Emerging and Persistent Pollutants in the Aquatic Ecosystems of the Lower Danube Basin and North West Black Sea Region—A Review

: The tremendous impact of natural and anthropogenic organic and inorganic substances continuously released into the environment requires a better understanding of the chemical status of aquatic ecosystems. Water contamination monitoring studies were performed for different classes of substances in different regions of the world. Reliable analytical methods and exposure assessment are the basis of a better management of water resources. Our research comprised publications from 2010 regarding the Lower Danube and North West Black Sea region, considering regulated and unregulated persistent and emerging pollutants. The frequently reported ones were: pharmaceuticals (carbamazepine, diclofenac, sulfamethoxazole, and trimethoprim), pesticides (atrazine, carbendazim, and metolachlor), endocrine disruptors—bisphenol A and estrone, polycyclic aromatic hydrocarbons, organochlorinated pesticides, and heavy metals (Cd, Zn, Pb, Hg, Cu, Cr). Seasonal variations were reported for both organic and inorganic contaminants. Microbial pollution was also a subject of the present review.


Introduction
With a total length of 2780 km, the Danube River crosses 10 countries and 4 capitals and eventually runs into the Black Sea through the Danube Delta, the largest European wetland [1]. Due to its biodiversity, The Danube Delta, together with the Razim-Sinoe lagoon, is stated as an UNESCO World Heritage Biosphere Reserve [2]. The Danube basin on Romanian territory is the largest, compared with other countries of the Danube River Basin [1].
Surrounded by six coastal countries-Bulgaria, Georgia, Romania, Russia, Turkey, and Ukraine-the Black Sea is one of the largest inland water basins. Being almost entirely isolated from the world's oceans, the Black Sea is the largest natural anoxic water basin in the world [3]. The Black Sea is also a reservoir for the contaminants from multiple sources, among which Danube, the Dniester, the Dnieper, and the Don are the most significant [3].
Due to the location and climatic and historical conditions, the Lower Danube and Black Sea basins constitute an unique ecosystem [4]. In Eastern Europe, the rapid development of small industry tourism activities and urban area, together with a decrease of the intensive

Contaminants of Emerging Concern (CECs)
Contaminants of emerging concern (CECs) is a general term for the organic pollutant(s) including: human and veterinary pharmaceuticals (PhACs), endocrine disruptors (EDs) as bisphenols and steroids hormones, personal care products (PCPs), illegal drugs, antifungals, biocides, pesticides, herbicides, surfactants, and nanomaterials [28]. The term of CECs characterizes classes of unregulated or not completely regulated chemicals [29]. CECs are generally chemicals previously known to be present in the environment but exhibiting new documented impacts, recombination of known chemicals or mixtures of chemicals which, in combination, are hazardous for the environment, pharmaceuticals, and pharmaceuticals metabolites [30]. The main contamination sources are untreated wastewater, the wastewater treatment plants, waste of medical centers, animals and livestock, fertilization practice with manure, poorly treated raw materials, and different industries [29,30]. Potential concerns of the environment contamination with CECs include abnormal physiological processes and reproductive impairment of aquatic biota, the development of antibiotic-resistant bacteria, and the potential increased toxicity of chemical mixtures [29,31].
Due to the large number and diversity, the continuous discharge and long-term persistence of CECs pose a significant challenge to the scientific community and policy regulators. Prioritization criteria have been set according to the occurrence, exposure routes, chemical properties, toxicological relevance as results of in vitro and in vivo studies, current regulator state, and current research [31]. REACH regulatory approach [32] and the NORMAN prioritization focused on eco-toxicity endpoints [33] are the most common methodologies applied in Europe. However, the lack of information on hazard and risk of CECs makes the prioritisation process a research field with many unknowns [34].

Contaminants of Emerging Concern (CECs)
Contaminants of emerging concern (CECs) is a general term for the organic pollutant(s) including: human and veterinary pharmaceuticals (PhACs), endocrine disruptors (EDs) as bisphenols and steroids hormones, personal care products (PCPs), illegal drugs, antifungals, biocides, pesticides, herbicides, surfactants, and nanomaterials [28]. The term of CECs characterizes classes of unregulated or not completely regulated chemicals [29]. CECs are generally chemicals previously known to be present in the environment but exhibiting new documented impacts, recombination of known chemicals or mixtures of chemicals which, in combination, are hazardous for the environment, pharmaceuticals, and pharmaceuticals metabolites [30]. The main contamination sources are untreated wastewater, the wastewater treatment plants, waste of medical centers, animals and livestock, fertilization practice with manure, poorly treated raw materials, and different industries [29,30]. Potential concerns of the environment contamination with CECs include abnormal physiological processes and reproductive impairment of aquatic biota, the development of antibiotic-resistant bacteria, and the potential increased toxicity of chemical mixtures [29,31].
Due to the large number and diversity, the continuous discharge and long-term persistence of CECs pose a significant challenge to the scientific community and policy regulators. Prioritization criteria have been set according to the occurrence, exposure routes, chemical properties, toxicological relevance as results of in vitro and in vivo studies, current regulator state, and current research [31]. REACH regulatory approach [32] and the NORMAN prioritization focused on eco-toxicity endpoints [33] are the most common methodologies applied in Europe. However, the lack of information on hazard and risk of CECs makes the prioritisation process a research field with many unknowns [34].
Among CECs, pharmaceuticals, PCPs, EDs, and pesticides (other than organochlorine pesticides) were more often monitored in the Danube River basin and represent the subject of the research in the present review.

