The effect of a reduction of nitrogen and phosphorus to eutrophic and mesotrophic conditions towards silver nanoparticle (AgNP) on freshwater green algae species

We investigated the sensitivity of algae towards silver nanoparticles with OECD test medium and lower nutrient concentrations under standard test conditions to improve comparability and to exclude any other confounding factor aside nutrient levels. Two unicellular freshwater microalgae Desmodesmus subspicatus and Raphidocelis subcapitata were chosen due to their status as standard test organisms for the algae growth inhibition test and the response to changes in nutrient supply was compared. The original medium was used as the reference (standard). For the other four media, the amount of either nitrogen or phosphorus in the medium was lowered from half (50%) to one-fourth (25 %) of that of the OECD guideline, resulting in the following media: 50% N, 25% N, 50% P and 25% P medium. As test substance, the OECD reference material NM-300K was used. For this reason, the characterization of AgNP was done using DLS and Absorption spectra (UV/vis). Actual silver concentrations and ionic silver concentrations were measured at the highest test concentration used (100 µg Ag L-1) in R. subcapitata treatments only to reduce the number of samples. All tests were run according to the OECD guideline 201 with sterilized 50 mL cell culture flask. Each medium was tested using the test conditions for culturing with 3 replicates. Test concentrations for both algae species were 0, 25, 50 and 100 µg Ag L-1 for OECD, 50% P and 25% P while for both N reductions, the silver concentrations were 0, 10, 25 and 100 µg Ag L-1. Samples for determining the algal density were taken at every 24 h.

本研究在标准试验条件下，采用OECD试验培养基（OECD test medium）并设置梯度降低的营养盐浓度，探究藻类对银纳米颗粒（silver nanoparticles, AgNPs）的敏感性，旨在提升试验结果的可比性，并排除除营养盐水平外的其他混杂因素干扰。本研究选取两种淡水单细胞微藻：斜生栅藻（*Desmodesmus subspicatus*）与羊角月牙藻（*Raphidocelis subcapitata*），二者均为藻类生长抑制试验的标准测试生物，以此对比二者对营养盐供给变化的响应。以原始OECD培养基作为对照（标准组）。其余四种培养基则分别将培养基中的氮（Nitrogen, N）或磷（Phosphorus, P）含量调整为OECD指南标准值的50%或25%，最终得到四种试验培养基：50%氮（50% N）、25%氮（25% N）、50%磷（50% P）及25%磷（25% P）培养基。本试验采用OECD标准参考物质NM-300K作为受试物。基于此，本研究采用动态光散射（Dynamic Light Scattering, DLS）与紫外-可见吸收光谱（UV/vis）对银纳米颗粒进行表征。为减少样本量，仅在最高试验浓度（100 µg Ag L⁻¹）下，对羊角月牙藻（*R. subcapitata*）处理组的总银浓度与离子银浓度进行了测定。所有试验均参照OECD 201号指南开展，采用灭菌后的50 mL细胞培养瓶进行培养。每种培养基均设置3次生物学重复，采用藻类培养的标准试验条件进行测试。针对OECD标准培养基、50%磷及25%磷培养基，两种藻类的受试银浓度均设置为0、25、50及100 µg Ag L⁻¹；而针对两种氮浓度降低的培养基，受试银浓度则设置为0、10、25及100 µg Ag L⁻¹。每24小时采集一次藻样，用于测定藻细胞密度。