Behavioural toxicity assessment of silver ions and nanoparticles on zebrafish using a locomotion profiling approach

• Developmental and behavioral toxicity of Ag+ and AgNPs is comparatively studied.
• This study presents a novel locomotion profiling approach in FET framework.
• Locomotion-based behavioral endpoints are sensitive biomarkers for sub-lethal toxicity.
• Use of dose-response ranking for multiple locomotion variables is commenced.
• Multivariate statistics eases the analysis of quantitative behavioral data

Zebrafish (Danio rerio) is not only a widely used species in the Fish Embryo Toxicity (FET) test but also an emerging model in behavioural ecotoxicology. By using automatic behaviour tracking technology, locomotion of developing zebrafish (ZF) larvae can be accurately recorded and potentially used in an ecotoxicological context to detect toxicant-induced behavioural alterations. In this study, we explored if and how quantitative locomotion data can be used for sub-lethal toxicity testing within the FET framework. We exposed ZF embryos to silver ions and nanoparticles, which previously have been reported to cause neurodevelopmental toxicity and behavioural retardation in early-life stages of ZF. Exposure to a broad range of silver (Ag+ and AgNPs) concentrations was conducted, and developmental toxicity was assessed using FET criteria. For behavioural toxicity assessment, locomotion of exposed ZF eleutheroembryos (120 hpf) was quantified according to a customised behavioural assay in an automatic video tracking system. A set of repeated episodes of dark/light stimulation were used to artificially stress ZF and evoke photo-motor responses, which were consequently utilized for locomotion profiling. Our locomotion-based behaviour profiling approach consisted of (1) dose-response ranking for multiple and single locomotion variables; (2) quantitative assessment of locomotion structure; and (3) analysis of ZF responsiveness to darkness stimulation. We documented that both silver forms caused adverse effects on development and inhibited hatchability and, most importantly, altered locomotion. High Ag+ and AgNPs exposures significantly suppressed locomotion and a clear shift in locomotion towards inactivity was reported. Additionally, we noted that low, environmentally relevant Ag+ concentrations may cause subordinate locomotive changes (hyperactivity) in developing fish. Overall, it was concluded that our locomotion-based behaviour-testing scheme can be used jointly with FET and can provide endpoints for sub-lethal toxicity. When combined with multivariate data analysis, this approach facilitated new insights for handling and analysis of data generated by automatized behavioural tracking systems.