Pharmaceuticals (PhACs)
Although the occurrence of PhACs has been documented since 20 years ago in the European environment, these chemicals are not included among those to be monitored [38]. Pharmaceutical compounds most often identified in the aquatic environment belong to several classes of human and veterinary antibiotics and human prescription and nonprescription drugs such as NSAIDs, β-blockers, blood lipid regulators, antiepileptics, analgesics, and antidepressants [15,39,40].
The occurrence of pharmaceuticals in the Lower Danube basin has been investigated since 2001. A number of PhACs, among which metronidazole, ambroxole, clotrimazole, paracetamol, and metamizole were monitored within the JDS1 sampling campaign (August-September 2001). From 2005, independent studies also reported the presence of PhACs in the Danube waters [41,42]. The next JDS 2 sampling campaign (September 2007) monitored NSADs ketoprofen, naproxen, ibuprofen, diclofenac, antiepileptic carbamazepine, caffeine, and sulfamethoxazole and reported high concentration of carbamazepine around Budapest and in Tisa and Sava tributaries [43]. After 2010, the number of published studies considerably increased and the studies have become more complex, comprising a higher number of compounds [10,15]. As the consequence of the improvements in analytical instrumentation sensitivity that have made it possible to detect extremely low concentrations, the number of pharmaceuticals substances detected in the environmental matrices has been dramatically increased [15,40].
A number of 14 published studies have been identified concerning qualitative and quantitative monitoring studies in Lower Danube basin including tributaries and the Danube Delta during 2010-2021 (Table 1). One publication describing a comprehensive study on the CECs on Dniester River was identified. No publication on the monitoring of pharmaceuticals in the North-West Black Sea coast was found. Environmental matrices such as surface water samples (13 publications), ground water (3 publications), drinking water (1 publication), and sediment (3 publications) were investigated. The majority of the studies are based on 'grab-sampling' for the surface water. For sediments samples, a gravity corer [44] or a steel hand bucket for the river bottom sites was used [39]. Solid phase extraction (SPE) has been used for analyte extraction, concentration and purification for the water samples. Ultrasonic-assisted extraction (UAE) followed by SPE purification was used in case of solid samples. Liquid chromatography (LC) was employed for analysis of PhACs in all selected studies.
Mass spectrometry (MS) and tandem MS/MS detection with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) represented the most common technique. High-resolution mass spectrometry (HRMS) was used in four studies, enabling a new acquisition approach as non-target Data Independent Acquisition (DIA) [26]. The selection of the monitored compounds in the listed studies (Table 1) was generally based on the occurrence in the aquatic environment, documented in previous reports or published data [10,39], frequency and magnitude of the pharmaceuticals usage [46], and updated priority substances [36,37].
More than 40 unregulated PhACs were reported in the listed publications (Table 1). Six studies reported the identification of metabolites for carbamazepine, aminophenazone, ibuprofen, metamizole, and others. The most reported antibiotic herein reviewed was sulfamethoxazole, followed by trimethoprim, and erythromycin. Among NSADs, the most reported was ibuprofen, followed by diclofenac. Substances found in fewer reports, but at very high concentrations were norephedrine (2633 ng/L in Dniester River), fluconazole (3390 ng/L in Dniester River) [26], and furosemide (444.63 ng/L in the Mures, River) [51].
Considering the studies on the Lower Danube River basin, the concentration of frequently monitored compounds in the surface water ranged as follows: The selection of the monitored compounds in the listed studies (Table 1) was generally based on the occurrence in the aquatic environment, documented in previous reports or published data [10,39], frequency and magnitude of the pharmaceuticals usage [46], and updated priority substances [36,37].
More than 40 unregulated PhACs were reported in the listed publications (Table 1). Six studies reported the identification of metabolites for carbamazepine, aminophenazone, ibuprofen, metamizole, and others. The most reported antibiotic herein reviewed was sulfamethoxazole, followed by trimethoprim, and erythromycin. Among NSADs, the most reported was ibuprofen, followed by diclofenac. Substances found in fewer reports, but at very high concentrations were norephedrine (2633 ng/L in Dniester River), fluconazole (3390 ng/L in Dniester River) [26], and furosemide (444.63 ng/L in the Mures, River) [51].
Considering the studies on the Lower Danube River basin, the concentration of frequently monitored compounds in the surface water ranged as follows:  Concentrations exceeding-EQS of carbamazepine, sulfamethoxazole, trimethoprim, diclofenac, and erythromycin were found in the Danube tributaries such as Morava river [38], Argeş river [10,44,49], Mureş river [51], and Jijia river [53], in sites that are heavily impacted by municipal or industrial wastewater discharges. Thus, the highest concentrations of carbamazepine (945 ng/L), sulfamethoxazole (204 ng/L) and diclofenac (255 ng/L) were reported in the Arges River, highly affected by the municipal and industrial discharges of the capital of Romania, the city of Bucharest. The concentration measured in the Danube River for the pharmaceuticals mentioned in the "Watch lists" [36,37] were below EQS.
Comparing with data on other river in Europe, including the upper Danube, concentrations of carbamazepine of 559 ng/L were reported in River Fyrisån (Sweden), 490 ng/L in river Grundlach (Germany), and 1670 ng/L in Ebro basin (Spain) [54]. High concentration of diclofenac of 930 ng/L was previously reported in upper Danube (Budapest, Hungary, September 2008) [55]. Sulfamethoxazole concentrations of 540 ng/L in surface water were recently reported in the upper Danube catchment area in Croatia [56].
However, the PhACs carbamazepine, diclofenac, and amoxicillin were previously listed as Danube basin-specific pollutants, derived within the EU-project SOLUTIONS [57].
Caffeine is also present in the Lower Danube and tributary waters. Both minimum (5.27 ng/L [47] and maximum concentrations (306 ng/L [46]) were reported along the Concentrations exceeding-EQS of carbamazepine, sulfamethoxazole, trimethoprim, diclofenac, and erythromycin were found in the Danube tributaries such as Morava river [38], Argeş river [10,44,49], Mureş river [51], and Jijia river [53], in sites that are heavily impacted by municipal or industrial wastewater discharges. Thus, the highest concentrations of carbamazepine (945 ng/L), sulfamethoxazole (204 ng/L) and diclofenac (255 ng/L) were reported in the Arges River, highly affected by the municipal and industrial discharges of the capital of Romania, the city of Bucharest. The concentration measured in the Danube River for the pharmaceuticals mentioned in the "Watch lists" [36,37] were below EQS.
Comparing with data on other river in Europe, including the upper Danube, concentrations of carbamazepine of 559 ng/L were reported in River Fyrisån (Sweden), 490 ng/L in river Grundlach (Germany), and 1670 ng/L in Ebro basin (Spain) [54]. High concentration of diclofenac of 930 ng/L was previously reported in upper Danube (Budapest, Hungary, September 2008) [55]. Sulfamethoxazole concentrations of 540 ng/L in surface water were recently reported in the upper Danube catchment area in Croatia [56].
However, the PhACs carbamazepine, diclofenac, and amoxicillin were previously listed as Danube basin-specific pollutants, derived within the EU-project SOLUTIONS [57].
Caffeine is also present in the Lower Danube and tributary waters. Both minimum (5.27 ng/L [47] and maximum concentrations (306 ng/L [46]) were reported along the Danube River near Novi Sad in two different studies. As this compound is efficiently removed by wastewater treatment plants, caffeine is a suitable marker of the presence of untreated wastewater. JDS3 reported a median concentration of caffeine of 93 ng/L in the Danube and 123 ng/L in the tributary [58].
Very high PhACs concentration was reported in the Dniester River (transboundary river between Ukraine and the Republic of Moldova) in a recent wide-scope screening study by Diamanti et al. [26]. In total, 40 PhACs compounds and their metabolites were determined in surface water samples. The highest total cumulative PhACs concentration was 26.1 µg/L (a total of 35 contaminants) in a site receiving wastewaters from Chisinau town and the pharmaceutical industry. A concentration of carbamazepine of 1981 ng/L (more than double that the maximum concentration reported in Argeş River) and 2858 ng/L for carbamazepine metabolite was reported in Byk River, Moldova [26]. Fluconazole concentration of 3390 ng/L and sulfamethoxazole of 1290 ng/L were measured for the same sampling site. Additionally, metabolites such as 4-acetamidoantipyrine (maximum concentration of 1611 ng/L) and 10,11-dihydro-10,11-dihydroxycarbamazepine (2858 ng/L) were detected in a higher concentration than the parent compounds [26], a fact that has been reported to occur in wastewater samples [51], and demonstrates the impact of uncontrolled discharge.
Concentrations of PhACs in the river sediments were much lower than the ones found in surface water samples in all reported studies.

