title: A case study to optimise and validate the brine shrimp Artemia franciscana immobilisation assay with silver nanoparticles: The role of harmonisation.
abstract: Brine shrimp Artemia sp. has been recognised as an important ecotoxicity and nanotoxicity test model organism for salt-rich aquatic environments, but currently there is still no harmonised testing protocol which would ensure the comparable results for hazard identification. In this paper we aimed to design the harmonised protocol for nanomaterial toxicity testing using Artemia franciscana and present a case study to validate the protocol with silver nanoparticles (AgNPs). We (i) revised the existing nanotoxicity test protocols with Artemia sp. (ii) optimised certain methodological steps based on the experiments with AgNPs and potassium dichromate (K2Cr2O7) as a soluble reference chemical and (iii) tested the optimised protocol in an international inter-laboratory exercise conducted within the EU FP7 NanoValid project. The intra- and inter-laboratory reproducibility of the proposed protocol with a soluble reference chemical K2Cr2O7 was good, which confirms the suitability of this assay for conventional chemicals. However, the variability of AgNPs toxicity results was very high showing again that nanomaterials are inherently challenging for toxicity studies, especially those which toxic effect is linked to shed metal ions. Among the identified sources for this variability were: the hatching conditions, the type of test plate incubation and the illumination regime. The latter induced variations assumingly due to the changes in bioavailable silver species concentrations. Up to our knowledge this is the first inter-laboratory comparison of the Artemia sp. toxicity study involving nanomaterials. Although the inter-laboratory exercise revealed poor repeatability of AgNPs toxicity results, this study provides valuable information regarding the importance of harmonisation of all steps in the test procedure. Also, the presented AgNPs toxicity case study may serve as a platform for further validation steps with other types of NMs.
authors.affiliation: Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia. Electronic address: monika.kos@bf.uni-lj.si.
authors.forename: Monika
authors.initials: M
authors.lastname: Kos
authors.affiliation: National Institute of Chemical Physics and Biophysics, Laboratory of Environmental Toxicology, Akadeemia tee 23, 12618 Tallinn, Estonia. Electronic address: anne.kahru@kbfi.ee.
authors.forename: Anne
authors.initials: A
authors.lastname: Kahru
authors.affiliation: Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia. Electronic address: damjana.drobne@bf.uni-lj.si.
authors.forename: Damjana
authors.initials: D
authors.lastname: Drobne
authors.affiliation: The Centre for Cellular & Molecular Biology, Habsiguda, Hyderabad, Telangana 500007, India. Electronic address: shashis@ccmb.res.in.
authors.forename: Shashi
authors.initials: S
authors.lastname: Singh
authors.affiliation: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia. Electronic address: gabriela.kalcikova@fkkt.uni-lj.si.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Kalčíková
authors.affiliation: Helmholtz-Centre for Environmental Research - UFZ, Department of Bioanalytical Ecotoxicology, Permoserstr. 15, 04318 Leipzig, Germany. Electronic address: dana.kuehnel@ufz.de.
authors.forename: Dana
authors.initials: D
authors.lastname: Kühnel
authors.affiliation: The Centre for Cellular & Molecular Biology, Habsiguda, Hyderabad, Telangana 500007, India. Electronic address: r@ccmb.res.in.
authors.forename: Rekulapelly
authors.initials: R
authors.lastname: Rohit
authors.affiliation: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia. Electronic address: andreja.zgajnar@fkkt.uni-lj.si.
authors.forename: Andreja Žgajnar
authors.initials: AŽ
authors.lastname: Gotvajn
authors.affiliation: Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia. Electronic address: anita.jemec@bf.uni-lj.si.
authors.forename: Anita
authors.initials: A
authors.lastname: Jemec
mesh_terms: D000818:Animals; D001156:Artemia / Q000187:drug effects*; D001681:Biological Assay / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects*; D053758:Nanoparticles / Q000737:chemistry* / Q000633:toxicity*; D015203:Reproducibility of Results; D012834:Silver / Q000737:chemistry* / Q000633:toxicity*; D018675:Toxicity Tests / Q000379:methods
chemical_list: D012834:Silver
keywords: Ag speciation; FP7 NanoValid; Illumination regime; Inter-laboratory exercise; Marine environment; Risk assessment