Ifremer, Laboratoire d' Écotoxicologie, rue de l'île d'Yeu, BP 21105, 44311 Nantes cedex 03, France.
Aquat Toxicol. 2013 Sep 15;140-141:380-8. doi: 10.1016/j.aquatox.2013.06.016. Epub 2013 Jul 13.
To investigate the ability of microalgae to develop stable, long-term resistance to herbicides, the marine microalga Tetraselmis suecica was exposed to the herbicide diuron (5 μg/L) for a 43-generation exposure period followed by a 12-generation depuration phase. During the first 25 generations, diuron-exposed cultures showed doubling times ranging from 1.95 to 2.6 days, which was 2 to 2.5-fold longer than control cultures. Between generations 25 and 38, during diuron exposure, two out of the three exposed cultures exhibited a spontaneous drop in doubling time. These results provided evidence of culture adaptation to diuron. To assess persistence of the diuron adaptation observed on growth performance, one of the adapted cultures (D3) was maintained for 12 months in unexposed conditions and then tested by a second, short-term exposure to diuron 5 μg/L, in parallel with a control culture (C1) for six generations. Flow cytometry analyses were used to monitor cell density, viability, morphology, relative chlorophyll content and intracellular reactive oxygen species (ROS) level. Under these conditions, diuron induced a strong increase of doubling time in exposed-C1 cultures (2.5-fold longer than unexposed-C1 cultures), but no significant increase occurred in exposed D3-cultures compared with unexposed D3- and unexposed C1-cultures, showing the persistence of adaptation in the previously-exposed strain D3. Intracellular ROS level showed the same trend. Significant differences were observed between these strains, with weaker effects of diuron on strain D3 compared with strain C1: forward scatter (FSC), representing relative cell size, decreased in exposed cultures (67.8% and 95% of the controls for C1 and D3, respectively), whereas FL3 as relative chlorophyll content increased in exposed cultures (115.6% and 108.6% of the controls for C1 and D3, respectively). Results of second exposure to diuron revealed that the adaptation of strain D3 had persisted after 12 months of depuration, as no growth impairment was observed. This study demonstrates the possible appearance of stable diuron resistance in microalgae in cases of strong, multigenerational chronic exposure to this herbicide in polluted environments.
为了研究微藻产生稳定、长期抗除草剂能力，将海洋微藻 Tetraselmis suecica 暴露于除草剂敌草隆（5μg/L）中进行了 43 代的暴露期，随后进行了 12 代的净化阶段。在最初的 25 代中，暴露于敌草隆的培养物的倍增时间范围为 1.95 至 2.6 天，比对照培养物长 2 至 2.5 倍。在 25 至 38 代之间，在暴露于敌草隆期间，三个暴露培养物中有两个表现出倍增时间的自发下降。这些结果提供了培养物适应敌草隆的证据。为了评估在生长性能上观察到的敌草隆适应的持久性，将一个适应培养物（D3）在未暴露的条件下维持了 12 个月，然后在与对照培养物（C1）平行进行了第二次短期暴露于 5μg/L 的敌草隆，进行了六代。流式细胞术分析用于监测细胞密度、活力、形态、相对叶绿素含量和细胞内活性氧物种（ROS）水平。在这些条件下，敌草隆诱导暴露于 C1 的培养物倍增时间明显延长（比未暴露于 C1 的培养物长 2.5 倍），但暴露于 D3 的培养物与未暴露于 D3 和未暴露于 C1 的培养物相比，没有明显增加，表明先前暴露的 D3 菌株的适应持久性。细胞内 ROS 水平也呈现出相同的趋势。这些菌株之间存在显著差异，与菌株 C1 相比，敌草隆对菌株 D3 的影响较弱：前向散射（FSC），代表相对细胞大小，在暴露于培养物中的降低（C1 和 D3 的对照分别为 67.8%和 95%），而 FL3 作为相对叶绿素含量在暴露于培养物中增加（C1 和 D3 的对照分别为 115.6%和 108.6%）。第二次暴露于敌草隆的结果表明，在净化 12 个月后，D3 菌株的适应仍然存在，因为没有观察到生长受损。这项研究表明，在污染环境中强烈、多代慢性暴露于这种除草剂的情况下，微藻可能会出现稳定的敌草隆抗性。
