## 2 Materials and Methods
The locations of the main inlets to the LVID include the Lis River, in Leiria (Arrabalde) and the mouth of its tributaries: Milagres Stream, Aroeira Stream and Lena River; and the perimeter outlet: the Lis River section, at the Bajanca bridge (Figure 1). Sampling downstream of the Lis River was always carried out on the seashore, to avoid the influence of brackish sea water. Water samples were collected in sterile plastic  
Figure 1: Location of the sampling sites (white figures, 1–8), the rice plots (green) in Block II (green dashed line) of Lis Valley Irrigation District (Source: Google Maps, [[https://www.google.pt/maps/](https://www.google.pt/maps/)]([https://www.google.pt/maps/](https://www.google.pt/maps/)), accessed on 6 July 2022). Legend: 1, LVID at upstream inlets (Lis River, at Arrabalde section, Lena River at the mouth and Milagres riverside mouth); 2, LVID inlet, at Aroeira riverside upstream section; 3, Block II inlet; 4, Paddies’ inlet; 5, Paddies (surface water, soil, and groundwater); 6, Paddies’ outlet; 7, Block II outlet; 8, LVID outlet (Lis River, at Bajanca bridge section). bottles and kept at 4 \({}^{\circ}\)C until microbiological processing, which was carried out up to four hours after collection. The physicochemical quality of these water samples was evaluated with a precalibrated in situ portable multiparametric probe (SmarTROLL RDO Handheld, Fort Collins, CO, USA), for the following parameters: pH, Electrical Conductivity (EC, \(\mu\)S/cm), Saturation of Dissolved Oxygen (SDO, %) and Total Dissolved Solids (TDS, ppm). Chloride (CL, mg/L) was evaluated with suppressed ion chromatography [15]. The SAR indicator was calculated from the measurement of the elements calcium, magnesium and sodium, evaluated by inductively coupled plasma optical emission spectrometry [16]. The results obtained were compared to the maximum recommended values (MRVs) of the Portuguese Irrigation Water Quality Standards [17]. The microbiological analysis of the water samples included the enumeration of Total Coliforms (TC), Fecal Coliforms (FC) and Fecal Enterococci (FE) by the dilution method with the multiple tube fermentation technique, with the results expressed by the Most Probable Number (MPN)/100 mL, according to the reference analytical methods [17,18]. The count of ampicillin-resistant _Enterobacteriaceae_ in the water samples was performed by the membrane filtration technique. Decimal dilutions of each sample were prepared in sterile 0.9% saline, from which a volume of 100 mL was filtered through a cellulose membrane of 0.45 \(\mu\)m pore size (Millipore, Bedford, MA, USA) under vacuum, with the filters placed aseptically on the surface of the selective medium for _Enterobacteriaceae_, Violet Red Bile Glucose (VRBG) agar (Oxoid, Hampshire, England) supplemented with 20 \(\mu\)g/mL sodium ampicillin (AppliChem, Darmstadt, Germany). After aerobic incubation of the plates at 37 \({}^{\circ}\)C for 24 to 48 h, isolated colonies of ampicillin-resistant _Enterobacteriaceae_ (Amp\({}^{\text{r}}\)) were counted [19]. Antibiograms were performed directly on the isolates in mixed culture, using the direct antimicrobial susceptibility tests, according to [PERSON] et al. [20], to determine the most resistant phenotypes present in a mixed population.