中国本土鱼类模式生物稀有鮈鲫研究应用的历史与现状

稀有鮈鲫(Gobiocypris rarus)是一种中国特有的小型鲤科鱼类,从1990年开始,中国科学院水生生物研究所以培育新的实验鱼为目的,对稀有鮈鲫开展了系列的实验动物化研究,并推动其在各研究领域中的应用。随着我国化学品环境管理制度的建立,稀有鮈鲫已逐渐成为我国化学品测试中最重要的受试鱼类。本文综述了稀有鮈鲫分类地位、分布、形态特征、早期发育、生长、繁殖、遗传、对环境的适应性等方面的生物学特点,分析了稀有鮈鲫作为模式生物的优点,介绍了稀有鮈鲫实验动物标准制定进展。随着实验动物标准化工作的深入以及基因组测序工作的开展,稀有鮈鲫将更广泛地应用于相关的研究和检定,成为一种我国具有代表性的本土模式生物。     

稀有鮈鲫作为化学品生态毒性测试鱼类模式生物的标准化实践

鱼类模式生物在化学品生态毒性测试中占有重要地位,中国尚没有合适的标准化本土实验鱼种。国家环境保护部第7号令《新化学物质环境管理办法》对本土受试生物用于生态毒性测试提出了要求。稀有鮈鲫(Gobiocypris rarus)是一种我国特有的小型淡水鲤科鱼类,具有成为标准试验鱼种的潜能。首个以稀有鮈鲫为标准供试生物的化学品鱼类急性毒性试验国家标准已经发布实施,其他测试方法的标准化工作正在持续进行中。本文阐述了研发稀有鮈鲫为标准供试生物的依据,对相关标准化研究工作进行了分析、归纳和总结,并提出了未来工作的方向和需求。     

稀有鮈鲫作为水生模式生物的研究及探讨

我国本土水生生物稀有鮈鲫(Gobiocypris rarus Ye et Fu,1983年)是一种优良的试验用鱼,具备成为模式生物必要条件。本文总结了稀有鮈鲫生物生态学研究情况,分析了稀有鮈鲫在多个应用领域与模式生物斑马鱼、青鳉存在的不足和差距,同时借鉴发达国家推动模式生物的实践经验,提出了针对性建议。     

稀有鮈鲫作为水生模式生物的研究及探讨

我国本土水生生物稀有鮈鲫(Gobiocypris rarus Ye et Fu,1983年)是一种优良的试验用鱼,具备成为模式生物必要条件。本文总结了稀有鮈鲫生物生态学研究情况,分析了稀有鮈鲫在多个应用领域与模式生物斑马鱼、青鳉存在的不足和差距,同时借鉴发达国家推动模式生物的实践经验,提出了针对性建议。     

氰戊菊酯对斑马鱼和稀有鮈鲫不同生命阶段的毒性效应

为探明氰戊菊酯对鱼类不同生命阶段的毒性效应,以斑马鱼和稀有鮈鲫为测试生物,评价了氰戊菊酯对斑马鱼和稀有鮈鲫不同生命阶段的急性毒性差异。研究发现,氰戊菊酯对斑马鱼和稀有鮈鲫胚胎的96 h-LC50值分别为0.901(0.664~1.22)和0.636(0.233~1.74)mg·L~(-1)。氰戊菊酯对稀有鮈鲫胚胎孵化有明显的促进作用,染毒48 h后,0.188和0.375 mg·L~(-1)氰戊菊酯对稀有鮈鲫胚胎的孵化率分别为88.9%和100%。氰戊菊酯对斑马鱼仔鱼、幼鱼和成鱼的96 h-LC50值分别为0.00340、0.0183、0.00487 mg·L~(-1),氰戊菊酯对稀有鮈鲫仔鱼、幼鱼和成鱼的96 h-LC50值分别为0.0520、0.00277、0.00345 mg·L~(-1)。因此,氰戊菊酯对斑马鱼不同生命阶段的毒性从高到低顺序依次为:仔鱼成鱼幼鱼胚胎,氰戊菊酯对稀有鮈鲫胚胎、仔鱼、幼鱼和成鱼的毒性为幼鱼成鱼仔鱼胚胎。试验结果表明,斑马鱼仔鱼对氰戊菊酯的敏感性高于稀有鮈鲫相应的生命阶段,相对于斑马鱼,稀有鮈鲫胚胎、幼鱼以及成鱼对氰戊菊酯的毒性作用更为敏感,说明氰戊菊酯对斑马鱼和稀有鮈鲫不同生命阶段的毒性效应差异较大。     

