Soil δ13C, δ15N and δ34S values are shaped by nutrient interactions rather than phosphorus alone in an 82-year fertilized grassland

Natural isotope ratios (δ 13 C, δ 15 N, and δ 34 S) are effective indicators of soil element cycling, yet the role of phosphorus (P) in regulating their variation under long-term fertilization remains unclear. Therefore, we compared an unfertilized Control, non-P fertilized plots: receiving Ca (as lime), CaN and P-fertilized plots: CaNP, CaNPKCl, and CaNPK 2 SO 4 in a meadow grassland (Rengen, Germany). Topsoil (0–10 cm) C:N:P:S range varied in line with fertilization, i.e., 109:8:1:1 (Control) to 45:4:2:1 (CaNPK 2 SO 4 ). In the topsoil, P-fertilized plots exhibited notably lower C:nutrient ratios than the Control. In contrast, all nutrient-addition treatments significantly enriched soil δ 13 C and δ 15 N compared to the Ca treatment, indicating accelerated organic matter C and N turnover. Whereas isotopic responses in the subsoil were weaker. By contrast, δ 34 S changed little across treatments except under CaNPK 2 SO 4 , suggesting a direct fertilizer effect. Across both depths, strong correlations ( p < 0.01) between δ 13 C and δ 15 N indicated tightly coupled C and N cycling, whereas δ 34 S was unrelated to either isotope . Redundancy analysis explained 55.4% of isotopic variation in topsoil and 61.2% in subsoil. Marginal tests identified TP and TS as significant explanatory variables in both soil layers, whereas TN was additionally significant only in topsoil. These results indicate that isotopic variation was better explained by coupled nutrient interactions, particularly involving P and S, and in topsoil additionally N. In conclusion, nutrient management in this P-limited grassland should adopt a balanced strategy rather than focusing on phosphorus alone.