)的生产和使用现已在欧洲(EC_1907/2006,2006年)和北美(美国环保局,2017年)受到限制。
Among PFASs, PFOS and PFOA are the two most ubiquitously distributed both in aquatic and terrestrial environment and are therefore of great concern for human and animal health (Ahrens and Bundschuh, 2014; Quinones and Snyder, 2009). PFOA and PFOS have been detected in both humans and animals, causing adverse effects on reproduction, development and immune response (DeWitt et al., 2012). In the environment, PFAS has been detected most commonly in the range of to level, however in highly contaminated areas such as at a fire-training area at Wurtsmith Air Force Base in USA it ranged from 3 to (Eschauzier et al., 2012; Moody et al., 2003) and at commercial airports in Sweden, levels between to were detected (Wennberg and Fridlund, 2015). A compilation of data from different studies estimated the release of PFAS into the aquatic environment in a range from to (Ahrens and Bundschuh, 2014). Studies on humans have shown that these chemicals cross the placental barrier, are transferred to the fetal circulation and also found in maternal milk (Kuklenyik et al., 2004; Midasch et al., 2007). The half-life of PFOS and PFOA in human serum is estimated to be 3.4 years and 2.7 years, respectively (Li et al., 2018). Estimated half-life in the aquatic environment due to hydrolysis has been reported to exceed 41 years for PFOS and 92 years for PFOA (Hekster et al., 2002).
在全氟辛烷磺酸和全氟辛酸中,全氟辛烷磺酸和全氟辛烷磺酰氨是在水生和陆生环境中分布最广的两种物质,因此对人类和动物的健康产生了极大的影响(Ahrens 和 Bundschuh,2014 年;Quinones 和 Snyder,2009 年)。已在人类和动物体内检测到全氟辛烷磺酸和全氟辛烷磺酸,它们会对生殖、发育和免疫反应造成不良影响(DeWitt 等人,2012 年)。在环境中,最常检测到的全氟辛烷磺酸含量在 之间,但在高污染地区,如美国沃兹密斯空军基地的消防训练区,全氟辛烷磺酸含量在 3 至 之间(Eschauzier 等人,2012 年;Moody 等人,2003 年),在瑞典的商业机场,检测到的全氟辛烷磺酸含量在 之间(Wennberg 和 Fridlund,2015 年)。不同研究的数据汇编估计,全氟辛烷磺酸在水生环境中的释放量在 之间(Ahrens 和 Bundschuh,2014 年)。对人类的研究表明,这些化学物质会穿过胎盘屏障,进入胎儿血液循环,并在母体乳汁中发现(Kuklenyik 等人,2004 年;Midasch 等人,2007 年)。据估计,全氟辛烷磺酸和全氟辛酸在人体血清中的半衰期分别为 3.4 年和 2.7 年(Li 等人,2018 年)。据报告,由于水解作用,全氟辛烷磺酸在水生环境中的半衰期估计超过41年,全氟辛酸则超过92年(Hekster等人,2002年)。
The toxicity of PFOS and PFOA has been studied in different model animals. The observed effects include mortality and immobilization, reproductive toxicity and immunotoxicity. However, little is known about their mode of action (Rainieri et al., 2017). Correlating gene expression profiles to physiological endpoints can provide better insights into the biological pathways and mode of action of the pollutants (Liu et al., 2014; Mao et al., 2013).
全氟辛烷磺酸和全氟辛酸的毒性已在不同的模式动物中进行过研究。观察到的影响包括死亡率和固定性、生殖毒性和免疫毒性。然而,人们对它们的作用模式知之甚少(Rainieri 等人,2017 年)。将基因表达谱与生理终点相关联,可以更好地了解污染物的生物途径和作用模式(Liu等人,2014年;Mao等人,2013年)。
