Name: protocol for acute and chronic ecotoxicity testing of the turquoise killifish nothobranchius furzeri
Uploaded: 2018-04-24T14:30:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri at JoVE.com

我们感谢 UAntwerpen 的球体小组和 Ugent 的作物保护部对水样进行分析。该项目的支持是由 PF/10/007 研究基金的卓越中心的生态和社会进化动态提供的。afg 胶 (11Q0516N) 和 ESJT (FWO-SB151323) 以博士和 TP (12F0716N) 为博士后研究员, FWO 佛兰德 (全宗 Wetenschappelijk Onderzoek)。

这里描述的所有方法都得到了 KULeuven 伦理委员会的批准。
1. furzeri的孵化和一般维护
<ol><li>准备鱼培养基 (pH 值 7), 温度为14摄氏度, 并加入纯化型 II 水, 加标准化盐, 电导率为600µS/厘米 (24 摄氏度)。</li>
	<li>从已存储在标准条件13下的 GRZ 实验室线中选择鸡蛋。选择 DIII 阶段的鸡蛋 (即准备孵化), 可识别的金色眼睛9 , 并轻轻地把它们与软镊子对塑料 2 L 坦克 (不超过大约30鸡蛋每罐)。 注: 为了有足够数量的健康测试有机体, 孵化两次尽可能多的卵作为所需的鱼幼虫数量。</li>
	<li>添加1厘米的鱼培养基在12摄氏度, 让水温逐渐收敛到室温 (24 °c)9。鱼将孵化在前12小时。</li>
	<li>24小时后, 喂养幼仔一种浓缩剂量的新鲜孵化的Artemianauplii (有关食物频率和数量的详细信息, 请参阅 Polačik et 等) 的育种协议 20169), 并将水深提高到5厘米添加鱼培养基。</li>
	<li>36小时后, 喂养幼仔另一种浓缩剂量的新鲜孵化的卤虫幼体和添加鱼培养基, 增加水深到10厘米。</li>
	<li>在恒温条件下孵化鱼容器 (例如在孵化器、气候室或温水浴中) 在 14 h:10 h 灯以下: 黑暗政权。</li>
	<li>在实验开始之前, 复杂的鱼容器 (没有鱼) 与暴露化合物, 填充他们的最高浓度的接触介质, 并留在一夜之间, 以限制转移有毒物质的容器在实际实验。</li>
	<li>48小时孵化后, 选择健康的浮力幼虫开始接触实验。放弃所谓的肚皮滑块, 无法填满他们的游泳膀胱, 因此有一个受损的浮力 (持续下沉到底部)。</li>
</ol>2. 短期曝光协议
注: 研究人员应针对每项治疗至少20次复制 (20 条鱼在单独的罐子里)。除完全控制处理外, 如果用溶剂制备该化合物的溶液, 则应包括溶剂控制。溶剂控制应包含在最高接触浓度中溶剂浓度的溶剂量。
<ol><li>将实验容器 (0.5 升玻璃罐) 贴上标签, 用适当的曝光介质 (不同的毒物浓度) 填充。添加化合物以获得正确的浓度。</li>
	<li>将幼虫 (48 小时孵化后) 分别转移到容器 (每容器1条鱼进行单独监测)。 注意: 鱼类被单独暴露, 以尽量减少社会互动的潜在混淆效应, 例如对食物和侵略的竞争。然而, 鱼被允许视觉上的互动, 根据道德标准的实验室动物使用。</li>
	<li>随访2周以上的急性暴露。在这段时间里, 用卤虫幼体每天两次喂鱼ad 随意, 每星期7天/周。</li>
	<li>每隔一天刷新培养基以维持水质, 并尽量减少复合降解的潜在影响。监测关键水分变量 (溶解氧水平应超过 80%, 电导率应介于600和700µS/厘米之间, pH 值介于7.8 和8.2 之间, 硬度 (如碳酸钙3) 介于350和450毫克/升之间, 位于最佳饲养条件范围内。N. furzeri 9)。在刷新培养基之前和之后取水样品, 以确定实际的复合浓度。</li>
	<li>端点<ol>
<li>检查鱼的死亡率, 压力 (例如反常行为: 游泳颠倒) 或疾病每天 (上午, 晚上)。查阅谢德et的发布。(1999)12了解有关死亡率、压力或疾病的观察的详细信息。</li>
		<li>使用剂量-响应曲线 (丽思和 Streibig, 2005) 在不同的时间点计算 LC50值。使用 r v3.2.3 (r 开发核心团队, 2016) 中的刚果民主共和国包中的drm函数或类似的统计方法。</li>
	</ol>
</li>
</ol>3. 慢性暴露协议
注: 在实验开始时, 至少瞄准25条鱼/条件, 以尽量减少性别比例偏倚的几率, 并考虑自然原因导致的潜在背景死亡率 (i. e. 与年龄有关的死亡率)。
<ol><li>孵化 (见1节)</li>
	<li>第一阶段 (孵化后2天-孵化后16天)<ol>
<li>按照 2.1-2.4 所述的协议执行</li>
		<li>在实验的第二阶段, 培养基会在一周内降解 (没有茶点, 见下文)。将所需的介质在大型惰性容器中存放一周, 以允许化合物的类似降解。</li>
