Exploring the impact of silver-based nanomaterial feed additives on green algae through single-cell techniques

Silver in its various forms, including dissolved silver ions (Ag⁺) and silver nanoparticles (AgNPs), is a promising alternative to traditional antibiotics, largely used in livestock as feed additives and could contribute to the decrease and avoidance of the development of antibiotic resistance. The present study aims to assess the potential ecotoxicity of a silver-based nanomaterial (Ag-kaolin), the feed supplemented with the nanomaterial and the faeces since the latter are the ones that finally reach the environment. To this end, green alga Raphidocellis subcapitata was exposed to the extracts of Ag-kaolin, supplemented feed, and pig faeces for 72 h, along with Ag⁺ and AgNPs as controls for comparison purposes. Given the complexity of the studied materials, single-cell techniques were used to follow the changes in the cell numbers and chlorophyll fluorescence by flow cytometry, and the accumulation of silver in the exposed cells by single cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Changes in cell morphology were observed by cell imaging multimode reader. The results revealed a decrease in chlorophyll fluorescence, even at low concentrations of Ag-kaolin (10 μg L^-1) after 48 h of exposure. Additionally, complete growth inhibition was found with this material like the results obtained by exposure to Ag⁺. For the supplemented feed, a concentration of 50 μg L^-1 was necessary to achieve complete growth inhibition. However, the behaviour differed for the leachate of faeces, which released Ag₂S and AgCl alongside Ag⁺ and AgNPs. At 50 μg L^-1, inhibition was minimal, primarily due to the predominance of less toxic Ag₂S in the leachate. The uptake of silver by the cells was confirmed with all the samples through SC-ICP-MS analysis. These findings demonstrate that the use of Ag-kaolin as a feed supplement will lead to a low environmental impact.