From diagnostic device to environmental contaminant: Leachate release from nano-enabled pregnancy tests and associated toxicity during early development stages of zebrafish (Danio rerio).

Single-use pregnancy test kits, which are widely used, are lateral flow assays (LFAs) that rely on gold nanomaterials (nAu) to produce a visual, rapid readout through colorimetric test lines. However, their environmental impact after disposal remains unclear. This study focused on assessing the release, physiochemical characteristics, and biological effects of nAu-containing leachates from two different pregnancy test kits with differing strip designs (dip-type, laminated vs. midstream, non-laminated, in a removable cassette). The leaching of chemicals from the strips into water was induced by placing used pregnancy test strips in water in different simulated landfill conditions (elevated temperature, low oxygen availability, and extended incubation times). The resulting leachates were characterised using UV-Vis spectroscopy, dynamic light scattering and transmission electron microscopy (TEM). Characterisation of the strips was done using attenuated total reflectance Fourier transform infrared spectroscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy. The selected LFAs released detectable amounts nAu, and visualization using TEM confirmed mean sizes of 8.2 ± 2.9 nm in PA(used) and 4.5 ± 2.0 nm for PB(used). FTIR of the strips confirmed that they are mainly composed of polymers such as polyethylene, adhesives, cellulose, and nitrocellulose. Following confirmation that the leachates contained nAu, it was important to assess the biological effects of these leachates. Zebrafish (Danio rerio) were used to conduct fish embryo acute toxicity tests in accordance with the OECD Test Guideline 236. Toxicity effects, including coagulation, oedema formation, deformities, and abnormal swimming behaviours, were significantly induced after exposure to low dose concentrations of the leachates; however, the extent of these effects varied between the LFA types. Neurobehavioral assessment using the optokinetic response assay indicated dose-dependent disrupted visual motor function in exposed larvae (96 hours post-fertilisation). These findings identify pregnancy tests as an overlooked source of contamination in aquatic environments, releasing nAu-containing leachates that can induce toxic effects and highlight the need for safe and sustainable-by-design materials.