Seasonal distribution, source identification, health and eco-toxicological risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in water and sediment from the Danube River in Hungary

Seasonal distribution, source identification, health and eco-toxicological risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in water and sediment from the Danube River in Hungary | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Seasonal distribution, source identification, health and eco-toxicological risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in water and sediment from the Danube River in Hungary Ruqayah Ali Grmasha, Csilla Stenger-Kovács, Osamah J. Al-sareji, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2765619/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Danube is one of the largest transboundary rivers word-wide, having several tributaries. The discharges from industrial activities and wastewater treatment facilities affect the river's aquatic environment. These discharges pose a serious risk to aquatic life by degrading the water and sediment quality. Therefore, 16 Polycyclic Aromatic Hydrocarbons (PAHs) compounds in six different locations were examined along the river over 12 months to investigate the temporal and special variations of the compounds in water and sediment. The findings highlighted a broad variance range in PAHs concentration in water within a year, ranging from 224.85 ng/L in summer to 365.87 ng/L in winter, whereas PAHs in sediment samples recorded values ranging from 316.72 ng/g in dry weight in summer to 422.98 ng/g in dry weight in winter. The overall results indicate that the putative anthropogenic sources of PAHs were of pyrolytic and pyrogenic origin, with pyrogenic sources being more prominent. Generally, except for Acenaphthylene and Fluorene concentrations, the eco-toxicological concerns for the aquatic environment of the Danube River do not pose a significant threat. In addition, the combined impact of the 16 PAHs pollutants in sediments suggests a low chance for negative biological impacts and low ecological risk. The total ILCR for both children and adults is more than 1/10 4 in all seasons, with the highest values recorded in spring followed by winter time, which becomes a matter of urgency. Earth and environmental sciences/Environmental sciences/Environmental chemistry Earth and environmental sciences/Environmental sciences/Environmental impact Danube River Water Diagnostic ratios Polycyclic aromatic hydrocarbons Sediment Risk assessment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Freshwater is a valuable and scarce resource for both individuals and ecosystems. The protection and preservation of the freshwater are increasingly important with the drive to promote food security and sustainable development growth agenda for life below water. The majority of the world's biggest cities were constructed on or near regions of freshwater, mostly rivers [ 1 ]. Most research on aquatic systems is primarily concerned with the effects of anthropogenic activities and natural phenomena, such as volcanism and biological processes, on both human health and ecology [ 2 ]. Population growth and corresponding increases in industrial, agricultural, and urban activities have increased the emission levels of different anthropogenic toxins into the ecosystem, among them Polycyclic Aromatic Hydrocarbons (PAHs) [ 3 , 4 ]. Due to their toxicity, persistence, bioaccumulation, and possible detrimental health impacts on living organisms, these compounds have attracted considerable attention globally [ 5 , 6 ]. Rivers, especially those in regions with heavy human activities, play a crucial role in transferring and transforming numerous contaminants and serve as an important connection between terrestrial and marine ecosystems [ 7 ]. Rivers are significant conduits for the entry of land-based natural materials and pollutants directly into the seas; approximately 85% of the materials from land are transferred to the sea in several ways, such as municipal and industrial effluents, atmospheric deposition, riverine runoff, urban and agricultural runoff, sewage outfalls, and petrogenic sources [ 8 – 10 ]. Among these materials, PAHs constitute a pervasive class of chemicals generated by the fusion of two or more aromatic rings of hydrogen and carbon atoms in diverse configurations [ 11 ]. Because PAHs possess concerning characteristics such as teratogenicity, carcinogenicity, and gene mutation, the United States Environmental Protection Agency (USEPA) and the European Union (EU) have identified 16 PAHs as priority pollutants among the hundreds of PAHs in the environment [ 12 – 14 ]. Due to their high octanol–water partition coefficient and hydrophobic lipophilicity, PAHs entering the water get adsorbed on suspended particulate matter and ultimately settle into the sediment [ 12 ]. Consequently, river sediments are susceptible to PAHs accumulation and release. Moreover, they are commonly used as an indicator for detecting probable emission sources and determining the exposure risk of PAHs to benthic biotas [ 15 ]. Therefore, the environmental fate and the possible ecological risk related to PAHs are serious matters of public concern [ 16 ]. As a result, PAHs have been the subject of environmental monitoring research for catchments worldwide. Numerous studies have employed water and sediment as important matrices for evaluating PAHs contamination in rivers [ 17 – 19 ], lakes [ 20 , 21 ], dams [ 22 ] as well as seas [ 23 , 24 ]. The Water Framework Directive (WFD) obligates the European Union member states to achieve a satisfactory quantitative and qualitative assessment of the status of all bodies of water [ 25 ]. Nonetheless, some bodies of water (for instance Danube River in Hungary) have not yet reached this objective, and emergent pollutants such as PAHs are not yet controlled [ 26 ]. Studies that have examined the PAHs level in the Danube River within the Hungarian regions can be categorized into two groups. The first group examined the upper area of the Danube River (i.e., the borders between Hungary and Slovakia) and the other group focused on the capital city (Budapest). Regarding the upper area of the Danube, Nagy, et al. [ 27 ] investigated the PAHs concentrations and distribution in the surface water and bed sediments of the Hungarian upper section of the Danube River as well as in the Moson Danube branch during the period of 2001 to 2010. They found that the concentrations of 16 PAHs in water samples ranged from 25 to 1208 ng/L, which were dominated by 2–3 rings of PAHs. The 16 PAHs concentrations in sediments ranged between 8.3 and 1202.5 ng/g in dry weight (dw). The same research group also continued the investigation within the upper part of the Danube River and its tributaries in the Autumn of 2012. The findings revealed that the total PAH concentrations in water samples ranged from 67 to 96 ng/L, and the PAHs concentrations ranged between 35.2 and 288.3 ng/g dw in sediments [ 28 ]. In another work, conducted in the upper part, the PAHs level in water from 2007 up to 2010 was reported to range between 25 and 357 ng/L [ 29 ]. The same group further investigated the PAHs concentration in water and sediments in the Moson Danube Arm and the upper area of the Danube River for the period 2014 to 2015 [ 30 ]. The total concentration of PAHs in water was found to vary from 16 to 133 ng/L, and the concentration in sediments varied in the range of 118 and 283 ng/g. Comparing the findings from the long-term research by Nagy, et al. [ 29 ] over the years, it can be noticed that concentrations of PAHs in water and sediment decreased dramatically. Moving to the second part of the PAHs level examination in the Danube River which was in the capital city, Visca, et al. [ 31 ] evaluated the contamination in terms of PAHs level in the Danube River (only water samples) passing through Budapest with three locations inside the city. The samplings were performed in April 2017, November 2017, and October 2018. It should be mentioned; however, that information about the PAHs range is missing[ 31 ]. Caracciolo, et al. [ 32 ] also conducted a study to monitor the PAHs level in Budapest at three different points without, however, revealing the coordinates of those sampling points. Their measurement was for PAHs level in water in April and November 2017. The finding revealed that total PAHs concentration ranged from 15.9 to 53.2 ng/L. The overall conclusion regarding the studies conducted for measuring the PAHs level in the Danube River within the framework frame of Hungarian borders is that most of these investigations were conducted in the upper part of the river, and with the latest sampling campaign in the period of 2014–2015 [ 30 ]. Other studies have reported PAHs level in the area of Budapest, however, with unclear conclusions due to missing information such as the PAHs ranges, sampling locations, and overlooking the sediments part, which is considered an important PAHs sink [ 31 , 32 ]. With a view to narrowing the gap in knowledge regarding PAHs concentration in the Danube River, the present investigation was designed to adequately investigate the spatial and temporal PAHs contamination of the river basin in Hungary. The main objectives are: 1) The spatial and temporal assessment of PAHs contamination in the Danube River; 2) The identification of the primary sources of PAHs contamination; 3) The evaluation of the eco-toxicological risk assessment of PAHs and; 4) The identification and qualification of the Incremental Lifetime Cancer Risk (ILCR), for both adults and children. 