Endocrine Disruptors (EDs)
Due to androgenic or estrogenic activities even at low concentrations, endocrine disruptors (EDs) can induce adverse effects on endocrine systems related to alterations in endocrine function and sexual development or altered fertility and reproductive behavior for the aquatic wildlife [21]. Steroids estrogens, as the natural hormones estrone (E1), 17β-estradiol (E2), and estriol (E3), as well as the synthetic hormones 17α-ethinyl estradiol (EE2) and diethylstilbestrol (DES), are of particular concern, being included in a European Union Water Framework Directive (WFD) "watch-list" [36]. EQS set levels of 0.4 ng/L for E1 and E2, and 0.035 ng/L for EE2, make their analysis extremely challenging.
The presence of EDs in surface waters was reported from early 2001, in Germany rivers (including Danube) with concentrations ranging from 0.15 to 3.6 ng/L E2 and 0.1 to 5.1 ng/L EE2 [62]. In 2003 in River Nene and River Lea (UK), concentrations of 0.9 ng/L mean E2 and 0.7 ng/L mean EE2 were reported [63] and in 2005 in Chesapeake Bay (SUA), in concentrations ranging from 1.9 to 6.0 ng/L, for E2 and 0.1-17 ng/L for E1 was founded [64].
Among previously studies on the Danube River concerned EDs, a paper published in 2011 reported the presence of the contaminants in sediments samples from the Upper Danube River (Germany) [21]. EDs nonylphenol and bisphenol A (BPA), as well as the natural estrogen E1, were frequently detected in the concentration range of 6.5-1364 ng/g sediment equivalent (SEQ) for nonylphenol, 1.2-22 ng/g SEQ for bisphenol A and 0.019-0.24 ng/g SEQ for E1 [21]. The JDS3 reported the detection of E2 in surface water from eight sites, in a maximum concentration of 0.029 ng/L [58]. In a recent study on rivers of the Carpathian Basin, concentration ranges of 0.018-3.13 ng/L for E2 and 0.005-0.124 ng/L for EE2 were reported in the Danube River in Slovenia, and a maximum concentration of 0.45 ng/L for E2 was found in Tisza tributary [9].
Seven publications were identified concerning the presence of EDs in Low Danube basin waters, one in the Danube Delta and one in the Romanian Black Sea Coast area ( Table 2). Surface water and sediments were analyzed within the listed studies.    Estrogenic hormones reported levels in the Lower Danube basin water ranged from 0.15 to 9.8 ng/L for E1 (with maximum value reported in Serbia [61]), from 1.5-3.3 ng/L for E2 (maximum value in the Argeş River [50]), from 0.37 to 4.8 ng/L for E3, and 0.5-3.8 ng/L for EE2 (with the maximum value in the Argeş River [50] (Figure 3, left side)). The values were higher than those reported by JDS3 [58], but comparable to other studies on European rivers [9,70]. Thus, concentration ranges of 0.17-7.3 for E1 was reported in the Iberian River (Spain), of 2.4-4.0 ng/L in Tagus River (Portugal) and of 2.5-49 ng/L in Körsch river (Germany) [70]. EE2 concentration range of 0.47-2.2 ng/L was reported in the Tagus river (Portugal) [70].
All four studies on the analysis of steroid hormones in surface water showed exceeding of the EQS for E1, E2 (0.4 ng/L), and EE2 (0.035 ng/L) both in the Danube [50,65,66] and in tributaries Sava River [65], Tisa River [61], and rivers Olt, Jiu, and Argeş on Romanian territory [50,66]. However, measuring EE2 could be very challenging due to the maximum acceptable method detection limit of 0.035 ng/L imposed by the regulation [45]. Thus, among the four mentioned studies, two of them reported higher limits of quantification (LOQ) [50,66].
Among bisphenol analogues, bisphenol A (BPA) is the most the most widespread in the aquatic environments. BPA was reported in five studies in the Low Danube basin including Delta and in one concerning the Romanian Black sea coast, in concentrations ranging from 0.6 to 693 ng/L ( Figure 3, right side). Seasonal variation of Bisphenol A in the Danube river was studied by Milanović in 2015 [20]. Lower concentration levels were reported in winter (maximum 33 ng/L, mean 6 ng/L), while in the summer, a maximum concentration of 693 ng/L (mean 220 ng/L) was registered due to an increase in the leaching of bisphenol A from plastic materials attributed to faster photo-and microbial degradation.
Significant lower concentrations were reported for other bisphenol analogues, such as BPC, BPE, and BPF. One publication reported the presence of bisphenols in the sediment on the Romanian Black Sea coast. A maximum concentration of 416 ng/L BPA (with mean of 165 ng/L) was measured in seawater and 10 ng/L (with of mean 6.3 ng/L) in algae [69]. Although a high concentration was measured in seawater, a maximum of 0.8 ng/L was determined in sediment, which is much lower than other reported data.
By comparison, lower concentrations of bisphenol A have been reported in other marine environments around the world. Thus, BPA average concentration found in sediments sampled in the north of Adriatic Sea (Venice Lagoon) was 44.89 ng/g, while those All four studies on the analysis of steroid hormones in surface water showed exceeding of the EQS for E1, E2 (0.4 ng/L), and EE2 (0.035 ng/L) both in the Danube [50,65,66] and in tributaries Sava River [65], Tisa River [61], and rivers Olt, Jiu, and Argeş on Romanian territory [50,66]. However, measuring EE2 could be very challenging due to the maximum acceptable method detection limit of 0.035 ng/L imposed by the regulation [45]. Thus, among the four mentioned studies, two of them reported higher limits of quantification (LOQ) [50,66].
Among bisphenol analogues, bisphenol A (BPA) is the most the most widespread in the aquatic environments. BPA was reported in five studies in the Low Danube basin including Delta and in one concerning the Romanian Black sea coast, in concentrations ranging from 0.6 to 693 ng/L ( Figure 3, right side). Seasonal variation of Bisphenol A in the Danube river was studied by Milanović in 2015 [20]. Lower concentration levels were reported in winter (maximum 33 ng/L, mean 6 ng/L), while in the summer, a maximum concentration of 693 ng/L (mean 220 ng/L) was registered due to an increase in the leaching of bisphenol A from plastic materials attributed to faster photo-and microbial degradation.
Significant lower concentrations were reported for other bisphenol analogues, such as BPC, BPE, and BPF.
One publication reported the presence of bisphenols in the sediment on the Romanian Black Sea coast. A maximum concentration of 416 ng/L BPA (with mean of 165 ng/L) was measured in seawater and 10 ng/L (with of mean 6.3 ng/L) in algae [69]. Although a high concentration was measured in seawater, a maximum of 0.8 ng/L was determined in sediment, which is much lower than other reported data.
By comparison, lower concentrations of bisphenol A have been reported in other marine environments around the world. Thus, BPA average concentration found in sediments sampled in the north of Adriatic Sea (Venice Lagoon) was 44.89 ng/g, while those reported for the lagoons in Po River Delta of 18.64 ng/g, and in the Kaštela Bay (Croatia) of 11.82 ng/L [71].

Pesticides (Other Than Organochlorine)
Pesticide is a broader term that covers herbicides, insecticides, nematicide, fungicides, plant growth regulator, defoliants, desiccants, and biocides. The pesticide pollution of surface waters or groundwater may have different pathways: surface run-off from farmyards, wastewater treatment plants, forestry, municipal use, grasslands and domestic gardens, or animal husbandry. Unlike pharmaceuticals, pesticides are designed to act against organisms (plants, insects) and have an inherent effect on the environment [28,44].
In the European Union, the presence of pesticides in water is regulated through the Directive 2006/118/EC [72], which refers to groundwater, the Directive 98/83/EC [73] on the quality of water intended for human consumption, and the general Framework of Water Directive 2000/60/EC [74]. EU standard acceptable concentration for pesticides in ground water and drinking water is 0.1 µg/L [73].
Within the present research, seven studies on pesticides in the surface water samples, and sediment in the selected area were identified (Table 3). Target compounds selection was based on literature reporting the occurrence of the contaminants and the list of priority substances under the WFD [35]. Complex monitoring studies consider also the abiotic transformation products of such compounds, which in some cases may be more toxic, persistent, and bioaccumulative than the parent compounds (e.g., metolachlor-ESA, 2hydroxypropazine, 2-hydroxysimazine, desethylterbuthylazine) [26,45,53].
Among the detected compounds in the Lower Danube basin, relevant concentrations were reported for carbendazim (in a range of 0.6-269 ng/L), atrazine (4-392 ng/L), metolachlor (80-150 ng/L), dimethoate (7-23 ng/L), and imazalil (2.5-80 ng/L) ( Figure 4). Griseofulvin was detected in the Danube Delta and Siret River [10]. Maximum concentrations were reported generally in tributary Tisa, Morava, and Siret River [10,39]. Higher concentration of dimethoate of 1222 ng/g was reported in Tisa River [39]. It is noteworthy that in the Dniester River, significantly higher concentrations than the Danube basin were reported, revealing the influence of untreated waters [26]. Thus, the concentrations of 4612 ng/L for metolachlor and 107 ng/L for imidacloprin recorded in the Prut River are over 30 times higher than the maximum concentrations in the Danube basin. Terbuthylazine maximum concentration of 2514 ng/L in the Dniester River is also significantly higher relative to those reported in the Danube (of 200 ng/L) and in the Prut river (of 41.4 ng/L). Concentration of carbandazim (of 755 ng/L) was almost three times higher than that reported for the Danube. A maximum concentration of diuron of 1197 ng/L was measured in the central part of Dniester basin (Moldova) [26]. For comparison reasons, worth mentioning are the maximum concentrations of imazalil of 409.73 ng/L and diuron of 150 ng/L measured in the Ebro River basin (Spain) [76].   In the Danube River basin, PNEC has been exceeded for carbendazim [77] and dimethoate [39]. In the Dniester River the value of PNEC was exceeded for acetochlor, carbaryl, dimethoate, diuron, imidacloprid, omethoate, metolachlor, terbuthylazine [26]. Most papers describe point surveys or seasonal monitoring, except the multi-year study of Antic' et al. [77], in which variations in concentration of pesticides as carbendazim, propazine, and dimethoate were attributed to their seasonal application during spring and to the rainfall above normal, leading to increased runoff.

