纳米材料和其他难物质藻类毒性试验的小型装置
Summary
我们使用使用 LED 垂直照明的装置，演示对困难物质（如彩色物质或纳米材料）的藻类毒性测试。
Abstract
生态毒性数据是欧洲和国际法规（例如 REACH）对化学品进行上市前和上市后注册的要求。藻类毒性试验经常用于化学品的法规风险评估。为了实现高可靠性和可重复性，制定标准化准则至关重要。对于藻类毒性测试，指南要求参数条件稳定且统一，如pH值、温度、二氧化碳水平和光强度。纳米材料和其他所谓的困难物质会干扰光，导致结果变化很大，妨碍了其监管的接受。为了应对这些挑战，我们开发了LEVITATT（藻类毒性试验LED垂直照明表）。该设置利用下面的 LED 照明，实现均匀的光分布和温度控制，同时最大限度地减少样品内阴影。该设置优化了生物量定量的样本量，同时确保 CO2 的充分流入，以支持藻类的指数级增长。此外，可定制测试容器的材料，以最大限度地减少吸附和挥发。在测试彩色物质或颗粒悬浮液时，LED 灯的使用还允许增加光强，而无需额外产生热量。紧凑的设计和最小的设备要求增加了在广泛的实验室中实施 LEVITATT 的可能性。在符合标准化ISO和经合组织藻类毒性测试指南的同时，LEVITATT还显示出两种参考物质（3，5-5-Dicholorophenol和K2Cr2O7）和三种纳米材料（ZnO、CeO2和BaSO4）的样品间变异性低于埃伦迈尔烧瓶和微蒂特板。
Introduction
藻类毒性试验是用于生成欧洲和国际法规（例如 REACH 1 和 TSCA（美国））对化学品上市前和上市后注册所需的生态毒性数据所需的三种强制性测试之一。为此，国际组织（如ISO和经合组织）制定了标准化藻类测试准则。这些测试标准和准则在 pH、温度、二氧化碳水平和光强方面规定了理想的测试条件。然而，在藻类试验期间保持稳定的测试条件实际上是困难的，结果存在一系列化学物质和纳米材料（通常称为”困难物质”）的可重复性和可靠性问题2。大多数现有的藻类毒性测试装置都位于孵化器内的轨道摇床上，其体积相对较大（100–250 mL）。这种设置限制了测试浓度和可复制的海藻培养和测试材料的数量。此外，这些设置很少具有均匀的光场，在大型烧瓶中还难以获得可靠的照明条件，部分原因是光强度在光传播得更远时呈指数级下降，部分由于烧瓶几何形状。替代设置包括塑料微刺激器3板，包含小样本量，不允许足够的采样量来测量 pH 值、额外的生物量测量、颜料提取或其他需要破坏性采样的分析。利用现有的纳米材料和形成彩色悬浮物的物质的藻类毒性测试的一个特别挑战是干扰或阻挡藻类细胞可用的光，通常被称为”阴影“4，5。,5测试材料和/或测试材料与藻类细胞之间的相互作用可能发生小瓶内的阴影，或者由于小瓶相对于彼此和光源的定位，在小瓶之间可能发生阴影。
该方法基于阿伦斯伯格等人提出的 小规模藻类毒性测试装置，允许按照经合组织2017和ISO 86928等标准进行测试。该方法得到进一步优化，以解决上述限制：1） 利用 LED 光技术确保均匀的光条件，产生最少的热量;2） 在保持恒定 pH、CO 2 水平的同时，为化学/生物分析提供足够的样品量;3） 允许使用多功能测试容器材料来测试挥发性物质或具有高吸附潜力的物质。
Protocol
1. 莱维特设置的描述 使用20 mL闪烁玻璃瓶（图1，插入1），允许光线穿透。或者，可以使用轻的可塑胶瓶。使用光度计量化光强。 在测试开始时至少使用 4 mL 测试悬浮液，以便对生物量进行定量，并在孵化期间和之后对纳米材料进行表征/定量（图 1，插入 2）。 将 20 mL 闪烁小瓶与盖（图1，插入 3）配合，其…
Representative Results
对参考物质进行初始测试，以确定藻类菌株的敏感性。经常用于R. 亚卡皮塔的参考物质是二氯酸钾和 3，5-二氯酚7，,8。图 3 和表 2显示了藻类测试的代表性结果，包括曲线拟合和统计输出，当 DRC 包装在 R 中应用于增长率时。 <img alt="Figure 3" class="xfigimg" src="/files/ftp_u…
Discussion
浮游植物将太阳能和二氧化碳转化为有机物，因此在水生生态系统中起着关键作用。因此，藻类生长率抑制试验是化学品监管风险评估所需的三项强制性水生毒性试验之一。在这方面，进行可靠和可重复的藻类毒性测试的能力是关键。使用 Erlenmeyer 烧瓶的测试设置引入了一系列变化和不便，如简介 中所述。为了规避这个问题，已经提出了微刺激板3。虽然微刺激板最大限度地减?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究由S前-纳米安全测试先进工具资助，根据Horizon 2020研究和创新计划授予协议760813。
Materials
| Acetone | Sigma-Aldrich | V179124 | |
| Ammonium chloride | Sigma-Aldrich | 254134 | |
| BlueCap bottles (1L) | Buch & Holm A/S | 9072335 | |
| Boric acid | Sigma-Aldrich | B0394 | |
| Calcium chloride dihydrate | Sigma-Aldrich | 208290 | |
| Clear acrylic sheet (40×40 cm) | |||
| Cobalt(II) chloride hexahydrate | Sigma-Aldrich | 255599 | |
| Copper(II) chloride dihydrate | Sigma-Aldrich | 307483 | |
| Ethylenediaminetetraacetic acid disodium salt dihydrate | Sigma-Aldrich | E5134 | |
| Fluorescence Spectrophotometer F-7000 | Hitachi | ||
| Hydrochloric acid | Sigma-Aldrich | 258148 | |
| Iron(III) chloride hexahydrate | Sigma-Aldrich | 236489 | |
| LED light source | Helmholt Elektronik A/S | H35161 | Neutral White, 6500K |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M9272 | |
| Magnesium sulfate heptahydrate | Sigma-Aldrich | 230391 | |
| Manganese(II) chloride tetrahydrate | Sigma-Aldrich | 221279 | |
| Orbital shaker | IKA | 2980200 | |
| Potassium phosphate monobasic | Sigma-Aldrich | P0662 | |
| Raphidocelis subcapitata | NORCCA | NIVA-CHL1 strain | |
| Scintillation vials (20 mL) | Fisherscientific | 11526325 | |
| Sodium bicarbonate | Sigma-Aldrich | S6014 | |
| Sodium hydroxide | Sigma-Aldrich | 415413 | |
| Sodium molybdate dihydrate | Sigma-Aldrich | 331058 | |
| Spring clamp | Frederiksen Scientific A/S | 472002 | |
| Thermostatic cabinet | VWR | WTWA208450 | Alternative: temperature controlled room |
| Ventilation pipe (Ø125 mm) | Silvan | 22605630165 | |
| Volumetric flasks (25 mL) | DWK Life Sciences | 246781455 | |
| Zinc chloride | Sigma-Aldrich | 208086 |
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Cite This Article
Skjolding, L. M., Kruse, S., Sørensen, S. N., Hjorth, R., Baun, A. A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances. J. Vis. Exp. (164), e61209, doi:10.3791/61209 (2020).