2. Materials And Methods 2.1 Chemicals All solvents used were High-performance liquid chromatography (HPLC)-grade and acquired from Fisher Chemical Co. (USA), with a minimum purity of 99%. Supelco (Bellefonte, PA, USA) supplied the reference standards (QTM PAH-Mix, 2000 µg/mL) of the 16 PAHs. Table S1 shows the abbreviation and chemical structure information of the considered 16 prioritized PAHs. Sigma-Aldrich, USA, supplied solid-phase extraction membranes (ENVI™-18 DSK SPE Disk, diameter 47 mm), Sodium sulfate anhydrous, Silica gel (desiccant ~ 2–5 mm), as well as PAH recovery standards. Sodium Sulfate Anhydrous and Silica gel were put in a furnace (FI 600 − 60, Borel) at a temperature of 500°C for 4 h to remove any moisture or organics before being transferred to a desiccator for storage until usage. In the experiments, Milli-Q water with a resistivity of 18.2 MΩ•cm at room temperature and a total organic carbon value of less than 5 ppb was utilized. Prior to each measurement, glassware was first cleaned using ultrasonic cleaners (Heidolph™, Fisher Scientific), washed afterwards with acetone, n-hexane, methanol, and dichloromethane to eliminate background pollutions and dried at 105°C before use. 2.2. Study area With a length of 2,780 km, the Danube is the second-longest river in Europe; its catchment area is 801,500 km 2 . The water of the Danube River Basin is used for a variety of reasons, including the production of drinking water, industrial and agricultural activity, leisure, hydroelectric power generation, and transportation. The length of the Hungarian Danube segment is 417 km (1850–1433 river kilometres (rkm)). Six locations were investigated along the proposed segment. Figure 1 depicts the sampling locations along the Danube River. Both S1 and S2 are representatives of the northern sites in our campaign in an area affected by both agricultural and industrial activities. Site 3 was taken at the heart of Budapest city, which happens to be situated downstream of a wastewater treatment plant, the second largest plant in the city (processing 180,000-200,000 m 3 /day of wastewater). Site 4 is located downstream of a polystyrene factory which is impact-resistant polystyrene and expandable polystyrene. Site 5 is located downstream of different industries, such as paper mill companies and electricity suppliers. Site 6 is located furthest south with respect to the other samples. This site was selected to be representative of the collective anthropogenic activities upstream. Table S2 shows the coordinates of the sampling sites. 2.3. Samples collection and pretreatment Throughout the study, the water and sediment samples were collected on a monthly basis, from February 2022 to February 2023, over a 12-calendar month period. Water samples were obtained at depths from 5 to 30 cm in 1 L brown glass containers that had been previously cleaned and then transported to the laboratory for analysis. Sediment samples were collected from the river bed at a depth 0–10 cm and were then placed in clean polyethylene bags and transferred to the laboratory for further processing. Sediment samples were dried at 25 o C and were ground and sieved through a 2 mm mesh to remove any roots, debris, or large particles. Finally, they were dry-frozen until the time of further use. 2.4. Extraction procedures of PAHs 2.4.1 Water sample extraction procedure Solid phase extraction (SPE) was used to extract one litter of filtered water samples for the purpose of water sample extraction. The water sample extraction followed the previously reported procedures [ 33 – 35 ]. An SPE membrane was pre-washed using six milliliter of dichloromethane (DCM) before conditioning (activation) with six milliliter of methanol and six milliliter of ultrapure water, and ten milliliter of methanol. A ten microliter of surrogate standard mixture solution was added to one litter of the water sample. This step was to enrich the sample, which was then passed through the SPE at a flow rate of 3 mL/min. After the extraction was finished, a vacuum pump was utilized to dry the column, and six milliliter of dichloromethane was added to soak it for 5 minutes before elution into a clean glass test tube. By using nitrogen, the eluate was concentrated to 0.5 mL prior to the addition of ten microliter of standard mixture solution. Then the samples were analyzed using gas chromatography-mass spectrometry (GC–MS) analysis. 