	</ol>
</li>
	<li>第二阶段 (孵化后16天)<ol>
<li>通过与化合物络合, 制备2升实验玻璃罐。用正确的曝光介质填充罐子, 并添加一个空气管通气罐子。在这些罐子里单独的把鱼放在实验的其余部分。允许视觉交互以适应道德标准。</li>
		<li>每星期刷新一次介质。将鱼与网转移到含有相同曝光介质的新罐子上。在每一周内每天抽取水样来监测化合物在每次浓度处理过程中的降解情况。如果测试多个压力源 (例如在不同温度下的化合物的毒性), 则计算每种处理的降解曲线。测量非生物参数 (pH 值, 温度, 溶解氧%, 电导率) 每周三次。</li>
		<li>从孵化后2天 (dph) 直到 23 dph, 每天喂鱼两次, 每周7天, ad 随意卤虫幼体。从 24 dph-37 dph, 补充ad libitumArtemia饮食与切碎的摇幼虫。从 38 dph, 每天喂鱼两次, 每周7天, ad 随意冷冻摇幼虫。</li>
	</ol>
</li>
	<li>端点<ol>
<li>每日检查鱼的死亡率, 压力或疾病12。</li>
		<li>为了确定生长, 每周测量身体大小 (9 dph-16 dph 21 dph-...) 通过把鱼转移到从他们的水库中填充培养基的培养皿中。采取 4-5 大小校准图片的鱼从上面 (在一个固定的高度) 和分析他们数字使用空间测量程序 (例如ImageJ)。 注: 对于成年鱼, 使用更高的培养皿, 以尽量减少处理压力, 保持所有的鱼淹没在整个测量过程中。</li>
		<li>对于男性成熟, 视觉检查鱼每天的着色从 15 dph 起。检查鳍的第一个迹象, 婚礼着色 (第二性特征)。使用第一天, 这是可见的作为一个代理的男性成熟时间。</li>
		<li>夫妇非奸鱼与男性相同的治疗组或非实验性男性每周三次从 30 dph 开始, 以确定女性成熟时间 (第一个卵子的一天)。为此, 请使用3.4.5 中描述的产卵协议。</li>
		<li>对于生育力, 夫妇成熟的女性与成熟男性3次/周从 30 dph 开始, 在他们的治疗使用交叉计划。<ol>
<li>为每对夫妇准备一个产卵池 (1 升), 使用暴露培养基从男性水族馆补充产卵基质 (细砂 < 500 µm)。</li>
			<li>将雄性和雌性转移到产卵池中, 让它们产卵两个小时。在此过程中尽量减少产卵容器周围的人类活动或干扰。</li>
			<li>然后, 将鱼轻轻地转移回原来的外壳容器, 不需要水的混合, 这将会在产卵基质中旋转卵。</li>
			<li>通过在500µm 网格上浇产卵基板来过滤卵。计数的鸡蛋和转移 (使用软镊子) 潮湿泥炭苔藓在培养皿中9,14。</li>
			<li>每天清除死卵。一个星期后, 密封的培养皿与密封膜, 并存储在一个温度控制孵化器在28摄氏度和14:10 小时的光: 黑暗循环, 立即发展到 DIII 阶段 (即准备孵化后约三周)。长期贮存时, 将卵存放在17摄氏度的恒定黑暗中, 卵子进入休眠阶段, 并保持多年存活。当从这些休眠卵子中招募鱼类进行实验时, 将休眠的卵转移到28°c 条件, 14:10 小时光: 黑暗周期约三周, 以允许发展到 DIII 阶段。</li>
		</ol>
</li>
		<li>测量成年鱼的临界热最大值 (CTmax) (用于性能15的度量值)。<ol>
<li>使用水浴, 以恒定的速度加热0.33 摄氏度/分钟, 并在其中的水不断循环。为每条鱼加1升水族馆。 注: 给定的空间限制在水浴, 有必要在几个系列工作。通过将 "系列" 作为随机因素进行统计分析时, 应考虑系列中条件之间的潜在差异。</li>
			<li>当水族馆的水达到实验饲养温度 (一般为28摄氏度) 时, 将鱼加入水族馆, 开始试验。使用数字温度计 (0.1 摄氏度) 监测 CTmax 浴1升水族馆中的温度每5分钟。</li>
			<li>当鱼无法保持 dorso 腹部直立位置或开始严重抽搐16,17时, 结束试验。测量 1 L 水族馆的温度, 这是临界最大温度。把鱼转回到实验房里恢复。</li>
		</ol>
</li>
		<li>在实验的最后一天测量鱼的重量 (0.1 毫克的准确度), 拍干并把它转移到一艘称量船上。注: 所有鱼类应在最后一次进食后四小时测量, 以规范肠道内的食物重量。</li>
		<li>用 0.1% Tricaine 弄死鱼。</li>
	</ol>
</li>
</ol>4. 跨代曝光协议
注: 要测量污染物对furzeri的跨代影响, 请遵循上文概述的第一代的慢性暴露协议。
<ol><li>每周两次, 检查生产的鸡蛋 (即第二代) 的发展, 储存在28°c 14:10h 光: 黑暗的周期条件通过检查培养皿的胚胎在 DIII 阶段 (见 Polačik et 等。20169)。当50多项父母治疗的复制被充分开发时, 在1.1 中根据该协议孵化他们。</li>
	<li>将健康的、浮力的鱼暴露在与父母鱼完全相同的设置和治疗中。</li>
</ol>

