Assay conditions matter: plate material and pH alter lithium responses in zebrafish embryos

Lithium demand is increasing due to its widespread use in rechargeable batteries, raising concerns about its release into aquatic environments. The bioavailability and toxicity of lithium under aquatic test conditions can be influenced by water chemistry and experimental conditions such as pH and exposure vessel material. This study investigated effects of plate material and pH on lithium concentrations and developmental, teratogenic, and behavioural responses in zebrafish embryos based on the OECD Test Guideline 236 - Fish Embryo Toxicity Test. Experiments were conducted in polystyrene and polypropylene plates under unmodified (pH 7.6) and acidified conditions (pH 6). Additionally, lithium speciation modelling was performed to evaluate potential changes in chemical speciation. No developmental and teratogenic changes were observed in exposed larvae. However, at higher target concentrations, measured lithium recoveries were higher in polypropylene plates, which was potentially attributed to higher inertness of polypropylene surfaces. Gene expression analysis further revealed material-dependent molecular responses, suggesting biological responses at the transcriptional level at low concentrations. Behavioural analyses showed reduced locomotor response at higher lithium concentrations, with stronger effects at pH 6. Speciation modelling demonstrated that lithium species distribution predominantly remained the same under all tested conditions, indicating that pH-related effects were unlikely driven by changes in lithium speciation. Protein-like compounds were formed under all experimental conditions, independent of plate material and pH, hinting towards in situ production and a limited role in binding large lithium fractions. Overall, our results show that experimental material and pH influence toxicity test outcomes and should therefore be carefully considered when designing aquatic toxicity tests.