Personal Care Products (PCPs)
This large category of emergent contaminants includes chemicals found in consumer products such as cosmetics, fragrances, disinfectants, antiseptics, UV filters, and insect repellents. Among the reviewed papers, fewer included PCPs monitoring.
Triclosan and triclocarban, highly used disinfectants in personal care products, were measured in surface water in 16 sites along the Romanian side of the Danube and its three main tributaries, Jiu, Olt, and Argeș rivers in 2014 in a range of 0.7-18.4 ng/L for triclorsan and 0.6-54 ng/L for triclocarban [50]. In a recent study on the Prut river, triclosan was reported in a range of 12.5-159 ng/L [26].
The occurrence of the 10 organic UV-filters on the North-Western Black Sea coast was reported for the first time in 2020 by Chiriac et al. [69]. High levels up to 5607 ng/L of BP3 (2 hydroxy-4-methoxy-benzophenone) were measured in the seawater. 234HBP (2,3,4-trihydroxybenzophenone) reached a maximum level of 824 ng/L and BP1 (2,4-tdihydroxybenzophenone) of 600 ng/L in seawater. Salicylate derivatives (ethylhexyl salycilate and homosalate) were also detected in high concentrations of 1286 ng/L and, respectively, 1262 ng/L in seawater, but reached a maximum value of 5823 ng/L of ethylhexy salicylate in sediments.
Insect repellent DEET (N,N-Diethyl-meta-toluamide) was monitored along the Danube River and its tributaries within JDS3. In the Lower Danube waters, the measured levels were lower than 10 ng/L, while maximum levels in the Morava (81 ng/L) and Arges (37 ng/L) tributaries were reported [44]. In the Dniester River basin, a maximum concentration of 345 ng/L was reported [26]. In the Danube River basin, PNEC has been exceeded for carbendazim [77] and dimethoate [39]. In the Dniester River the value of PNEC was exceeded for acetochlor, carbaryl, dimethoate, diuron, imidacloprid, omethoate, metolachlor, terbuthylazine [26]. Most papers describe point surveys or seasonal monitoring, except the multi-year study of Antic' et al. [77], in which variations in concentration of pesticides as carbendazim, propazine, and dimethoate were attributed to their seasonal application during spring and to the rainfall above normal, leading to increased runoff.

Personal Care Products (PCPs)
This large category of emergent contaminants includes chemicals found in consumer products such as cosmetics, fragrances, disinfectants, antiseptics, UV filters, and insect repellents. Among the reviewed papers, fewer included PCPs monitoring.
Triclosan and triclocarban, highly used disinfectants in personal care products, were measured in surface water in 16 sites along the Romanian side of the Danube and its three main tributaries, Jiu, Olt, and Arges , rivers in 2014 in a range of 0.7-18.4 ng/L for triclorsan and 0.6-54 ng/L for triclocarban [50]. In a recent study on the Prut river, triclosan was reported in a range of 12.5-159 ng/L [26].
The occurrence of the 10 organic UV-filters on the North-Western Black Sea coast was reported for the first time in 2020 by Chiriac et al. [69]. High levels up to 5607 ng/L of BP3 (2 hydroxy-4-methoxy-benzophenone) were measured in the seawater. 234HBP (2,3,4-trihydroxybenzophenone) reached a maximum level of 824 ng/L and BP1 (2,4tdihydroxybenzophenone) of 600 ng/L in seawater. Salicylate derivatives (ethylhexyl salycilate and homosalate) were also detected in high concentrations of 1286 ng/L and, respectively, 1262 ng/L in seawater, but reached a maximum value of 5823 ng/L of ethylhexy salicylate in sediments.
Insect repellent DEET (N,N-Diethyl-meta-toluamide) was monitored along the Danube River and its tributaries within JDS3. In the Lower Danube waters, the measured levels were lower than 10 ng/L, while maximum levels in the Morava (81 ng/L) and Arges (37 ng/L) tributaries were reported [44]. In the Dniester River basin, a maximum concentration of 345 ng/L was reported [26].
Low PFOA levels ranging from 0.6-1.0 ng/g were reported in 2019 in sediment core from the Iron Gate I Reservoir, the largest impoundment on the Danube River, at the boundary between Serbia and Romania. [44].  [53].
The most frequently reported contaminants of emerging concern in the study area for the target period of time were: pharmaceuticals (carbamazepine, sulfamethoxazole, and diclofenac, trimethoprim, and caffeine), bisphenol A, estrogens, caffeine, pesticide (atrazine, carbendazim, metolachlor), and PCP-like triclosan ( Figure 5).

Persistent Organic Pollutants
Persistent organic pollutants (POPs) are non-polar organic compounds with high stability and high bioaccumulation properties, toxic at threshold level [78]. The potentially most-hazardous POPs include: industrial by-products dioxins and dibenzofurans, organochlorinated pesticides (OCPs) (dichlorodiphenyltrichloroethane and its four isomers

Persistent Organic Pollutants
Persistent organic pollutants (POPs) are non-polar organic compounds with high stability and high bioaccumulation properties, toxic at threshold level [78]. The potentially most-hazardous POPs include: industrial by-products dioxins and dibenzofurans, organochlorinated pesticides (OCPs) (dichlorodiphenyltrichloroethane and its four isomers (DDTs), hexachlorocyclohexanes (HCHs), aldrin, dieldrin, endrin, chlordane, hep-tachlor, toxaphene, mirex, hexachlorobenzene) and chemicals resulting from industrial processes such as polychlorobiphenyls (PCBs), brominated flame retardants (BFRs), and polycyclic aromatic hydrocarbons (PAHs). POPs fall under incidence of the Stockholm Convention [79] [81] and are consistently monitored in the water bodies and reported as Σ16PAHs (US EPA PAHs). Similar, a set of six indicator PCBs (indicator Σ6PCBs) was recommended by the EU for assessing the pollution by PCBs [82].
Due to high hydrophobic nature and low solubility in water, POPs can be adsorbed on sediment particles or water-suspended particles, leading to the accumulation and concentration in different compartments of the aquatic ecosystems (water, sediment, biota) [2]. International standardized methods are available for the quantification of POPs in surface water or sediments [43]. Solid phase microextraction and liquid-liquid or solid-liquid extractions are the methods employed for the sample processing phase. Due to the complexity of the POPs' nature, multi-methods allowing detection and quantification of a large number of contaminants, such as gas chromatography-mass spectrometry (GC-MS) or gas chromatography coupled with an electron capture detector (GC-ECD), are generally used in the monitoring programs.
During 2010-2021, 16 studies were identified on POPs for the selected area (Table 4). Among POPs, PAHs were investigated in 10 studies, PCBs in 5, and OPCs in 11. Most studies were focused on the analysis of sediment or top soil (12 publications) and surface water (four studies). Although POPs analysis in aquatic organisms' tissues is highly relevant for the assessment of the water ecosystem pollution [2], only two studies considered fish as matrix. Previously investigation of highly persistent PCBs and DDTs in fish from Danube River and the Black Sea were performed by Covaci, (2006) [2] and Stoichev, (2007) [83].
Most of the reviewed studies describe complex monitoring programs over several years [84][85][86]. The Danube Delta was investigated in 2 publications, the Black Sea in 4, the Dniester basin in 1, and the Danube basin in 11. Data systematized in Table 4 show values for total concentrations of Σ16PAHs in sediment ranging from 70 to 6983 µg/kg, for ΣPCBs from 0.3-74 µg/kg and for ΣDDTs from 0.7 to 61.7 µg/kg ( Figure 6). Compared with other data on river sediments in Europe, including the upper Danube, the concentrations of Σ16PAHs (US EPA PAHs) in the sediment samples from the Lower Danube basin were higher than those reported for Danube in Hungary (8.3-1202 µg/kg) [87], Tiber River in Italy (157.8-271.6 µg/kg) [88], or from Durance River in France (57-1527 µg/kg) [89], but lower than those reported in the Ammer River in Germany (112-22,900 µg/kg) [90] and Ría de Arousa in Spain (45-7901 µg/kg dry wt.) [91].
Data of PCB in sediments and fishes collected in the Danube Delta in 2001 have been reported [2]. In sediments, ΣPCBs < 2 µg/kg and ΣDDTs in the range 0.9-17 µg/kg were found. Compared with this, more recent data on sample collected in 2009-2011 show a dramatic increase in Σ6PCBs level ranging from 27.3 to 74 µg/kg and Σ 3DDTs from 0.4 to 29.1 µg/kg [92].   ported by another study for a sampling site in Murighiol near the pontoon of the supply ship. A maximum value of 414 μg/kg for ΣPCBs was reported in the same sapling point (Danube Delta, Murighiol, near the pontoon of the supply ship) [92]. Concentrations of PAHs ranging from 12.2 to 260 ng/L [96] and of PCBs from 3 to 13 ng/L in Lower Danube basin waters were reported ( Figure 6). Much lower values of PAHs in water (5-72 ng/L) and of PCBs (0.005 to 0.016 ng/L) were reported for the middle stretch of the Danube river between the cities of Vienna and Bratislava [104]. Overall, data suggest a considerable increasing of the organic pollution in the Danube Delta area. However, regarding the data in Table 4, it is difficult to draw a conclusion due to the heterogeneity of the studies. For example, while the data provided by studies on PAH pollution in the Black Sea coastal area are comparable (a range of 82-6983 μg/kg was reported by Ţigănuş et al. [102] and of 304-5611 μg/kg by "The Black Sea state of the environment" report [103]), for the Prut and Dniester rivers, no comparison can be made because the analyzed matrix is different (topsoil and river sediments). The same, in the Danube Delta, a values range of 329-1093 μg/kg was reported for Σ 16PAHs in sediment by "The Black Sea state of the environment" report [103], while Vosniakos et al. outlined only a maximum value of 24570 μg/kg for Σ12PAHs corresponding to a sampling site near the supply ships pontoon [92].

