## 3 Results and Discussion
### Changes in Soil Cu
A similar range of Cu release was also measured in calcareous organic soil (44.37 mg kg\({}^{-1}\), 33.5%), but a much lower concentration was measured in the calcareous conventional soil (16.59 mg kg\({}^{-1}\), 15.7%), which was sampled in the Vipava Valley wine region. Trace metal elements may be incorporated in many forms of organic matter, including living organisms, organic coatings on inorganic particles, and biotic detritus [62]; this organic material tends to be degraded, leading to the release of sorbed metals. The amount of Cu released in our study from the oxidizable step (Step 3) was generally in the same range of previously published results, where extraction percentages ranged from 29.2 to 51.3% for most samples [19; 63; 64]. In our case, the largest Cu release from Step 3 was measured in acidic conventional soils (39.30 mg kg\({}^{-1}\), 27.4%) and the lowest was measured in calcareous conventional soils (13.37 mg kg\({}^{-1}\), 11.5%). Similar results were already reported by Alva and colleges [65], where the major total Cu release was for the low-pH soils. Regarding the residual fraction (Step 4), a larger proportion was found in the calcareous conventional soil (66.0% as mean value) than acidic organic, calcareous organic, and acidic conventional, with 43.1, 42.5, and 35.7%, respectively. The reason why most of the metals in our study were bound to the residual fraction (Step 4) (crystalline lattice of primary and secondary minerals) is due to the specific distribution of fungicide-derived Cu in soil aggregates after application (ageing processes). In the first 8 weeks after the application of Cu oxychloride [66], the metal ions in the soil transferred from the soil outer sphere (from available Step 1 fraction) to the soil inner sphere (to the Fe-Mg oxides, Step 2 fraction). Later, the metal ions from Cu oxychloride could form highly stable complexes with organic matter [67], and, finally, after 15 months of soil incubation of Cu oxychloride in soil aggregates, Cu ions were mainly bound to the Fe-Mn oxyhydroxic surface and the crystalline structure of the primary and secondary minerals [21]. This means that most of the total Cu measured in our soils was of older origin and therefore bound to the less available soil fraction. In fact, according to the latest report (from 2017), Cu oxychloride and Cu hydroxide are the most commonly used chemical forms of Cu-based plant protection products in Slovenian viticulture (9.3 t y\({}^{-1}\)) [68]. The results of the sequential extraction procedure (SEP) reveal that there is also an obvious difference in Cu fractionation between the sampling sites of Stajerska and Vipava Valley. Although we decided to take samples based on similar chemical properties of the soils (level of Cu, TOC, and pH) when selecting the sampling sites at the beginning of the study, the results of the Cu fractionation show a clear influence of the sampling site. However, the incubations generally resulted in a statistically significant reduction in Cu released in the first step (Step 1) of the sequential extraction compared to soil samples before treatment (\(p<0.05\)), but there were no statistical differences between the different climate change simulations (moisture, temperature, or combined treatments) (Table 2 and Figure S2). The binding of Cu to the different soil fractions did not change as consequence of both temperature or drought stresses in both conventional and organic vineyard soils, or that changes were too small for the variability of the analytical method to become statistically significant. The only exception was for Step 3 (oxidizable) in the calcareous organic soil, where all treatments, in particular the drought stress, caused a significant reduction of extractable Cu.