三唑酮对斑马鱼和稀有鮈鲫不同生长阶段的急性毒性比较

为探明三唑酮对鱼类不同生长阶段的毒性效应,以斑马鱼和稀有鮈鲫为测试生物,检测了三唑酮对斑马鱼和稀有鮈鲫不同生长阶段的急性毒性差异。研究发现,三唑酮对斑马鱼和稀有鮈鲫胚胎为低毒,其96 h-LC50值分别为21.1(14.4~31.0)和14.2(9.65~20.9)mg·L~(-1);高浓度的三唑酮对斑马鱼和稀有鮈鲫胚胎的孵化有明显的抑制作用,染毒96 h后,23.7 mg·L~(-1)三唑酮组斑马鱼胚胎的孵化率为4.17%,22.5 mg·L~(-1)三唑酮组稀有鮈鲫胚胎的孵化率为33.3%。三唑酮对斑马鱼仔鱼、幼鱼和成鱼为低毒,其96 h-LC50值分别为24.8、21.3、13.1 mg·L~(-1),三唑酮对稀有鮈鲫仔鱼、幼鱼和成鱼为中毒,其96 h-LC50值分别为9.96、7.89、6.89 mg·L~(-1)。因此,三唑酮对斑马鱼和稀有鮈鲫不同生长阶段的毒性效应排序一致,毒性从高到低顺序依次为:成鱼幼鱼仔鱼。试验结果表明,相对于斑马鱼,稀有鮈鲫对三唑酮的毒性作用更为敏感,三唑酮对稀有鮈鲫不同生长阶段的毒性均高于斑马鱼相应生长阶段的毒性。     

磷酸三(2,3-二氯丙基)酯阻燃剂对稀有鮈鲫的毒性效应

为研究新型阻燃剂磷酸三(2,3-二氯丙基)酯(TDCPP)的毒性效应,以稀有鮈鲫(Gobiocypris rarus)为实验生物,采用半静态实验方法,分别进行96 h和28 d的染毒,研究TDCPP对稀有鮈鲫的急性毒性和慢性毒性效应,并通过对稀有鮈鲫脑组织中与神经纤维的生长、发育、轴突再生等相关基因mRNA表达量的检测,初步探讨了TDCPP的神经毒性作用。结果表明：TDCPP对稀有鮈鲫的96 h-LC₅₀为2.99(2.20～3.38) mg·L^-1,根据化学物质对鱼类毒性分级标准TDCPP属于剧毒性。经0.9、1.5、2.1和2.7 mg·L^-1 TDCPP染毒28 d,与对照组相比,暴露组稀有鮈鲫肝脏及脑组织中SOD和GSH-Px的活性均受到抑制,且随着TDCPP的暴露浓度增加,其抑制作用显著增强;暴露组稀有鮈鲫脑组织中与神经纤维的生长、发育相关的微管蛋白α/β、神经丝NF-M以及关键蛋白GAP-43等基因的mRNA表达量,与对照相比均发生显著下调。可知,TDCPP暴露可以诱发稀有鮈鲫神经毒性作用。     

