Exploring the Impacts of Antibiotics in Manure on Soil Nitrogen Dynamics and Plant Growth in Grasslands

Animal manure often contains antibiotic residues due to the prevalent use of these compounds in animal husbandry. After manure application, these residues could potentially affect soil microorganisms and plant growth, yet their impacts on soil nitrogen (N) dynamics in grasslands remain largely unexplored. We hypothesized that applying manure containing antibiotics would shift soil N dynamics by affecting plant morphology and certain N-cycling microbial guilds such as symbiotic N-fixing bacteria. To test this, we conducted a 64-day greenhouse experiment using field soil and including four plant treatments (no plants, ryegrass monoculture, clover monoculture, and a ryegrass-clover mixture) and three fertilizer treatments (antibiotic-free manure, manure containing oxytetracycline, and manure containing sulfadiazine). We measured nitrous oxide (N₂O) emissions, N content in the shoot and root biomass, and antibiotic uptake in plant shoots, and used the δ¹⁵N technique to estimate symbiotic N fixation of clover. We also sampled soils at the end of the experiment to measure plant-available N pools (ammonia and nitrate) and the abundance of symbiotic N-fixing bacteria (by nifH gene). Our results showed that antibiotics in manure did not significantly alter soil N₂O emissions, soil N pools, or plant aboveground N in any plant community. Both compounds were barely been taken up in plant shoots. However, both antibiotics significantly reduced root biomass in clover monocultures. Despite this root growth inhibition, N fixation (both aboveground and belowground) in clover monoculture was unaffected by both antibiotics. Interestingly, analysis of variance suggested that antibiotics in manure could lead to a higher abundance of nifH gene in soil than that of antibiotic-free manure in clover monoculture. In summary, although overall soil N dynamics were not impacted by antibiotics in manure, root growth inhibition in clover monoculture suggests varying grassland species susceptibilities to antibiotic stress. Our results also suggest that clover may adapt to antibiotic stress by modifying plant-microbe interactions. This study calls for further research on long-term environmental impacts of antibiotic residues in grasslands.