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This study aims at assessing the effect of limestone mining by-product from the Irati Formation on soil calcium, magnesium, and sulfur contents, as well as their accumulation in the onion biomass. The study was carried out in a greenhouse. A Eutrophic Haplic Planosol (SXe) and a Eutrophic Yellow Argisol (PAe) soil were used. The treatments included the control, limestone by-product, dolomitic limestone, calcium (Ca) + magnesium (Mg) carbonates, and Ca + Mg carbonates + gypsum. Onions were grown in pots containing 10 dm3 of soil kept at a moisture level close to the field capacity. The onion biomass harvest (bulb + leaves) was performed 77 days after transplanting. Afterwards, the soil macronutrient content and the dry onion biomass were determined, as well as its production. The by-product application increased Mg2+ content in both soils (+74% in SXe and + 182% in PAe) and Ca2+ and sulfate (S-SO42-) contents, respectively, in the PAe (+62%) and SXe (+124%). There was an increase in S accumulation due to the onion biomass cultivated in both soils (about 85%). Regarding Ca and Mg accumulations, no difference was observed in the by-product treatment when compared to the control. Therefore, it is possible to infer that the limestone mining by-product from the Irati Formation is potentially useful as a source of secondary macronutrients and proves to be a clean environmental solution for soil fertilization, increasing the sustainability of the local production system. [ABSTRACT FROM AUTHOR]