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The guideline monitors soil microbial nitrogen transformation over 28 days, potentially missing long-term effects of persistent pesticides. Additionally, nitrate alone may be not sensitive enough to detect disruptions in microbial functions. We investigated whether functional gene analysis could provide a more sensitive bioindicator of pesticide impact. To compare this method with the standard test, we conducted a microcosm experiment following the OECD 216 experimental setup. To capture long-term effects beyond the typical test period, we extended the incubation duration to 56 days. Four different concentrations of the persistent fungicide boscalid were added based on predicted environmental concentration. We also assessed microbial responses to fungicide exposure by measuring classical soil microbial parameters. According to the standard test, boscalid had no harmful long-term effects on soil microbiota. In contrast, our analysis of functional genes found an overall reduction in the acid phosphatase-encoding <jats:italic>phoN<\/jats:italic> gene abundance on Day 56, and correspondingly, in acid phosphatase activity in the highest fungicide treatment. Simultaneously, we observed a tendency towards lower fungal abundance based on measured copy numbers of an ITS region of nuclear ribosomal DNA (rDNA) and increased cumulative CO<jats:sub>2<\/jats:sub> production. These results indicate a fungicide-related response of the microbial community and impaired microbial phosphorus cycling. Extending the experimental period to 56 days revealed long-term effects that would have otherwise been undetected under the typical 28-day test duration.<\/jats:p>","DOI":"10.1007\/s10646-025-02920-w","type":"journal-article","created":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T10:57:32Z","timestamp":1751021852000},"page":"1312-1324","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Quantification of soil microbial functional genes as potential new method in environmental risk assessment of pesticides"],"prefix":"10.1007","volume":"34","author":[{"given":"Fabian","family":"Stache","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franziska","family":"Ditterich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zuzana","family":"Hochmanov\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jakub","family":"Hofman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Poll","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen","family":"Kandeler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,6,27]]},"reference":[{"key":"2920_CR1","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/j.apsoil.2016.05.005","volume":"106","author":"A \u00c1lvarez-Mart\u00edn","year":"2016","unstructured":"\u00c1lvarez-Mart\u00edn A, Hilton SL, Bending GD, Rodr\u00edguez-Cruz MS, S\u00e1nchez-Mart\u00edn MJ (2016) Changes in activity and structure of the soil microbial community after application of azoxystrobin or pirimicarb and an organic amendment to an agricultural soil. 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