2.4.2 Sediment sample extraction procedure The freeze-dried sediment samples were pulverized using a mortar and pestle and were sieved using a 100-mesh to remove large particles. Sediment samples dried and homogenized were put into brown glass vials for further laboratory analysis. The sediment sample extraction followed the previously reported procedures [ 33 – 35 ]. A sample of 2 grams was precisely weighed out. Five milliliters of acetone/n-hexane (1:1, v/v) and a standard surrogate solution were added to the test tube. Afterwards, they were vortexed for 60 seconds, followed by 15 minutes of ultrasonic extraction in a water bath. The test tubes were then centrifuged for 20 minutes at 2000 rpm to separate the solid and liquid phases. Using a Pasteur pipette, the supernatant was transferred into another clean test tube. Then, five milliliters of a 1:1 mixture of acetone and n-hexane was added to each sample. In one test tube, the extracts were mixed, and activated copper was added for desulfurization. Then, sodium sulfate anhydrous was applied to eliminate water, followed by a concentration step to 0.5 milliliters using a nitrogen-blowing concentrator. Finally, an internal standard solution was added for the GC/MS analysis. As described in previous work by the present authors [ 36 ], PAHs were measured using GC–MS (gas chromatography mass spectrometry) type Agilent 6890 N 5975C mass selective detector, USA system. Helium was utilized as a carrier gas at 1.5 ml/min using an HP-5MS gas chromatography column (30 m 0.32 mm 0.25 µm). The selective ion scanning (SIM) mode was utilized for quantitative analysis. The injector temperature was set to 300 o C. The oven temperature was programmed as follows: the initial temperature was set to 100°C for one minute, after which it increased to 300°C at a rate of 8°C per minute and remained at 300°C for 39 minutes. Triplicate measurements were recorded for each sample resulting in a relative standard deviation of less than 10.2%. 2.4.3 QC/QA In this study, the quality controls included triplicate samples, matrix spike standards, calibration standards, a procedural blank, and detection limits. Prior to each measurement, the glassware was first cleaned using ultrasonic cleaners, washed afterwards with acetone, n-hexane, methanol, and dichloromethane to eliminate background pollution and dried at 105°C before use. Utilizing the dry weight approach, the concentration of 16 PAHs in sediment samples was determined. The lowest detection limits (LOD) were determined using analyte concentration and a 3-fold signal-to-noise ratio [ 37 , 38 ]. The range of LOD for water was between 0.02 and 0.59 ng/L, while for sediment was between 0.37 and 0.96 ng/g in dw. PAHs recovery was determined by spiking water and sediment samples with standard solutions. A procedure blank (solvent), a spiked blank (standards added to solvent), and sample triplicates were conducted for each sample. Analysis of method blanks proved the absence of detectable PAH contamination. The recovery ranges for 16 PAHs in water, and sediment samples were 91.5%± 4.3–104.1% ± 7.6% and 86.8%± 5.5–99.2% ± 6.2%, respectively. For water samples, the recovery ranges of the spiking standards were 92.8% ± 4.5–109.7% ± 6.9%, and for sediment samples, they were 90.5% ± 8.3–97.4%± 4.4%. For recovery, the concentrations of 16 PAHs were adjusted. Reference and blank samples were measured to confirm the accuracy of the analysis. Moreover, each sample was measured three times, resulting in a relative standard deviation between 1.7% and 10.4%, which is within the acceptable limit (< 25%). Mean values are presented for the measurements. The data in this study were tested to the Kolmogorov-Smirnov normality at a significance level of 0.05. 3. Results And Discussion 3.1. PAHs spatial distribution and seasonal variation in water and sediment 3.1.1. Water Figure 2 shows the 16 identified PAHs concentrations in the water of the Danube River. The total PAHs contents in water for all seasons were ranging from 224.85 (in summer) to 365.87 ng/L (in winter). The seasonal concentration variation of PAHs was 283.10-541.91 ng/L in winter, 198.65-404.37 ng/L in spring, 160.03-324.63 ng/L in summer, and 228.26-378.19 ng/L in autumn. The concentrations of LMWPAHs (low molecular weight PAHs, 2–3 rings): Nap, Acy, Ace, Fl, Phe, and Ant) in water samples of the Danube River were greater than the concentrations of HMWPAHs (high molecular weight PAHs 4–6 rings): Flu, Pyr, BaA, Chr, BaP, DBA, BkF, BbF, IND and BghiP, and in all four seasons. The HMWPAHs recorded the highest values during the summer season (Fig. 2 ), being congruent with