**Background:** Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that accumulate in soil and pose carcinogenic risks. Microbial degradation is a key remediation pathway, influenced by nutrient availability, soil pH, organic carbon content, and microbial community composition. The effect of long-term combined organic and mineral fertilization on PAH dynamics in soil remains poorly understood.
**Methods:** Soil samples were collected in 2015 from a long-term field experiment established in 1986 in Bałcyny, Poland, on Haplic Luvisols developed from sandy loam. The experiment compared eight mineral fertilization regimes (N₀P₀K₀ through N₂P₁K₂MgCa) applied with or without manure (40 t ha⁻¹ every other year). Spring barley was grown in the eighth crop rotation cycle (sugar beets, spring barley, maize, spring wheat). Soil was sampled at 0–30 cm depth on four dates: 22 April (BBCH-10), 18 May (BBCH-23), 8 August (after harvest), and 15 September (after skimming). The 16 US EPA priority PAHs were quantified by GC/MS. Microbial counts were determined by serial dilution plating on selective media. Enzymatic activities (dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase) were measured spectrophotometrically. Statistical analyses included repeated measures ANOVA, Kruskal-Wallis tests, and principal component analysis (PCA).
**Key Results:** Total PAH content varied significantly across sampling dates (p < 0.05): highest in May (484.6 µg kg⁻¹) and lowest in August (194.8 µg kg⁻¹). LMW PAHs followed the same pattern (May: 384.7 µg kg⁻¹; August: 119.8 µg kg⁻¹), while HMW PAHs peaked in September (158.3 µg kg⁻¹) and were lowest in August (75.0 µg kg⁻¹). Manure-amended soil had higher total PAHs than mineral-only soil across all dates. Manure increased organic carbon and total nitrogen content, and boosted microbial counts: organotrophic bacteria (1.7-fold), ammonifying bacteria (1.4-fold), nitrogen-fixing bacteria (1.4-fold), actinobacteria (2-fold), and fungi (32%). Enzymatic activity was also higher with manure: dehydrogenases (1.8-fold), catalase (17% increase), acid phosphatase (1.7-fold), and alkaline phosphatase (2.3-fold). Rising nitrogen rates (up to 90 kg ha⁻¹) increased organotrophic bacteria (1.4-fold) and acid phosphatase activity (20%) but suppressed dehydrogenases (8% decrease at 30 kg N) and alkaline phosphatase (13% decrease at 90 kg N). Liming increased soil pH, reduced hydrolytic acidity, and enhanced catalase (1.4-fold), urease, and alkaline phosphatase (2-fold) activities. PCA revealed that the first two principal components explained 65% (April), 58% (May), 59% (August), and 71% (September) of total variance, with strong correlations between organic carbon, total nitrogen, microbial counts, enzymatic activities, and PAH content.
**Clinical Implications:** This study provides evidence that long-term manure application, while beneficial for soil fertility and microbial/enzymatic activity, can increase PAH accumulation in agricultural soil. The total PAH concentrations remained below the IUNG threshold for non-contaminated soil (<600 µg kg⁻¹), suggesting that balanced fertilization does not necessarily lead to hazardous contamination. The seasonal variation in PAH levels, driven by weather and microbial activity, underscores the importance of timing in soil monitoring and remediation strategies. These findings inform agricultural practices aimed at optimizing soil health while minimizing persistent organic pollutant accumulation.