<ol>
	<li>European-Chemicals-Bureau. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TAPIR Three point three-A Project for the Information Requirements of REACH. Final Report-2 August 2005. Scoping study on the development of a Technical Guidance Document on information requirements on intrinsic properties of substances (RIP 3.3-1). , (2005).</li><li>Philippe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+sensitivity+to+copper+of+a+promising+ecotoxicological+model+species,+the+annual+killifish+Nothobranchius+furzeri.">Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.</a> Ecotoxicol Environ Saf. , 26-35 (2017).</li><li>Noyes, P. D., Lema, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forecasting+the+impacts+of+chemical+pollution+and+climate+change+interactions+on+the+health+of+wildlife.">Forecasting the impacts of chemical pollution and climate change interactions on the health of wildlife.</a> Current Zoology. 61 (4), 669-689 (2015).</li><li>Consommateurs, S. S. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Health &amp; Consumer Protection Directorate-General European Commission. 4, (2002).</li><li>Commission, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+document+on+aquatic+ecotoxicology.+Under+Council+directive+91/414/EEC.">Guidance document on aquatic ecotoxicology. Under Council directive 91/414/EEC.</a> SANCO/3268/2001 Rev 4. 2002b. , (2002).</li><li>. Ecological Effects Test Guidelines, OPPTS 850.1500 Fish life cycle toxicity Available from: <a target="_blank" href="https://www.epa.gov/sites/production/files/2015-07/documents/850-1500.pdf">https://www.epa.gov/sites/production/files/2015-07/documents/850-1500.pdf</a> (1996)</li><li>Philippe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+sensitivity+to+copper+of+a+promising+ecotoxicological+model+species,+the+annual+killifish+Nothobranchius+furzeri.">Acute and chronic sensitivity to copper of a promising ecotoxicological model species, the annual killifish Nothobranchius furzeri.</a> Ecotoxicol Environ Saf. , (2017).</li><li>Ankley, G. T., Villeneuve, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fathead+minnow+in+aquatic+toxicology:+past,+present+and+future.">The fathead minnow in aquatic toxicology: past, present and future.</a> Aquatic Toxicology. 78 (1), 91-102 (2006).</li><li>Pola&#269;ik, M., Bla&#382;ek, R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+breeding+of+the+short-lived+annual+killifish+Nothobranchius+furzeri.">Laboratory breeding of the short-lived annual killifish Nothobranchius furzeri.</a> Nature Protocols. 11 (8), 1396-1413 (2016).</li><li>Reichwald, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Sex+Chromosome+Evolution+and+Aging+from+the+Genome+of+a+Short-Lived+Fish.">Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.</a> Cell. 163 (6), 1527-1538 (2015).</li><li>Valenzano, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Turquoise+Killifish+Genome+Provides+Insights+into+Evolution+and+Genetic+Architecture+of+Lifespan.">The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.</a> Cell. 163 (6), 1539-1554 (2015).</li><li>Shedd, T. R., Widder, M. W., Toussaint, M. W., Sunkel, M. C., Hull, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+annual+killifish+Nothobranchius+guentheri+as+a+tool+for+rapid+acute+toxicity+screening.">Evaluation of the annual killifish Nothobranchius guentheri as a tool for rapid acute toxicity screening.</a> Environ. Toxicol. Chem. 18 (10), 2258-2261 (1999).</li><li>Platzer, M., Englert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nothobranchius+furzeri:+a+model+for+aging+research+and+more.">Nothobranchius furzeri: a model for aging research and more.</a> Trends Genet. 32 (9), 543-552 (2016).</li><li>Watters, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+and+distribution+of+Nothobranchius+fishes.">The ecology and distribution of Nothobranchius fishes.</a> J Am Killifish Assoc. 42, 58-61 (2009).</li><li>Op de Beeck, L., Verheyen, J., Stoks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+magnifies+the+impact+of+a+pesticide+in+a+warming+world+by+reducing+heat+tolerance+and+increasing+autotomy.">Competition magnifies the impact of a pesticide in a warming world by reducing heat tolerance and increasing autotomy.</a> Environ Pollut. 233, 226-234 (2018).</li><li>Patra, R. W., Chapman, J. C., Lim, R. P., Gehrke, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+three+organic+chemicals+on+the+upper+thermal+tolerances+of+four+freshwater+fishes.">The effects of three organic chemicals on the upper thermal tolerances of four freshwater fishes.</a> Environ. Toxicol. Chem. 26 (7), 1454-1459 (2007).</li><li>Beitinger, T. L., Bennett, W. A., McCauley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+tolerances+of+North+American+freshwater+fishes+exposed+to+dynamic+changes+in+temperature.">Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature.</a> Environ Biol Fishes. 58 (3), 237-275 (2000).</li><li>Cellerino, A., Valenzano, D. R., Reichard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+bush+to+the+bench:+the+annual+Nothobranchius+fishes+as+a+new+model+system+in+biology.">From the bush to the bench: the annual Nothobranchius fishes as a new model system in biology.</a> Biological Reviews. , (2015).</li></ol>