稀有鮈鲫成体神经发生相关基因的分子克隆及序列分析

成体神经发生是脊椎动物中广泛存在的一种生物学特征。成体硬骨鱼类的脑展现出强烈的神经活性以及出色的脑修复能力,这使得硬骨鱼类成为研究成体神经发生和脑修复的一个理想的模型。本文克隆了成体稀有鮈鲫(Gobiocypris rarus)脑组织中神经发生及脑修复相关的hes5、pax6、sox11和prox1基因的部分c DNA序列并进行了序列分析。序列分析结果表明,pax6和sox11基因片段与斑马鱼(Danio rerio)对应的基因片段的同源性最高,分别为97%和94%;hes5基因与鲤鱼(Cyprinus carpio)相对应的基因片段的同源性最高,为92%;prox1基因在物种间的同源性最低。基于稀有鮈鲫和已知物种相应基因的核苷酸序列构建了系统发育树,发现稀有鮈鲫prox1基因与其他硬骨鱼类的亲缘关系最远。本文为进一步开展神经毒性类化学品对水生生物鱼类的成体神经毒性作用机制研究提供了分子生物学基础。     

三氯酚暴露致稀有鮈鲫肝脏损伤的比较蛋白质组研究(Proteomic Analysis of Hepatic Protein Profiles in Rare Minnow （Gobiocypris rarus） Exposed to Trichlorophenol （TCP）)

为探讨2,4,6-三氯酚（2,4,6-TCP）暴露致鱼类肝脏损伤的毒性效应,以稀有鮈鲫（Gobiocypris rarus）为模式生物,TCP暴露浓度为1、10、100μg·L-1,进行流水暴露实验（28d）,利用二维凝胶电泳方法（2-DE）,结合质谱分析技术（MALDI-TOF-MS）,对TCP致稀有鮈鲫肝脏损伤的蛋白表达谱进行了研究. 结果显示,共发现35个差异蛋白点,其中11个蛋白被成功鉴定（C.I.>95%）. 对鉴定的蛋白根据GO Terms 进行功能分类,发现它们主要参与脂类代谢与转运、氧化应激以及蛋白修复和氧化磷酸化等生物学过程。以上结果表明,TCP对稀有鮈鲫具有明显的肝脏毒性效应.     

三氯酚暴露致稀有鮈鲫肝脏损伤的比较蛋白质组研究

为探讨2,4,6-三氯酚(2,4,6-TCP)暴露致鱼类肝脏损伤的毒性效应,以稀有鮈鲫(Gobiocypris rarus)为模式生物,TCP暴露浓度为1、10、100μg·L-1,进行流水暴露实验(28d),利用二维凝胶电泳方法(2-DE),结合质谱分析技术(MALDI-TOF-MS),对TCP致稀有鮈鲫肝脏损伤的蛋白表达谱进行了研究.结果显示,共发现35个差异蛋白点,其中11个蛋白被成功鉴定(C.I.>95%).对鉴定的蛋白根据GOTerms进行功能分类,发现它们主要参与脂类代谢与转运、氧化应激以及蛋白修复和氧化磷酸化等生物学过程。以上结果表明,TCP对稀有鮈鲫具有明显的肝脏毒性效应.     

评估斑马鱼胚胎毒性试验在农药危害筛选中的应用

考虑到人类社会中大量使用化学物质,想要准确有效地评估这些化学物质对人类和生态受体的潜在风险,研发有效的手段和方法是至关重要的。鱼胚胎急性毒性试验是其中一种工具,已表现出与幼鱼急性毒性标准试验的预测结果高度吻合,而幼鱼试验对资源消耗更多。然而,也有证据表明,对于某些类型的化学物质,包括神经毒素,鱼类胚胎的敏感性低于幼鱼。本文利用已发表的斑马鱼胚胎毒性数据,与3种常用检测用鱼幼鱼(虹鳟鱼、蓝鳃太阳鱼、羊鲷)的半致死浓度50%(LC₅₀)数据进行比较,研究了鱼类胚胎对农药危害评估的效用。在将农药视为单因素的情况下,斑马鱼胚胎和幼鱼毒性数据相关性较差,差异显著(r²=0.28;p2=0.64;p 精选自Glaberman, S., Padilla, S. and Barron, M. G. (2017), Evaluating the zebrafish embryo toxicity test for pesticide hazard screening. Environmental Toxicology and Chemistry, 36: 1221–1226. doi: 10.1002/etc.3641
详情请见http://onlinelibrary.wiley.com/doi/10.1002/etc.3641/full
     

