## 2 Materials and Methods
### Roof Runoff Sample Analysis  
The roof runoff samples collected from downpipes of roof gutters were collected into 250 mL vessels, for the purpose of cation (with acidification with 10 \(\upmu\)L of concentrated HNO\({}_{3}\)), anion (without acidification), and acid digestion analyses. Regarding microbial analysis, the samples of roof runoff were collected in sterile 250 mL glass bottles. The values of pH and conductivity were measured _in situ_ in non-acidified roof runoff samples, using a Thermo Scientific Orion Star A329 Portable Multiparameter Meter, in accordance to the ISO 10523:2008 [46] and ISO 7888:1985 [47] protocols. The roof runoff samples were transported to the laboratory and immediately subjected to further procedures. Back in the laboratory, the samples were filtered by 0.45 \(\upmu\)m membrane filters and stored at 4 \({}^{\circ}\)C before further analysis. The concentrations of nitrate nitrogen (N-NO\({}_{3}\)) and phosphate phosphorus (P-PO\({}_{4}\)) were determined by the spectrometric method, using spectrophotometer UV Vis Camspec M501, based on the ISO 13395:1996 [48] and ISO 6878:2004 [49] protocols, respectively. The contents of chlorides were determined by titration--Mohr's method according to the ISO 9297:1989 protocol [50]. The acid digestion of the roof runoff samples was conducted in a closed vessel device, using temperature control and a microwave oven (Milestone ETHOS lab station), according to United States Environmental Protection Agency (US EPA) Method 3015A [51]. A total of 5 mL of 65% HNO\({}_{3}\) was added to 45 mL of each roof runoff sample, then samples were digested in a microwave oven, according to a 2-step program: 1 st (10 min, up to 160 \({}^{\circ}\)C) and 2 nd (10 min, up to 165 \({}^{\circ}\)C), with 1000 Watts in each step. The concentrations  
Figure 1: Sampling area: A—location of Kraków city at the map of Poland; B—sampling sites (KR1-KR5, Mogilany (MOG)) on the map of Kraków (four main districts in the city are marked with different shades of grey).  
of the analyzed cations, without and after acid digestion, were determined in the roof runoff samples, using an inductively coupled plasma-optical emission spectroscopy (ICP-OES) emission spectrometer, with inductive coupled plasma (Plasma 40 Perkin Elmer) and an ICP-MS mass spectrometer (ELAN 6100 Perkin Elmer), based on the ISO 11885:2007 [52] and ISO 17294-2:2016 [53] protocols, respectively.  
Quantitative assessments of bacteria and fungi presence in roof runoff were conducted with the serial dilution method, complemented by the Petri dish streaking and spreading according to ISO 6222:1999 [54] and ISO 8199:2018 [55] protocols. The roof runoff samples were diluted ten-fold with physiological saline (10\({}^{-1}\), 10\({}^{-2}\), 10\({}^{-3}\), and 10\({}^{-4}\)), then 0.1 mL of each dilution was transferred to nutrient agar plates (BTL, Poland) for bacteria and to word agar plates (BTL, Poland) for fungi, and finally precisely spread on media surface, with a sterile spreader. All plates were incubated at room temperature (22 \({}^{\circ}\)C) until complete colonies were formed (if no new colonies were observed during the following three days, incubation was recognized to be completed). Then, the colonies observed at each plate were calculated, with regard to the recommendations of [PERSON] et al. [56] and the _Bacteriological Analytical Manual_ of the United States Food and Drug Administration [57]. If confluent growth was observed, or the number of colonies did not fall within the range of 25-250, the data were not taken into account. Finally, the results obtained for particular samples were averaged and converted into colony-forming units (CFU) of either bacteria, or fungi, respectively, as existing in 1 mL of roof runoff. All parameters analyzed and the analytical methods applied are gathered in Table S1.