title: A new, sensitive marine microalgal recombinant biosensor using luminescence monitoring for toxicity testing of antifouling biocides.
abstract: In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC(50)) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.
authors.affiliation: Université Pierre et Marie Curie Paris 06, UMR 7621 Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls sur Mer, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Sanchez-Ferandin
authors.forename: Fanny
authors.initials: F
authors.lastname: Leroy
authors.forename: François-Yves
authors.initials: FY
authors.lastname: Bouget
authors.forename: Fabien
authors.initials: F
authors.lastname: Joux
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D000460:Chlorophyta / Q000235:genetics* / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004202:Disinfectants / Q000032:analysis*; D004237:Diuron / Q000032:analysis; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D049449:Luminescence; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012623:Seawater / Q000737:chemistry*; D014227:Triazines / Q000032:analysis; D014873:Water Pollutants / Q000032:analysis*
chemical_list: D004202:Disinfectants; D011993:Recombinant Fusion Proteins; D014227:Triazines; D014873:Water Pollutants; C103341:irgarol 1051; D004237:Diuron; D008156:Luciferases; D018844:Cyclin-Dependent Kinases