## 2 Material and Methods  
### Water Sampling and Analysis of Physico-Chemical Parameters  
To assess the spatial variation of contaminants, water samples were collected in the period 22 June-1 July 2020 from a total of 32 sites located in the connected system Danube River-Danube Delta-Black Sea in SE part of Romania (Figure 1). The freshwater sampling started from Ostrov (at the border with Silistra/Bulgaria), and continued until the Danube flows into the sea through its 3 arms (Chilia--Musura bay, Sulina, and Sf. Gheorghe); the seawater sampling was done in Sacalin and 7 representative points of the Black Sea, Romanian littoral. The geographical coordinates of the sampling points were recorded, and samples were coded sequentially as 1-32 (Figure 1). The target sites included the main localities on the lower, pre-deltaic, deltaic, and maritime sector of the system, points of confluence with the main tributaries Siret and Prut, protected areas and several border sites. The expeditions were organized by the project teams of Danube Delta National Institute for Research and Development, Tulcea (DDNI), and Dunarea de Jos University of Galati (UDJG), Romania, by boat and cars. Surface water samples were collected according to European standards [37; 38], from the water column (0-60 cm) for both monitoring points located along the Danube and in the coastal area. For the Danube, samples were taken from the navigable channel area, as this area captures the maximum degree of homogenization of pollutants, and in the case of the coastal area, sampling was carried out at depths of 1.5-2 m, at a distance of 15-25 m from the shoreline. The containers used were prepared in advance, and at the time of sample collection, they were rinsed well with surface water [12]. For dissolved nutrients (nitrates, nitrites, ammonia) and total phosphorus analysis, the samples were taken in 1 L polyethylene containers, labeled from 1 to 32, stored at a temperature of 2-5\({}^{\circ}\), to minimize deterioration prior to chemical analysis. For organic nitrogen analysis, the water samples were preserved by adding 1 mL of concentrated sulfuric acid per 100 mL of sample, for acidulation at pH \(<\) 2. After collecting and preserving the samples, the bottles were sealed to ensure the integrity of the samples. During transport, the samples were protected from light and excessive heat, as the quality of the sample can change rapidly due to gas exchange, chemical reactions, and metabolism of the organisms. Figure 1: The target area in Romania and coordinates of the sampling sites in the Lower Danube River, Danube Delta, and Black Sea Basin. The samples were kept at a temperature of 2-5 \({}^{\circ}\)C (refrigerated box) [37]. The same principle of sample acidification was also used for the preservation of heavy metals, for which 1 mL of superpure nitric acid was added to a volume of 100 mL of the sample. The samples selected for microbiological analysis were transported to UDJG by car on the same day of collection. For nutrient (N, P) compounds, the measurements of the total forms of nitrogen and phosphorus and dissolved forms of nitrogen (nitrite, nitrate, ammonia) and dissolved phosphorus were performed by validated procedures at DDNI, in a chemistry laboratory accredited since 2005, according to the ISO/IEC 17025:2017, General requirements for the competence of testing and calibration laboratories [39], using molecular absorption spectrometry with the aid of a UV-VIS Lambda 650 Perkin Elmer spectrophotometer and ISO standards, and their quality was controlled through the analysis of blank samples and control standards [12]. All the reagents were of analytical grade quality. For quality assurance, flow charts were made with the specific certified reference materials. The N species determined and reported in this study as nitrogen are referred to as N-NO\({}_{3}\), N-NO\({}_{2}\), N-NH\({}_{4}\), and total nitrogen (N total), expressed in mg/L.