Metals
Heavy metals pollution is a significant environmental hazard for invertebrates, fish, and humans due to their toxicity, persistence, and bioaccumulative nature [104,105]. Their natural sources include corrosion of the metal-containing rocks, soil erosion, and volcanic eruptions, while principal anthropogenic sources include industrial emissions, mining, smelting, foundries, and agricultural activities using pesticides, insecticides, and fertilizers [105]. While, naturally, trace elements in sediments are mainly associated with silicates, anthropogenic pollution leads to the release into the environment of more mobile and reactive elements [106]. Environmentally relevant heavy metals and metalloids include Cr, Ni, Cu, Zn, Cd, Pb, Hg, and As [107]. As the studies on the metal content are more numerous compared with those on other pollutants, for the present review a selection of 28 representative studies was carried out in terms of area, matrix, and analyzed elements (Table 5).  [92].
Concentrations of PAHs ranging from 12.2 to 260 ng/L [96] and of PCBs from 3 to 13 ng/L in Lower Danube basin waters were reported ( Figure 6). Much lower values of PAHs in water (5-72 ng/L) and of PCBs (0.005 to 0.016 ng/L) were reported for the middle stretch of the Danube river between the cities of Vienna and Bratislava [104]. Overall, data suggest a considerable increasing of the organic pollution in the Danube Delta area.
However, regarding the data in Table 4, it is difficult to draw a conclusion due to the heterogeneity of the studies. For example, while the data provided by studies on PAH pollution in the Black Sea coastal area are comparable (a range of 82-6983 µg/kg was reported byŢigănuş et al. [102] and of 304-5611 µg/kg by "The Black Sea state of the environment" report [103]), for the Prut and Dniester rivers, no comparison can be made because the analyzed matrix is different (topsoil and river sediments). The same, in the Danube Delta, a values range of 329-1093 µg/kg was reported for Σ 16PAHs in sediment by "The Black Sea state of the environment" report [103], while Vosniakos et al. outlined only a maximum value of 24570 µg/kg for Σ12PAHs corresponding to a sampling site near the supply ships pontoon [92]