Previous studies have shown that the is for PFOS and for PFOA for Daphnia magna (Ji et al., 2008), while the is for PFOS and for PFOA (Li, 2009). In a multigenerational study on Daphnia magna, it was observed that reduced fecundity was not observed below and that the time to first brood increased with exposures above (Jeong et al., 2016). In another study on Daphnia magna it was observed that PFOS at concentrations above and PFOA at concentrations above resulted in inhibition of reproduction (Yang et al., 2019).
先前的研究表明,对于大型蚤而言,全氟辛烷磺酸的 ,全氟辛酸的 (Ji 等人,2008年),而全氟辛烷磺酸的 ,全氟辛酸的 (Li,2009年)。在对大型蚤进行的一项多代研究中,观察到在 以下未观察到繁殖力降低的情况,而随着暴露量超过 ,第一次育雏的时间会增加(Jeong 等人,2016 年)。在另一项关于大型蚤的研究中观察到,全氟辛烷磺酸浓度高于 和全氟辛酸浓度高于 会导致繁殖受到抑制(Yang 等人,2019 年)。
In the present study, the aim was to determine both physiological effects (acute toxicity, fecundity, hatching, growth, longevity and lipid metabolism) on Daphnia magna and compare these effects with the toxicogenomic responses. In order to make these comparisons we choose to use concentrations of PFOS and PFOA that had earlier been observed to have effects on Daphnia magna, Japanese medaka and C. elegans (Jeong et al., 2016; Kang et al., 2019; Stylianou et al., 2019).
本研究旨在确定对大型水蚤的生理影响(急性毒性、繁殖力、孵化、生长、寿命和脂质代谢),并将这些影响与毒原反应进行比较。为了进行这些比较,我们选择使用早先已观察到对大型水蚤、日本青鳉和秀丽隐杆线虫有影响的全氟辛烷磺酸和全氟辛酸浓度(Jeong 等人,2016 年;Kang 等人,2019 年;Stylianou 等人,2019 年)。
Daphnia magna is widely distributed throughout the planet in all types of freshwater systems and is a part of the food chain of freshwater ecosystems and is therefore an excellent model system to study aquatic toxicology (Koivisto, 1995). Daphnia magna reproduces both by asexual parthenogenesis under favorable conditions and sexual production of dormant encapsulated eggs (ephippia) under unfavorable environments (Hebert, 1978). It is considered a representative species among the aquatic invertebrates and is used in standardized toxicity tests as pollution indicator species (Brown and Thompson, 1982; Le et al., 2016). In the present study, we analyzed the effects of PFOS and PFOA at different concentrations on Daphnia magna. Physiological endpoints were analyzed and compared to the gene expression from several signaling pathways including stress response, immune response, respiration, lipid metabolism, and reproduction. The results show a strong correlation between the physiological responses and the regulation of genes related to these physiological pathways. Analysis of toxicogenomic endpoints of PFOS and PFOA, on Daphnia magna, is important for improving our understanding of the effects elicited by these compounds and to develop adverse outcome pathway maps.
大型蚤广泛分布于地球上的各种淡水系统中,是淡水生态系统食物链的一部分,因此是研究水生毒物学的极佳模式系统(Koivisto,1995 年)。大型水蚤在有利条件下通过无性孤雌生殖繁殖,在不利环境下通过有性繁殖产生休眠包裹卵(ephippia)(Hebert,1978 年)。它被认为是水生无脊椎动物中的代表性物种,在标准化毒性试验中被用作污染指示物种(Brown 和 Thompson,1982 年;Le 等人,2016 年)。在本研究中,我们分析了不同浓度的全氟辛烷磺酸和全氟辛酸对大型蚤的影响。我们对生理终点进行了分析,并与包括应激反应、免疫反应、呼吸、脂质代谢和繁殖在内的几种信号通路的基因表达进行了比较。结果表明,生理反应与这些生理途径相关基因的调控之间存在很强的相关性。分析全氟辛烷磺酸和全氟辛酸对大型蚤的毒性基因组学终点,对于提高我们对这些化合物引发的影响的认识和开发不良后果途径图非常重要。

