Impact of plant protection product applications on soil microbial nitrogen cycle function not fully captured by gene quantification

The widespread use of plant protection products (PPPs) in agriculture raises concerns about their long-term impact on soil health and nitrogen (N) cycling. Current regulatory assessments focus mostly on single active ingredients and microbial mineralisation, ignoring the complexities of formulated PPPs and their influence on microbial functions. We investigated the effects of realistic PPP application scenarios on soil N cycling using a controlled incubation experiment with increasing PPP intensities, measuring potential nitrification (PN), denitrifying enzyme activity (DEA), and N₂O reduction capacity (NRC), alongside molecular analyses of key microbial genes involved in N-cycling. Functional assays were more sensitive to PPP exposure than gene abundances, indicating severe disruptions to N cycling. Among measured processes, PN was the most PPP-sensitive, showing substantial reductions across treatments. DEA and NRC were also strongly inhibited, exhibiting complex temporal patterns. While gene abundances were less responsive, there were significant positive correlations between the gene abundance of archaeal and bacterial ammonia monooxygenase (amoA) and PN, as well as between nitrite reductase (nirK) and DEA. Our findings underscore the importance of updated risk assessments that integrate both molecular and functional indicators. We propose a tiered approach, using gene quantification as an initial screening tool, followed by functional assays to capture biologically relevant changes. Post-registration monitoring of PPP mixtures under field conditions is likewise essential to address cumulative and long-term impacts. Overall, this study highlights the vulnerability of soil N cycling to PPP exposure and provides a framework to enhance environmental risk assessments aimed at safeguarding soil ecosystem functions.