Metals
Heavy metals pollution is a significant environmental hazard for invertebrates, fish, and humans due to their toxicity, persistence, and bioaccumulative nature [104,105]. Their natural sources include corrosion of the metal-containing rocks, soil erosion, and volcanic eruptions, while principal anthropogenic sources include industrial emissions, mining, smelting, foundries, and agricultural activities using pesticides, insecticides, and fertilizers [105]. While, naturally, trace elements in sediments are mainly associated with silicates, anthropogenic pollution leads to the release into the environment of more mobile and reactive elements [106]. Environmentally relevant heavy metals and metalloids include Cr, Ni, Cu, Zn, Cd, Pb, Hg, and As [107]. As the studies on the metal content are more numerous compared with those on other pollutants, for the present review a selection of       Biosphere   Surface waters were analyzed in 17 studies, sediments in 16, and biota (fish species, mollusks, plankton, aquatic plants, and microalgae) in 15 studies. A number of six complex studies considered water, sediments, and biota. As environmental pollution with heavy metals is well regulated, sampling, sample preparation, and analysis are generally carried out according to specific standards, e.g., ISO 5667-3:2018 [131] for water sampling, sample preservation, transport, and storage, ISO 5667-13:2011 [132] for sediments sampling, ISO 15587-1/2:2002 [133,134] for water sample preparation, ISO 17294-2:2016 [135] concerning the ICP-MS method, ISO 11047:1998 [136] concerning flame and electrothermal AA for analysis.
The most frequently reported metals, in the following decreasing order, were: Pb, Cd, Ni, Cr, As, Cu, Zn, Hg, Mn, Fe, Co, Al. The reported values show large variation for all matrices (Figure 7). Among the selected studies, 17 studies concerned the Lower Danube basin in Serbia, Bulgaria, Romania, and Republic of Moldova, 6 studies-the Danube Delta, 4 studiesthe Black Sea coasts (in Romania and Bulgaria), and 2-the Dniester River (Ukraine).
The most frequently reported metals, in the following decreasing order, were: Pb, Cd, Ni, Cr, As, Cu, Zn, Hg, Mn, Fe, Co, Al. The reported values show large variation for all matrices (Figure 7). Regarding spatial distribution of metal concentrations in water, it can be observed that, in general, the highest concentrations were reported for tributaries, followed by those in the Danube Delta biosphere Reserve, the lowest concentrations being reported in the Danube River.
Thus, for Cd, a concentration range of 0.002-1.33 μg/L was reported in the Danube River, values of 50 μg/L were measured in the tributary Morava River, Serbia [109], and of 14.90 μg/L in the tributary Yantra River, Bulgaria [111] as a maximum of 11.05 μg/L in the Biosphere Reservation of the Danube Delta [123]. Similarly, Cu reported concentrations varying within a range 2.65-10.1 μg/L, except the maximum value of 112.3 μg/L in the Danube, reported in the Danube in sampling sites corresponding to a large industrial city (Galaţi town) [117]. In the tributary Tisa River, a concentration of 70 μg/L was measured [109], as a value of 6.7 μg/L was reported in the Danube Delta [126]. Although lower concentrations of metals in seawater have generally been reported for Cu, a maximum value of 30.66 μg/L was measured by Jitar et al. [128].
Cr concentrations ranged from 0.21 μg/L to 9 μ/L in the Danube River. A maximum concentration of 64 μg/L was reported in Olt River [107], as comparable maximum concentrations of 81.  Regarding spatial distribution of metal concentrations in water, it can be observed that, in general, the highest concentrations were reported for tributaries, followed by those in the Danube Delta biosphere Reserve, the lowest concentrations being reported in the Danube River.
Thus, for Cd, a concentration range of 0.002-1.33 µg/L was reported in the Danube River, values of 50 µg/L were measured in the tributary Morava River, Serbia [109], and of 14.90 µg/L in the tributary Yantra River, Bulgaria [111] as a maximum of 11.05 µg/L in the Biosphere Reservation of the Danube Delta [123]. Similarly, Cu reported concentrations varying within a range 2.65-10.1 µg/L, except the maximum value of 112.3 µg/L in the Danube, reported in the Danube in sampling sites corresponding to a large industrial city (Galaţi town) [117]. In the tributary Tisa River, a concentration of 70 µg/L was measured [109], as a value of 6.7 µg/L was reported in the Danube Delta [126]. Although lower concentrations of metals in seawater have generally been reported for Cu, a maximum value of 30.66 µg/L was measured by Jitar et al. [128].
Cr concentrations ranged from 0.21 µg/L to 9 µ/L in the Danube River. A maximum concentration of 64 µg/L was reported in Olt River [107], as comparable maximum concentrations of 81.24 µg/L [122] and 78.25 µg/L [124] were measured in the Biosphere Reservation of the Danube Delta. A similar trend was observed for Hg, with concentration range of 0.001-0.117 µg/L in the Danube and a maximum value of 68.15 µg/L reported in the Yantra River, Bulgaria [117]. Low concentrations of Pb are generally reported in the Danube river in a range of 0.003-3.81 µg/L with the exception of a maximum value of 21.4 µg/L reported near the Galaţi town by Ionita et al. [117]. However, considerably higher concentrations of 48.04 µg/L [122] and 34 µg/L [124] of Pb were reported in the Danube Delta Biosphere reserve. For As, concentration range of 0.5-16.96 µg/L was reported in the Danube River, a maximum of 10.1 µg/L in the tributary Yantra River [111], and a maximum of 16.25 µg/L in the Danube Delta [124].
Regarding the concentrations of metals in sediments, in general, the lowest concentration values were reported for the Danube Delta followed by those in the Danube River.
Significantly higher values were reported for tributary, especially where sampling was carried out from reservoirs or between of shipping locks.
Thus, a concentration range of 1.96-126.52 mg/kg Cu in sediments was measured in the Danube River [85] and 40 mg/kg in the Danube Delta [126], whereas, a maximum concentration of 263 mg/kg was reported in the Begej canal [84]. A similar trend was observed for Pb, Hg, Cd, As, Zn, and Cr. For example, concentration ranges of 0.42-84.75 mg/kg for Pb [85] and of 0.02-0.690 mg/kg for Hg [110] were reported in the Danube river, and maximum values of 47.45mg/kg for Pb and 0.99 mg/kg for Hg were reported by Gati et al. in the Sf. Gheorghe Branch of the Danube Delta [121], while a maximum value of 263 mg/kg Pb was measured in the Begej canal [84] and 1.7 mg/kg Hg in the Olt river reservoirs [115].
Comparable maximum values were reported in the Danube River and Danube Delta for Cd (1.33 mg/kg [85] and 1.34 mg/kg respectively [121], and Zn (217 mg/kg [85] and 209.8 mg/kg [123]), whereas, maximum values of 3.26 mg/kg Cd and 975 mg/kg Zn were measured in the Begej Canal [84]. For As, a concentration range of 1.06-16.96 was reported in the Danube river, a maximum concentration of 20.55 mg/kg was measured in the Sf. Gheorghe Branch of the Danube Delta [121], and 43 mg/kg in the Begej canal [84].
Four multi-annual studies were identified concerning the presence of metals in water, sediment, and fish [85,111,123,130]. A descendant trend in the concentrations of Pb and Zn in water samples from the Somova-Parcheş aquatic complex (Danube delta) between 2007-2012 was reported by Burada et al. [123] and attributed to ''reducing emissions from the surrounding industrial activity". In the lower section of the Danube River (km 375-km 175), mostly homogeneous evolution in time of metals concentrations in sediments was reported by Radu et al. in a six-year study [85]. There were seasonal and age-dependent dynamics of Cu and Zn in different freshwater fish in Dniester and Prut for the period between 2005 and 2010 [130].
The concentrations of heavy metals in sediments revealed seasonal variation and significant differences between the sampling sites [107,113,123]. The bioaccumulation capacity of these pollutants was studied by determining metals in microalgae [127] aquatic plants [128], plankton [122], mollusks [129], and fish [117,137]. However, the diversity of the species studied, differences in the expression of the results (dry weight or wet weight), or different target analytes led to a difficult comparative analysis of the results. Nevertheless, the studies showed correlations between the heavy metals concentrations in water, sediments and the biota, especially for As, Cd, Pb, Cu, Ni, Cr, Hg, Co, and Zn, with various bio-concentration factors (BCF) depending on the biota species [109,123,125,128,129].
Exceeding of the maximum allowable concentration of heavy metals in surface waters according to the Directive 2013/39/EU [35] and Romanian legislation (Order 161/2006) [138] concerning environmental quality standards (EQS) for priority substances in the field of water policy were frequently reported. Cd concentrations exceeded the maximum EQS of 1.5 µg/L in the tributaries West Morava (reported value of 50 µg/L) [109] and Yantra (reported value 14.9 µg/L) [111], in the Danube river near Galati (km 150) and Tulcea (km 71) town (reported values of 15.7 50 µg/L and 18.4 50 µg/L respectively) [117], and in the Biosphere Reservation of Danube Delta (reported range of 3.5-10.5 µg/L) [123]. Pb concentrations exceeded the EQS of 14 µg/L in the Danube river near Galati and Tulcea towns (reported value of 21.6 µg/L and 14.6 µg/L respectively) [117] and in the Danube Delta Biosphere Reserve (maximum value of 48.06 µg/L) [122]. For Hg, concentrations exceeding the EQS of 0.07 µg/L were reported in the tributary Yantra River (reported value 68.15 µg/L) [111], in the tributary Olt river (maximum 1.5 µg/L) [107], and in the Danube river (reported value of 0.117 µg/L) [112].  [124]. Concentrations of Cu, Cr, Co, and As exceeding the maximum allowable value of EQS according to national regulations (Order 161/2006) were reported in the Danube River [113,117,118,128], in the tributary Olt [107] and Prut rivers [130], and in the Danube Delta [122,123].
However, the values of heavy metal concentrations reported for water and sediment in the Lower Danube basin were significantly lower than those reported elsewhere. Mean concentration of 623.32 mg/kg for As, 2005.94 mg/kg for Pb, 151.09 mg/kg for Cd, 375 mg/kg for Cr, and 4.65 for Hg in sediments were recently reported in Watershed of Southwestern Ethiopia [139], which is much higher than any concentration value reported for the Lower Danube basin. Comparable values for Pb, As, and Cd but lower for Hg than in the Lower Danube basin were reported in rivers from Southern Italy [140]. In the sediments from Jarama River (central Spain) average concentrations of 55.59 mg/kg for Cu, 135.6 for Zn, 15.83 mg/kg for Ni, 1.15 mg/kg for Cd, and 35.77mg/kg for Pb were reported [141]. Higher values for Cu were reported in the Lower Danube basin by Radu et al. [85]. The measured concentration in the Danube basin for Zn, Ni, Cd and Pb exceeded the values reported for Jarama River in several studies [84,85,113,119,120,123].
The few studies conducted for water and sediments in the North West Black Sea do not allow extensive comparative analysis. A recent report on the heavy metal pollution over the last 20 years in the Baltic Sea [142] revealed concentration ranges of 28-90 mg/kg for Pb, 0.5-1.3 mg/kg for Cd, 1-4 mg/kg for Ni, 20-380 mg/kg for Cu, and 1.2-5.5 mg/kg for Co, in sediments in open sea, which are higher than those reported in the publications included in this review. In a comprehensive study on the heavy metal pollution of sediments from a coastal area of the central western Adriatic Sea [143], average concentrations of 63 mg/kg was reported for Ni, 14.4 mg/kg for Cu, 61.5 mg/kg for Cr, and 12 mg/kg for Pb, which higher than the values reported for the Black Sea is as well. In a recent study [144], the assessment of sediments quality concerning the heavy metals Cd, Cr, Cu, Pb, Zn and Mn was carried out in 2019 and 2020 for the Romanian part of MONITOX Network (32 sampling points in the system of Danube river-Danube Delta-Black Sea: Lower Danube RO-BG, Lower Danube RO, Lower Prut RO-MD border, Danube Delta RO-UA border, Danube Delta-RO and Black-Sea area-RO) using both single indices and integrated indices. The research revealed that sediments from the Black Sea area were much less contaminated with heavy metals than those from the Lower Danube (Romania), attributed to the historical pollution resulting from anthropogenic activities [144].