literature studies from other countries [ 39 ]. In Fig. 3, the PAHs profile in water and sediment is presented. Ace was found to be the most predominant component, with levels between 16 and 47.86 ng/L, followed by Nap, Acy, FI, Ant, and Phe with values between 11.9 and 45 ng/L, 15.5 and 44 ng/L, 7.17 and 40.9 ng/L, 10.5 and 40.2 ng/L, and 11 and 36 ng/L, respectively. The highest HMWPAHs in water samples were Bghip (11.4–28.3 ng/L), IND (8.25–17.99 ng/L), DBA (9.66–17.88 ng/L), BaP (9.13–17.5 ng/L), BkF (7.29–17.2 ng/L), BbF (9.56–17.1 ng/L), Pyr (7.81–14.99 ng/L), Chr (10.16–14.70 ng/L), Flu (5.99–11.05 ng/L), and BaA (8.2-10.13 ng/L). The concentrations of ∑PAHs in the water of the Danube River were greater in the winter, spring, and autumn seasons compared to the summer season, which was attributed to the lower temperatures during these sampling times. The variation in the patterns of PAHs in water throughout various sample periods (Fig. 2 ) is likely related to connected with the PAHs molecular weight and degradation, which is more evident during warm seasons [ 40 ]. In contrast to HMWPAHs (4–6 rings) which have a poor water solubility and dissolution rate and are, hence, more resistant to decomposition, LMWPAHs (2–3 ring PAHs) are more degradable and soluble during warm seasons. The 2–3 rings PAHs are higher in cold seasons, and 4–6 rings PAHs are higher in the hot season (Fig. 3); this is because of the reduction in the content of LMWPAHs in water. Furthermore, the current data reveal that PAHs concentration in water also varies across the sampling sites. The highest concentrations of PAHs in water were found at the S3 sampling location during cold seasons (Fig. 2 ), in conjunction with high amounts of LMWPAHs, indicating a local origin of recently generated LMWPAHs resulting from atmospheric deposition caused by vehicular and industrial emissions from various plants. The ∑PAHs concentrations in the water samples in all seasons (ranges from the lowest of 224.85 in summer to the highest of 365.87 ng/L in winter) are higher than those found in the Danube River and tributaries (67–96 ng/L) from the territory of Hungary [ 28 , 29 ], Danube River (16–133 ng/L) [ 30 ], Danube River measurements ( 15.9 to 53.2 ng/L) [ 32 ]. Contrarily, these concentrations are lower than those found in the Raba River (the largest Danube tributary in Hungary), with a range between 41–437 ng/L [ 27 ]. These variations are likely due to the difference in the selected sampling sites. This study deliberately selected sites where the potential source of contamination lies ahead. Generally, these concentrations are also lower than those found in other countries such as the Tiber River in Italy, Humen River, Bai Chao and Chaobai Rivers in China, with PAHs range of 10.3–951.6 ng/L, 311.13–1012.80 ng/L, and 55–882 ng/L, respectively [ 18 , 41 , 42 ]. In addition, it was similar to those reported in China by Chen, et al. [ 43 ] in the Yinma River (23.2-386.9 ng/L). 3.1.2 Sediment The concentrations of PAHs in sediments are presented in Fig. 4 . PAHs concentrations in all four seasons for the sediment samples were ranging from 316.72 in summer to 422.98 ng/g dw in winter. The concentration variation of PAHs within seasons is as follows: 313.78-622.78 ng/g in winter, 312.75-595.41 ng/g in spring, 215.16-465.49 ng/g in summer, and 311.34-491.78 ng/g in autumn. The variance of PAHs in the sediments of the Danube River indicates that PAHs levels vary with sampling sites, indicating the anthropogenic sources along the Danube River, as well as with seasons, given that the samples were gathered at various periods and places. Contrarily to the reported concentrations of PAHs in water, HMWPAHs are more prevalent than LMWPAHs in sediments of the Danube River (Fig. 3), which is in accordance with other studies [ 17 , 44 ]. LMWPAHs had greater water solubility owing to lower octanol-water coefficients and were more volatile than HMWPAHs, which makes them record the lowest concentrations values in sediment samples [ 45 ]. In contrast, HMWPAHs have low water solubility, greater partitioning coefficients, and high hydrophobicity in aqueous conditions [ 46 ]. BaP, Chr, BbF, and BkF are the most predominant PAHs in sediments samples, with records ranging between 28.79 and 52.47 ng/g, 16.16 and 50.82 ng/g, 22.94 and 42.62 ng/g, as well as 14.97 and 42.42 ng/g, respectively-followed by BkF, Pyr, Bghip, and BaA with values between 26.32–36.85 ng/g, 11.41-36.00 ng/g 13.34–33.81 ng/g, and 10.17–32.88 ng/g, respectively. Consistent with water samples, S3 recorded the highest PAH concentration (Fig. 4 ). IND and Flu had the lowest concentrations with values ranging