毒死蜱对我国南方稻区水域中12种淡水鱼的毒性

毒死蜱作为稻田常用农药,普遍存在于稻区沟渠、池塘和河流中,从而对生活在其中的鱼类具有潜在风险。通过短期暴露试验,比较了毒死蜱在纯水、水-沉积物体系中对淡水鱼的毒性效应,进一步研究了毒死蜱在不同鱼体内的生物富集作用,以及对鱼脑Ach E活性的影响。试验结果表明:毒死蜱对12种淡水鱼均表现为高毒或剧毒,最敏感的是太阳鱼,但体系中沉积物的存在会通过吸附作用降低农药对鱼类的毒性;毒死蜱在鱼体内表现为中等或高富集性,其中斑马鱼的富集系数最大;毒死蜱对鱼脑Ach E酶活性有明显抑制作用,其中以虹鳟最敏感。研究结果为稻田常用农药对水生态环境中鱼类安全的风险性评价提供了科学依据。     

Utility of the QSAR modeling system for predicting the toxicity of substances on the European inventory of existing commercial chemicals∗

Many chemicals are in common commercial use for which no information on the environmental fate or toxicity exists. Recent legislation requires that many substances be assessed for their toxicity to aquatic organisms within a very short time and determine which of these chemicals need to be studied in greater detail. It would be impossible to measure the acute and chronic effects of all of these compounds on a single organism, let alone a battery of different types of organisms, communities or ecosystems. Initially, the chemicals on the European Inventory of Existing Commercial Chemical Substances (EINECS) need to be screened and relative hazard to the environment determined. In response to OECD directives, there has been a great deal of activity by government and industry scientists. At the International Workshop on Advances in Environmental Hazard and Risk Assessment it was concluded that quantitative structure activity relationships (QSAR) could and should be used in the hazard assessment process. Papers published in that volume outline the advantages, disadvantages, limitations, advances and research requirements.

The QSAR, structure‐activity based chemical modeling and information system, which was developed by the US‐Environmental Protection Agency was used to predict the acute toxicity of 113 substances from the “Old Substances”; list of the German government to the four commonly used aquatic toxicity test organisms: Daphnia magna (DM), fathead minnow (FHM), rainbow trout (RBT), and blue‐gill sunfish (BG).

Of these compounds the QSAR system predicted the acute toxicity of 87 substances towards fathead minnow. For the other three species examined the QSAR system could be used to predict toxicity for 78 compounds.

The predicted toxicities were compared to observed toxicities of compounds which have been evaluated and stored in the “Aquire”; data base. Observed toxicity values were available for at least one species for 38 compounds. The toxicities of some compounds are well predicted while those of other compounds were not well predicted. Overall, the QSAR system accurately classified the acute toxicity ranges of 50%, 64%, 56% and 56% of the compounds investigated for DM, FHM, RBT and BG, respectively. Of the compounds studied 10 were very poorly predicted, of these the QSAR system overpredicted the toxicity of three, while underpredicting the toxicity of seven. Of these seven compounds, five contained amino groups.     