2. Materials and methods

2.1. Chemicals 2.1.化学品

PFOS (CAS No. 1763-23-1) and PFOA (CAS No. 335-67-1) (Fig. 1) were purchased from Sigma (USA) with stated purities in excess of . To obtain experimental exposure concentration i.e. and PFAS were dissolved in dimethyl sulfoxide (DMSO; Sigma). The final assay concentration of DMSO was .
全氟辛烷磺酸(化学文摘社编号:1763-23-1)和全氟辛酸(化学文摘社编号:335-67-1)(图 1)购自 Sigma(美国),纯度超过 。为了获得实验暴露浓度,即 ,将 PFAS 溶解在二甲基亚砜(DMSO;Sigma)中。DMSO 的最终检测浓度为

PFOS 全氟辛烷磺酸
CAS No.  化学文摘社编号
Mol wt. 500.13 摩尔重量 500.13
CAS No. 335-67-1 化学文摘社编号 335-67-1
Mol wt. 414.07 摩尔重量 414.07
Day 14 第 14 天
  • PFOS  全氟辛烷磺酸
  • PFOS  全氟辛烷磺酸
PFOS   全氟辛烷磺酸
Fig. 2. PFOS and PFOA impede Daphnia magna ephippia hatching, growth and lifespan. Twenty ephippia were exposed in triplicate to 1,10 and PFOS and PFOA and hatching success were recorded at (A). Similarly, 20 neonates ("24-h old) were exposed to PFOS and PFOA for 7 and 14 days and body and tail length were measured (B, C, D and E). Daphnia magna neonates ("24-h old) were exposed to PFOS and PFOA and the lifespan was recorded (F). One-way ANOVA followed by Dunnett post-test ("p ; * ; ***p p 0.001 ). .
图 2.全氟辛烷磺酸和全氟辛酸阻碍大型蚤蜕膜孵化、生长和寿命。将 20 只蜉蝣暴露于 1、10 和 的全氟辛烷磺酸和全氟辛酸中,一式三份,在 (A)记录孵化成功率。同样,20 只新生幼体("24 小时龄")分别暴露于全氟辛烷磺酸和全氟辛酸 7 天和 14 天,并测量了体长和尾长(B、C、D 和 E)。将大型蚤新生儿("24 小时龄")暴露于全氟辛烷磺酸和全氟辛酸并记录其寿命(F)。单因素方差分析,然后进行邓尼特后检验("p ; * ; ***p p 0.001 )。 .

2.2. Daphnia magna maintenance

Daphnia magna ephippia were purchased from MicroBioTests Inc. (Belgium), and were activated by rinsing in tap water and hatched by incubating for 72-90 in standard freshwater (MicroBioTests Inc) at a temperature of under continuous illumination of . The artificial media, which was used for Daphnia magna culture included and , with a hardness of . The media was aerated for by bubbling air through a tube connected to an air pump. Newly hatched neonates ( old) were pre-feed with a suspension of Spirulina microalgae before exposure. For long term exposures, Daphnia magna cultures were maintained in controlled laboratory conditions, at a temperature of and a photoperiod cycle of light/10 h dark. Daphnia magna were fed a mixture of Spirulina microalgae and yeast (Saccharomyces cerevisiae) at a concentration of cells day and cells day, respectively. Fifty percent of treatment water was changed every other day. and dissolved oxygen (DO) were measured before starting and during exposure and remained in the range of and DO . Similarly, salinity, nitrite and nitrate were measured throughout the experiment and was maintained at , below and below respectively.
大型蚤(Daphnia magna ephippia)购自 MicroBioTests Inc.(比利时),用自来水冲洗活化,在标准淡水(MicroBioTests Inc)中孵化 72-90 ,温度为 ,持续光照 。用于大型蚤培养的人工培养基包括 ,硬度为 。培养基通过与气泵相连的管子鼓气,以便 。新孵化的新生蚤( )在暴露前预先投喂螺旋藻微藻类悬浮液 。对于长期暴露,大型蚤培养物保持在受控实验室条件下,温度为 ,光周期为 光/10 小时暗。给大型水蚤喂食螺旋藻微藻和酵母(酿酒酵母)的混合物,浓度分别为 cells day 和 cells day。每隔一天更换 50%的处理水。在开始接触前和接触期间测量 和溶解氧 (DO),并保持在 和 DO 的范围内。同样,在整个实验过程中对盐度、亚硝酸盐 和硝酸盐 进行了测量,并分别保持在 以下。

2.3. Hatching, acute toxicity and gene expression analysis

To determine the effects of PFOS and PFOA on hatching success, ephippia were rinsed with tap water and exposed in triplicates ( ) in standard fresh water containing DMSO (control) or PFOS and PFOA ( 1,10 and ). The ephippia were exposed in 6 well plates (BD Falcon, USA). In order to reduce binding of PFAS to the plates, the plates were pretreated with the exposure solutions for .
为确定全氟辛烷磺酸和全氟辛酸对 孵化成功率的影响,用自来水冲洗鳗鲡,并以三重复 ( ) 的方式将鳗鲡置于含有 DMSO(对照)或全氟辛烷磺酸和全氟辛酸 ( 1,10 和 ) 的 标准淡水中。表皮藻暴露在 6 孔板(BD Falcon,美国)中。为了减少 PFAS 与平板的结合,用暴露溶液对平板进行预处理,
For survival assay, Daphnia magna neonates ( old) were exposed as outlined above with 5 animals in each well with 5 replicates. Mortality was recorded at 6,24 and 48 h. Daphnia magna were considered dead when there was no mobility and no organ movement under the microscope. For qRT-PCR analysis, 20 Daphnia magna neonates ( old) were exposed to 1,10 and PFOS and PFOA in 6 well plates for . Following exposure Daphnia magna were collected, snap frozen using liquid nitrogen and the samples were stored at until analyzed.