作者没有什么可透露的。

这项工作描述了一种新的生物测定方法, 使用Nothobranchius furzeri(一种新兴的模型有机体) 来研究毒物和其他压力源的个体和组合的长期效应。提出的协议已成功地应用于测量物种对一系列有毒物质 (铜、镉、34-dichloroaniline 和毒死蜱) 的敏感性。由于它的快速生命周期, 这个脊椎动物模型允许评估致死和跨代的影响在四月内。使用这种鱼类作为毒性筛选模型的另一个主要优点是它生产抗旱蛋。这...

将一组物种的敏感度配置为战略性地选定的毒物被描述为欧洲范围立法 (注册、评估、授权和限制化学品) 的1 。急性或短期毒性试验主要用于这一目的, 因为它们给出了一个物种的敏感性的快速迹象。然而, 在它们的自然环境中, 生物体暴露在较长的时间内, 而完整的生命周期, 甚至几代可能会影响到2。此外, 受污染环境中的有机体通常会一次暴露于多个压力源, 这可能相互作用, 可能导致协同效应3。因此, 基于急性单源压力毒性试验计算的安全浓度可能低估了有毒物质在自然环境中的实际风险。因此, 最好还要研究欧洲委员会4、5和环保局 (联合致死) 所倡导的与环境有关的有毒物质的长期和几代效应。国家环境保护局)6,7。特别是在脊椎动物的研究中, 由于脊椎动物的寿命比无脊椎动物模型生物体长, 所以在从事慢性和几代人的暴露研究时, 劳动力、金钱和时间方面的成本很高。因此, 根据研究的问题, 选择最合适的鱼类模型有机体是可取的。此外, 还应提供广泛的脊椎动物种类, 以检验跨物种的反应的普遍性, 以便能够根据最敏感的物种来调整规章。目前, 有必要制定新的、有效的协议, 以短寿命周期为特征的脊椎动物模型物种, 以降低在脊椎动物身上进行慢性和多代暴露的成本7,8。
绿松石鳉鱼Nothobranchius furzeri 是一个有趣的鱼模型, 用于这种长期暴露实验, 因为它的成熟时间短和生命周期 (生成时间少于4周 9).这意味着与其他鱼类模型7相比, 可以在短时间内研究与生态相关的终点, 如成熟时间和繁殖力。此外, 这些鱼生产抗旱的休眠蛋, 在标准条件下储存数年后仍然可行, 从而消除了对连续养殖的需要9。在生态的研究中, 这也意味着复制鱼可以在完全相同的时刻孵化, 导致所有动物的时间同步, 甚至在不同时间生产的鸡蛋批次中。我们建议使用实验室 GRZ 应变进行暴露实验。这种菌株在实验室条件下表现良好, 是纯合的 (除了性染色体), 基因组的特征很好10,11。
在生态研究中, 选择合适的测试浓度范围是很重要的。为此, 可以使用几种互补方法。标称浓度范围可以基于相关物种的灵敏度, 如Nothobranchius guentheri12。或者, 该范围可以基于标准鱼模型的灵敏度, 如斑马鱼 (斑马斑马)2 , 对大多数毒物具有可比性的敏感性 (菲利普et 。(回顾))。结合上述两种选择, 应进行范围发现实验, 选择标称浓度范围。对于急性试验, 研究人员应瞄准100% 的死亡率, 中间死亡率和0% 的死亡后24小时接触毒物的浓度治疗。对于慢性试验, 最好在两周内进行范围发现实验, 以验证在这个参考期内, 测试浓度最高的条件下的幼虫死亡率是否不超过10%。
该议定书可以作为基线, 在furzeri上对水上污染物进行急性和长期接触, 检查压力源在个体和细胞层面的潜在影响。它还可用于执行多源压力研究, 以适应更高的生态相关性, 混合不同的有毒化合物或研究污染与其他自然压力源 (例如捕食) 之间的交互作用或人为压力源 (例如因气候变化而变暖)。

<table><tbody><tr><td>purified water Type 1 (milli Q)</td><td>Millipore</td><td></td><td></td></tr><tr><td>Sea Salt</td><td>Instant Ocean</td><td></td><td></td></tr><tr><td>2L plastic tank</td><td>SAVIC</td><td></td><td>Always separate material for control and toxicity treatments</td></tr><tr><td>1L plastic tank (spawning)</td><td>Avamoplast</td><td></td><td>Always separate material for control and toxicity treatments</td></tr><tr><td>nets</td><td>Aqua bilzen</td><td></td><td>Always separate material for control and toxicity treatments</td></tr><tr><td>2L glass jars</td><td>Sepac-Flacover</td><td></td><td>Always separate material for control and toxicity treatments</td></tr><tr><td>0,5L glass jars</td><td>Sepac-Flacover</td><td></td><td>Always separate material for control and toxicity treatments</td></tr><tr><td>Artemia eggs</td><td>Ocean Nutrition</td><td></td><td></td></tr><tr><td>chironomus</td><td>Ocean Nutrition</td><td></td><td>frozen</td></tr><tr><td>tricaine</td><td>Sigma aldrich</td><td></td><td></td></tr><tr><td>petri