PFOS潜在替代品全氟丁基磺酸钾对不同营养水平水生生物的毒性

全氟丁基磺酸钾(PFBSK)作为全氟辛基磺酸(PFOS)潜在的替代品,极易溶于水,主要存在于水体中,因而其水生毒性的研究十分重要。采用OECD 201、OECD 202、OECD 203和OECD 211标准试验方法,研究了PFBSK对羊角月牙藻(Pseudokirchneriella subcapitata)、大型溞(Daphnia magna)和中国本土鱼种稀有鮈鲫(Gobiocypris rarus)的急性毒性效应以及对大型溞繁殖的影响。组合多终点急慢性水生生物毒性结果：PFBSK的急性毒性终点均大于100 mg·L^-1,大型溞繁殖试验的无观察效应浓度(NOEC)为571 mg·L^-1,最低可观察效应浓度(LOEC)为981 mg·L^-1。按GHS分类导则,PFBSK未表现出急性毒性和慢性毒性。与之相比,PFOS则对水生生物表现出毒性,黑头软口鲦(Pimephales promelas)为最敏感物种,其96 h-LC₅₀为4.7 mg·L^-1;大型溞繁殖试验的NOEC为12 mg·L^-1。按GHS分类导则,属于中等毒性物质。可见,PFBSK较PFOS水生毒性明显降低。     

Ammonia modeling for assessing potential toxicity to fish species in the Rio Grande, 1989-2002.

Increasing volumes of treated and untreated human sewage discharged into rivers around the world are likely to be leading to high aquatic concentrations of toxic, unionized ammonia (NH3), with negative impacts on species and ecosystems. Tools and approaches are needed for assessing the dynamics of NH3. This paper describes a modeling approach for first-order assessment of potential NH3 toxicity in urban rivers. In this study daily dissolved NH3 concentrations in the Rio Grande of central New Mexico, USA, at the city of Albuquerque's treated sewage outfall were modeled for 1989-2002. Data for ammonium (NH4+) concentrations in the sewage and data for discharge, temperature, and pH for both sewage effluent and the river were used. We used State of New Mexico acute and chronic NH3- N concentration values (0.30 and 0.05 mg/L NH3-N, respectively) and other reported standards as benchmarks for determining NH3 toxicity in the river and for assessing potential impact on population dynamics for fish species. A critical species of concern is the Rio Grande silvery minnow (Hybognathus amarus), an endangered species in the river near Albuquerque. Results show that NH3 concentrations matched or exceeded acute levels 13%, 3%, and 4% of the time in 1989, 1991, and 1992, respectively. Modeled NH3 concentrations matched or exceeded chronic values 97%, 74%, 78%, and 11% of the time in 1989, 1991, 1992, and 1997, respectively. Exceedences ranged from 0% to 1% in later years after enhancements to the wastewater treatment plant. Modeled NH3 concentrations may differ from actual concentrations because of NH3 and NH4+ loss terms and additive terms such as mixing processes, volatilization, nitrification, sorbtion, and NH4+ uptake. We conclude that NH3 toxicity must be considered seriously for its potential ecological impacts on the Rio Grande and as a mechanism contributing to the decline of the Rio Grande fish community in general and the Rio Grande silvery minnow specifically. Conclusions drawn for the Rio Grande suggest that NH3 concentrations may be high in rivers around the world where alkaline pH values are prevalent and sewage treatment capabilities are poorly developed or absent.     

Water Quality Criteria for European Freshwater Fish

1. For water pollution control purposes, the concentration-addition model for describing the joint effects of mixtures of toxicants on aquatic organisms is appropriate; in this model the contribution of each component in the mixture is expressed as a proportion of the aqueous concentration producing a given response in a given time (e.g. p 96-h LC50).

2. Examination of available data using this model shows that for mixtures of toxicants found in sewage and industrial effluents, the joint acutely-lethal toxicity to fish and other aquatic organisms is close to that predicted, assuming simple addition of the proportional contribution from each toxicant. The observed median value for the joint effect of these toxicants on fish is 0.95 of that predicted, and the corresponding collective value for sewage effluents, river waters, and a few industrial wastes, based on the toxicity of their constituents, is 0.85, while that for pesticides is 1.3.