Microbiological Pollution
The microbiological contamination of the surface water is one of the most significant health-related problems in the Danube region [145]. The available data show that both the upstream and downstream reaches on the Danube are microbiological contaminated [146]. The main sources of contamination are uncontrolled raw sewage, discharge of untreated or inadequately treated wastewaters, and impact by diffuse sources of agricultural land and pastures [146].
Bacteria are ideal sensors for indicating microbial pollution of surface water bodies due to their rapid response to changing environmental conditions. Faecal coliforms, particulary Escherichia coli as the predominant species, and intestinal enterococci are parameters for assessing faecal pollution (standard faecal indicator bacteria (SFIB)), also showing the potential presence of pathogenic bacteria, viruses, and parasites [146]. Monitoring of the standard microbiological parameters is mandatory by legislation in the field of waters bodies intended for drinking water, irrigation, and bathing according to the Water Directive [74], Urban Wastewater Treatment Directive (European Council, 1991) [147], the Bathing Water Directive (European Parliament and Council, 2006) [148], and the Drinking Water Directive (European Council, 2020) [149]. However, no regulatory values concerning microbial faecal pollution for river water in Europe are currently set.
In order to assess the water quality in the Danube River basin, riparian countries use different methods for microbiological analysis. The method ISO 9308-2:2012 commonly used for the enumeration of E. coli and coliform bacteria in water is based on the growth of target organisms in a liquid medium and calculation of the "Most Probable Number" (MPN) of organisms by reference to MPN tables [150].
Studies published in 2014 [151] and 2017 [145] by Kirschner et al. on the microbial faecal pollution along the Danube River based on the results obtained within The Joint Danube Surveys (JDS) 2001, 2007, and 2013 reveal human faecal pollution as the primary pollution source along the whole river. The lowest Danube section showed low to moderate E. coli pollution levels [145,151]. However, sites downstream from Russenski Lom (rkm 488, Bulgaria, 46,900 MPN/100 mL) and Arges (rkm 429, Romania, 3100 MPN/100 mL) were demonstrated as the most polluted tributaries of the whole river basin. The tributaries Iskar and Jantra (Bulgaria) showed low E. coli pollution, while in Romania, Siret and Prut were critically polluted. Concerning the Low Danube, the section between Novi Sad and Velika Morava (110-2300 MPN/100 mL) showed moderate pollution values, while downstream from Zimnicea/Svistov (rkm 550) exhibited high faecal pollution (27,700 MPN/100 mL) [145].
As only the determination of the SFIB does not provide information regarding the pollution source, microbial source tracking approaches have become appropriate tools for determining the origin of microbial faecal pollution in different water ecosystems [145]. The most common methods are based on the quantitative polymerase chain reaction (qPCR) detection of host-associated Bacteroidetes populations. The human-associated faecal marker (BacHum) expressed as marker equivalents (ME) was detected in 92.4% of all the investigated Danube samples and in 100% of all the tributary samples. Furthermore, statistical analysis revealed a significant correlations between both E. coli and enterococci and human BacH marker [145]. The BacHum concentrations in the whole Danube River ranged from 250 to 1.3 ×10 6 ME/100 mL, with the highest concentration measured downstream from Arges (Romania). Tributary Rusenski Lom (Bulgaria), followed by Arges (Romania) showed the highest BacHum concentrations (4.5 × 10 6 ME/100 mL, corresponding to Arges river) [145]. In contrast to BacHum marker, the animal associated markers (BacR for ruminant, and Pig2Bac for pig) were of minor importance along the whole Danube River and major tributaries, except the Danube Delta, where the highest Pig2Bac concentration (6.9 × 10 3 ME/100 mL) was measured in the Sulina arm and Jantra tributary with the highest BacR concentration (2.9 × 10 3 ME/100 mL) [145].
Apart from study related to JDSs, very few studies concerning microbiological pollution on the Lower Danube basin were identified.
An industrial area of the Danube, near Galati town (rkm [155][156][157][158], was investigated from a microbiological point of view for a period of four months, from June to September 2010 [152]. The lowest value for total coliforms of 4.5 × 10 2 MPN/mL was measured in July, while the highest value of 2 × 10 4 MPN/mL in August. For faecal coliforms, the lowest pollution (4.5 × 10 2 colony-forming unit (CFU)/mL) was recorded in July, while the highest value (20 × 10 3 CFU/mL) in August. Maximum E. coli pollution was recorded in September (6.4 × 10 3 CFU/mL) [152].
A total of 32 different sites of MONITOX network were selected in the Lower Danube region and Romanian Black Sea coast for a recent study conducted in June 2019 and June-July 2020, concerning microbiological pollution in the Black Sea Basin [153]. Heterotrophic bacteria and total coliforms were used as microbiological indicators. The coliform bacteria were identified in all water samples ranging from 130 CFU/100 mL (Ostrov) and 250,000 CFU/100 mL (Calarasi/Silistra) in the Lower Danube sector and from 10 CFU/100 mL (Izmail) to 70,000 CFU/100 mL (Sfantu Gheorghe arm in the Danube Delta). The heterotrophic bacteria ranged from 155 CFU/mL (downstream Braila town) to 6080 CFU/mL (upstream Siret river) in the Lower Danube sector and from 111 CFU/mL (Black Sea, Mangalia town) to 17,000 CFU/mL (Sfantu Gheorghe arm) in the Danube Delta-Black Sea area [153]. The study enabled a comparison of microbiological contamination of surface water in the periods before (2019) and after the (2020) COVID-19 lockdown, demonstrating a decrease of bacteria load in 2020 in all the samples collected from Black Sea coast, Danube branches, Danube-Black Sea confluence, and Danube River downwards of Galati town. For these water samples, an ongoing work is carried out in the frame of the EU-funding project BSB27-MONITOX for several CECs at "Dunarea de Jos" University of Galati, Romania using a high-resolution mass spectrometry technique.
Only a few studies on fecal pollution in the North West coast of the Black Sea were conducted in the last 10 years. Comparison between faecal and organic pollution of water samples from the Black Sea area, Romania and water samples from the Aegean Sea, Kavala, Greece were presented by Vasile et al. in 2020 [154]. Higher values of fecal indicators were measured in Black Sea compared with the Aegean Sea coast. Thus, total mesophilic aerobic bacteria in Black Sea area (Romania), ranged from 1.11 × 10 2 to 1.70 × 10 4 CFU/mL and of coliforms between 250 MPN/mL to 7000 MPN/mL. In Aegean Sea water, no coliforms were found and the number of heterotrophic bacteria was 1.50 × 10 CFU/mL [154].
Among the study on the eastern part of the Black Sea, a recent one investigating bacterial pollution along coastal areas in Turkey, between May 2017 and February 2018, revealed a high degree of contamination in the study area [155]. Total coliform levels ranged from 1.0 × 10 3 CFU/100 mL to 3.14 × 10 8 CFU/100 mL. The fecal coliform levels ranged from 2.0 × 10 2 CFU/100 mL to 9.04 × 10 7 CFU/100 mL. Bacteriological pollution increased in all sites in summer months [155]. A comparison of the results for the seawater in Romania and Turkey is difficult due to different methods and different expressions of the results.