dishes</td><td>VWR</td><td></td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td></td><td></td></tr><tr><td>pipettes</td><td>MLS</td><td></td><td></td></tr><tr><td>tweezers</td><td>FST</td><td></td><td></td></tr><tr><td>500 &amp;micro;m mesh sieve</td><td>/</td><td></td><td>self-made</td></tr><tr><td>microcentrifuge tube (2ml)</td><td>BRAND</td><td></td><td>To store fish in freezer</td></tr><tr><td>glass vials</td><td>Sigma aldrich</td><td></td><td>For water analysis</td></tr><tr><td>weighing boat</td><td>MLS</td><td></td><td></td></tr><tr><td>Jiffy 7c pellets</td><td>Jiffy</td><td></td><td></td></tr><tr><td>water bath</td><td>Gilac</td><td></td><td>for Ctmax</td></tr><tr><td>liquid nitrogen</td><td>Air liquide</td><td></td><td></td></tr><tr><td>digital thermometer</td><td>Testo AG</td><td>testo 926</td><td></td></tr><tr><td>HETO therm heater</td><td>Anker Schmitt</td><td></td><td></td></tr><tr><td>calibrated balance</td><td>Mettler-Toledo AG</td><td></td><td></td></tr><tr><td>camera</td><td>/</td><td></td><td></td></tr><tr><td>platform for camera</td><td>/</td><td></td><td>self-made</td></tr><tr><td>Multiparameter kit</td><td>HACH</td><td></td><td></td></tr><tr><td>Freezer (-80&amp;deg;C)</td><td>Panasonic Ultra low temperature freezer</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fysio&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homogenisation buffer</td><td>VWR</td><td></td><td>0.1&amp;nbsp;M TRIS&amp;ndash;HCl, pH 8.5, 15&amp;nbsp;% polyvinyl pyrrolidone, 153&amp;nbsp;&amp;micro;M MgSO4&amp;nbsp;and 0.2&amp;nbsp;% Triton X-100</td></tr><tr><td>chloroform:methanol</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>glyceryl tripalmitate</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>amyloglucosidase</td><td>Sigma Aldrich</td><td>A7420</td><td></td></tr><tr><td>glucose assay reagent</td><td>Sigma Aldrich</td><td>G3293</td><td></td></tr><tr><td>Biorad protein dye</td><td>VWR</td><td></td><td></td></tr><tr><td>96-well microtiter plate</td><td>Greiner Bio-one</td><td></td><td></td></tr><tr><td>384 microtiter plates</td><td>Greiner Bio-one</td><td></td><td></td></tr><tr><td>2 ml glass tubes</td><td>Fiers</td><td></td><td>For fat analysis</td></tr><tr><td>2,5ml eppendorf tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>homogeniser</td><td>Ultra-turrax TP 18/10</td><td></td><td></td></tr><tr><td>photospectrometer</td><td>Infinite M200 TECAN</td><td></td><td></td></tr><tr><td>heater for glass tubes</td><td>Hach COD REACTOR</td><td></td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf Centrifuge 5415 R</td><td></td><td></td></tr><tr><td>Incubator</td><td>Bumako</td><td></td><td></td></tr></tbody></table>