3. The less-than-predicted effect of commonly-occurring toxicants in some mixtures may be partly attributable to small fractions of their respective LC50 values having a less-than-additional effect. However, recent research has shown that for some organic chemicals which have a common quantitative structure-activity relationship (QSAR), their joint action as determined by acute toxicity is additive at all concentrations.

4. The few (unpublished) data available for the long-term lethal joint effect on fish of toxicants in mixtures suggest that they may be markedly more than additive, a phenomenon that needs confirmation and further investigation.

5. In the few studies on the sub-lethal effects on fish (eg growth), the joint effect of toxicants has been consistently less-than-additive which suggests that as concentrations of toxicants are reduced towards the levels of no effect, their potential for addition is also reduced. There appear to be no marked and consistent differences between the response of different species to mixtures of toxicants.

6. Field studies have shown that reasonably accurate toxicity predictions based on chemical analysis can be made if the waters which are polluted are acutely lethal to fish, and that a fish population of some kind can exist where the median 2 p t LCSOs (rainbow trout) is < 0.2. It is not known whether this condition is equivalent to a C p NOEC of 4.0 (ie the sum of the individual fractions of the NOEC for the species present), or to a NOEC of < 1.0 for each individual toxicant (i.e. fractions of the NOEC are not summed).

7. In general, the joint effect of the common toxicants on lethal and sub-lethal responses of fish is not explained by variations in the uptake of the individual toxicants concerned; this may not apply for those chemicals with a common QSAR, although there is little experimental evidence in this field.

8. There is an immediate need for more empirical studies on the joint effect of mixtures of toxic units of individual components, and the relation between long- and short-term lethal and non-lethal joint effects. This applies to mixtures of commonly occurring toxicants as well as to mixtures of organic chemicals with a common QSAR. The data obtained should be reinforced by studies on the mechanisms of interaction of toxicants. More field studies which relate water quality to the structure and productivity of fish populations are also required, involving direct measurements of fractional toxicity of the river water wherever possible.

9. The concentration-addition model appears to be adequate to describe the joint effect of commonly-occurring constituents of sewage and industrial wastes, and for tentative predictions of the joint effect on fish populations of toxicants present at concentrations higher than the EIFAC recommended values. However, concentrations lower than the EIFAC recommended values may make an increasingly lesser contribution to the toxicity of mixtures of toxicants and there may be a need to adjust the tentative water quality criteria downwards where two or more toxicants are present at concentrations close to these values. For toxicants with a common QSAR, their additive joint action may necessitate the setting of water quality criteriafor this group as a whole and not on the basis of individual compounds. However, too little is known of their precise joint action where the combined concentration produces a sub-lethal response.     

环境中金属生物有效性的预测模型——生物配体模型研究进展

生物配体模型(BLM)是一种用于预测环境中金属生物毒性的机理性模型.模型理论起源于自由离子活度模型(FIAM)和鱼鳃络合模型(GSIM),考虑了自由金属离子的活度以及自然环境存在的其他离子(如Ca2+、Na+、Mg2+、H+)、非生物配体(如可溶性有机质、氯化物、碳酸盐、硫酸盐)和生物配体的竞争.目前,在水生生态系统中,基于鱼鳃络合模型的框架基础,通过生物化学实验手段并结合数学方法,建立了预测铜、锌、银、镍对Rainbowtrout(虹鳟鱼)、Fatheadminnow(黑头呆鱼)和Daphniamagna(水蚤)的急、慢性毒性的BLM版本,并积极探索其在陆地生态系统中的应用.虽然生物配体模型在实验室模拟条件下取得了较为满意的结果,但其中包含着一些假设,在实际应用中还具有一定的局限性,尤其是陆地生态系统生物配体模型的发展还需要做许多研究工作.本文主要论述了生物配体模型的理论基础、实现手段和应用情况,讨论了生物配体模型的优势和局限性并对其未来研究方向进行了展望.