General Overview
Continuous monitoring over the last two decades, either through point surveys or complex surveillance programs covering several years, has led to changes and continuous updates of the legislation (e.g., Commission Decisions EU 2018/840 [26] and 2020/1161 [45]), to the development of large substances databases (e.g., NORMAN [60]), and to new approaches in pollutant prioritization and risk assessment methodology [33]. The literature on monitoring programs of organic and inorganic pollutants published during 2010-2021 for the Lower Danube basin and North West Black Sea region show that this research area has been extended in the last year ( Figure 8 due to the higher concern of political entities and, on the other hand, the development of more sensitive analytical techniques. Siret river) in the Lower Danube sector and from 111 CFU/mL (Black Sea, Mangalia town) to 17000 CFU/mL (Sfantu Gheorghe arm) in the Danube Delta-Black Sea area [153]. The study enabled a comparison of microbiological contamination of surface water in the periods before (2019) and after the (2020) COVID-19 lockdown, demonstrating a decrease of bacteria load in 2020 in all the samples collected from Black Sea coast, Danube branches, Danube-Black Sea confluence, and Danube River downwards of Galati town. For these water samples, an ongoing work is carried out in the frame of the EU-funding project BSB27-MONITOX for several CECs at "Dunarea de Jos" University of Galati, Romania using a high-resolution mass spectrometry technique. Only a few studies on fecal pollution in the North West coast of the Black Sea were conducted in the last 10 years. Comparison between faecal and organic pollution of water samples from the Black Sea area, Romania and water samples from the Aegean Sea, Kavala, Greece were presented by Vasile et al. in 2020 [154]. Higher values of fecal indicators were measured in Black Sea compared with the Aegean Sea coast. Thus, total mesophilic aerobic bacteria in Black Sea area (Romania), ranged from 1.11 × 10² to 1.70 × 10 4 CFU/mL and of coliforms between 250 MPN/mL to 7000 MPN/mL. In Aegean Sea water, no coliforms were found and the number of heterotrophic bacteria was 1.50 × 10 CFU/mL [154].
Among the study on the eastern part of the Black Sea, a recent one investigating bacterial pollution along coastal areas in Turkey, between May 2017 and February 2018, revealed a high degree of contamination in the study area [155]. Total coliform levels ranged from 1.0 × 10 3 CFU/100 mL to 3.14 × 10 8 CFU/100 mL. The fecal coliform levels ranged from 2.0 × 10 2 CFU/100 mL to 9.04 × 10 7 CFU/100 mL. Bacteriological pollution increased in all sites in summer months [155]. A comparison of the results for the seawater in Romania and Turkey is difficult due to different methods and different expressions of the results.

General Overview
Continuous monitoring over the last two decades, either through point surveys or complex surveillance programs covering several years, has led to changes and continuous updates of the legislation (e.g., Commission Decisions EU 2018/840 [26] and 2020/1161 [45]), to the development of large substances databases (e.g., NORMAN [60]), and to new approaches in pollutant prioritization and risk assessment methodology [33]. The literature on monitoring programs of organic and inorganic pollutants published during 2010-2021 for the Lower Danube basin and North West Black Sea region show that this research area has been extended in the last year ( Figure 8 due to the higher concern of political entities and, on the other hand, the development of more sensitive analytical techniques. Most of the papers focused on the active substances and only a few on the metabolites or biodegradation products [15,26,39,49,53]; -Studies are repetitive, which is helpful in terms of pollutants dynamics but, on the other hand, not only the most frequently studied substances should be considered but also those with high risk and relevance for the environment; -Most of the authors reported occurrence of CECs without justifying the selection of compounds. Criteria as ''substances that commonly have been detected" or "ubiquitous presence" were appealed. Priority substances listed by the EU regulations or NORMAN databases were mentioned in four papers [26,44,51,77]; -Insufficient attention was paid to the natural variability of the aquatic environment, leading to inadequate data collection (e.g., substances that are susceptible to degradation caused by sunlight exposer or absorption of the pollutant on suspended particulate matter were rarely discussed). One paper concerning CECs analysis in suspended particulate matter was identified [45]; - The majority of the reviewed studies concerning CECs monitoring were based on 'grab-sampling' often with no intra-day repetition. The limitations of such an approach results in snap shots data on pollutant concentration for a specific point in time.
Composite sampling that considered flow fluctuations was performed in 1 of 24 studies [15]; furthermore, chemical stability of the target analytes during storage until analysis was investigated in only one study [45]; -Few antibiotics are usually monitored in the studies cited in Table 1 (pharmaceuticals) despite the risk posed to aquatic and terrestrial organisms and possible occurrence of bacteria resistance; - The analytical approach of targeted screening with low resolution mass spectrometry (e.g., triple quadrupoles) used in the majority of reviewed studies resulted in numerous substances such as metabolites or transformation products going undetected. Among 24 studies concerning CECs monitoring, tentative identification using HRMS-MS was carried out in one publication [26]. Multiresidue methods allowing targeted (quantitative) and non-targeted (qualitative) screening should become standard procedures for CECs analysis as well as combining analytical methods with metabolomics for the identification of uncommon chemicals, metabolites, and degradation product(s); - To determine and predict trends, multivariate statistical methods (factor analysis of principal component analysis (PCA) were applied as well as indicators of pollution status, as Hazard Quotient (HQ), Enrichment Factor (EF), Geo-accumulation index (Igeo), and Ecological risk index (RI) were determined in several papers concerning persistent pollutants as PAHs, OCPs, and metals [2,46,51,84,100,107,121,144]; -Regarding CECs, the basis for risk assessment was rarely discussed. Risk coefficients (RQ) value based on the ratio of the Predicted/Measured Environmental Concentration (PEC/MEC) and Predicted No-Effect Concentration (PNEC) was performed in three papers for endocrine disruptors [61,68,69] and in one for pharmaceuticals, pesticides, and other CECs [26]; the fate of pesticides in sediments and risk assessment according to their physico-chemical properties was discussed in one paper [44]; -Spatial distribution of the contaminants was highlighted in several publications [45,49,53,61,100]; -Pollution emission sources were investigated for PAHs [96], OPCs [94], pharmaceuticals [51,53], and heavy metals [106,126,128,156]. Untreated and inadequate treated waste water was demonstrated as being the main source of organic pollution in the low Danube basin. The metals pollution is associated with industrial and municipal sources; -Seasonal variations were reported for all contaminants classes, probably due to the temperature related processes of biotransformation and absorption. Similar phenomena were reported for pharmaceuticals in Swedish aquatic environment [157] and for herbicide and insecticide in surface waters in Spain [158]; - The Dniester River is one of the less-studied rivers in Europe; -No report was identified concerning pharmaceuticals residues in seawater or sediments for the North-West Black Sea coast; -Studies were heterogeneous and, generally, did not allow comparisons; - The pollution level in the Lower Danube basin was in agreement with other European rivers such as the Rivers Elbe (Germany) [159], Lis (Portugal) [160], or Po (Italy) [161]; -Future research should be conducted in the investigation on the effect of emerging pollutants mixtures to different biological systems, on the development of bacterial resistance, and the fate of CECs in the environment (transport, bioaccumulation, degradation). Effective wastewater treatment and reliable fate and toxicity assessment are needed.

Conclusions
It is unrealistic to believe that monitoring and screening programs of today can embrace all known pollutants. However, in recent years, important steps have been taken toward improving analytical methods, risk-assessment approaches, and regulatory bases.
The publications herein reviewed revealed the occurrence and spatial distribution of persistent and emerging micropollutants in surface waters, sediments, and biota in the Low Danube basin and North West Black Sea region. The current situation of these aquatic environments is of great importance in light of the recent EU Directives.
This review showed that pharmaceuticals were determined in the area of study in the following decreasing order of concentrations: carbamazepine >sulfamethoxazole >diclofenac >trimethoprim >ibuprofen. Regarding pesticides, the highest concentrations were reported for carbendazim >metalochlor >atrazine. The reported metals, in the following decreasing order of concentration, were: Fe > Zn > Cu > Pb > Cr > Ni > As > Cd > Hg.
These findings show that further studies concerning the fate and bioaccumulation capacity of the contaminants in different environmental compartments (water, sediment, and biota) are needed in order to predict their possible impact to non-target organisms.