protocol for acute and chronic ecotoxicity testing of the turquoise killifish nothobranchius furzeri

鳉鱼Nothobranchius furzeri 是生态领域新兴的模型有机体, 它在急性和慢性毒性试验中的适用性已经证明。总体而言, 该物种对有毒化合物的敏感性与其他模型物种的敏感程度相同。
这项工作描述了对furzeri的单一和联合应激作用的急性、慢性和几代生物鉴定的协议。由于它的成熟时间短和生命周期, 这脊椎动物模型允许研究终点, 如成熟时间和生育力在四月内。跨代全生命周期暴露试验可以在8月内进行。由于这一物种生产的鸡蛋具有抗旱性, 并且在多年内仍然可行, 所以不需要这种物种的现场养殖, 但在需要时可以招募个人。这些协议旨在测量生命历史特征 (死亡率、生长、繁殖力、体重) 和临界热最大值。

furzeri的严重暴露结果与不同的铜浓度 (如在2.5.2 中计算) 显示 cleardose 响应关系 (图 1)。随着毒物浓度的增加, 死亡率增加。LC50值随着时间的推移而减少, 这意味着随着浓度的降低, 50% 的复制死前会有更多的时间通过。有关furzeri对铜的急性和慢性暴露的详细结果, 以及与其他物种相比, 该物种的灵敏度比较, 我们指的是菲利普et</...

Watch this Scientific Journal Video about Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri at JoVE.com

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

在这项工作中, 我们描述了一种急性, 慢性和几代生物测定, 以研究单一和联合压力源对绿松石鳉鱼Nothobranchiusfurzeri 的影响。本协议旨在研究生命史特征 (死亡、生长、繁殖力、体重) 和临界热最大值。

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

protocol-for-acute-chronic-ecotoxicity-testing-turquoise-killifish

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

7.4K 次观看.  University of Leuven. 在这项工作中, 我们描述了一种急性, 慢性和几代生物测定, 以研究单一和联合压力源对绿松石鳉鱼Nothobranchiusfurzeri 的影响。本协议旨在研究生命史特征 (死亡、生长、繁殖力、体重) 和临界热最大值。

视频: Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

科研

环境科学

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

<meta charset="utf8"></head><body>评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

使用计算机辅助视频和数字图像分析量化鱼游泳行为应对水铜的急性接触

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

神经科学

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

生物学

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

摘要

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在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估