from 17.01–29.38 ng/g and 9.00-21.45 ng/g, respectively. LMWPAHs that had the highest concentrations are Ant (13.21–24.86 ng/g), FI (11.90–23.30 ng/g), and Phe (14.59–23.21 ng/g), while Acy, Nap, and Ace scored the lowest concentration ranges of 11.67–18.46 ng/g, 14.01–19.23 ng/g, and 11.20–22.00 ng/g, respectively. No distinctive patterns were identified for PAHs in sediments samples during the different seasons indicating the sediments independence of temporal variations. Sediment pollution assessed by total PAHs concentrations may be categorized as follows: (A) low polluted (less than100 ng/ g), (B) moderately polluted (between101 and 1000 ng/g), (C) highly polluted (between 1001 and 5000 ng/g), and (D) very polluted (more than 5000 ng/g) [ 47 ]. Consequently, the pollution levels of the sediments from the Danube River can be categorized as low pollution. The seven carcinogenic PAHs (CPAHs) account for most of the total PAHs ranging from 181.09 in summer to 240.02 ng/g in winter, with the CPAHs exhibiting the same spatial distribution as ΣPAHs in S3. Figure 5 shows the 7CPAHs distribution in water and sediment along the Danube River in all seasons. Variations in the spatial distribution of PAH concentrations in sediments can be attributed to various factors [ 4 , 21 , 44 , 48 ], such as (1) discharge of untreated municipal wastewater, traffic emissions, industrial activities, and fuel consumption; (2) various hydrodynamic systems related to meteorological conditions that can stimulate resuspension and re-deposition of the sediments; (3) alteration in sediment textural characteristics based on spatial properties of sampling sites and (4) the presence of redox conditions in sediments and PAH biodegradation. The PAHs maximum concentration in site 3 sampling locations can be due to the position of S3 inside Budapest and downstream of the wastewater treatment plant. The PAHs concentrations in wastewater are a significant cause of concern for the industry. PAHs are regarded as hazardous components because of their highly toxic and polluting possibilities, which can persist for many decades in the environment, and their carcinogenic, genotoxic, and mutagenic effects, which can cause irreparable harm to individuals' health [ 49 , 50 ]. PAHs pollution can occur in wastewater effluent when they are not eliminated, and as a result, they can enter river water from these sources [ 51 – 53 ]. The concentrations of PAHs in sediment samples from the Danube River (316.72 in summer − 422.98 ng/g, dw in winter) were higher than those detected in the Hungarian upper section of the Danube River and its tributaries (35.2 to 288.3 ng/g) [ 28 ] and Danube River and Moson Danube Arm (Hungary) (118–283 ng/g ) [ 30 ] but lower than those reported in Hungarian upper section of the Danube River and the Moson Danube branch (8.3 to1,202.5 ng/g) [ 27 ]. Generally, the measured PAHs concentrations in the present study were comparable to those detected in Soltan Abad River, Iran (180.3–504.0 ng/g) as well as Ovia River, Nigeria (5.25-573.33 ng/g) [ 39 , 54 ]. Followed by the lower comparison to the concentrations that were reported by Liu, et al. [ 55 ] in Rivers in Shanghai, China (248.89–36198.23 ng/g). 3.2 Sources identification ratios of PAHs in water and sediment samples Based on the average concentrations of individual PAHs in water and sediments for each sampling season (winter, spring, summer, and autumn), the following diagnostic ratios were determined to identify the dominant sources and the related emission routes. The precise meaning of each ratio related to Flu/(Flu + Pyr), LMW/HMW, Flu/Pyr, BaA/(BaA + Chr), IND/(IND + BghiP), IND/BghiP, and BaA/(BaA + Chr) with the determined ratios are given in Table S3 and Table S4 for water and sediments, respectively. According to the literature, a ratio of BaA/(BaA + Chr) less than 0.2 suggests that PAHs are mostly generated from petrogenic inputs (liquid fuel discharges), a ratio between 0.2 and 0.35 shows that PAHs are sourced from mixed sources (petrogenic/pyrogenic), and a ratio greater than 0.35 indicates that PAHs are primarily formed from pyrogenic - combustion of solid fuel - natural sources such as biomass and coal. A ratio of Flu/ (Flu + Pyr) less than 0.4 indicates that PAHs originate from petrogenic inputs, a ratio between 0.4 and 0.5 indicates that they are derived from pyrolytic (burning of liquid fossil fuels and crude oil, vehicles), and a ratio greater than 0.5 indicates that they are sourced from pyrogenic - combustion of solid fuel. A ratio of IND/ (IND + BghiP) lower than 0.2 indicates that PAHs originate from petrogenic inputs, a ratio between 0.2 and 0.5 indicates that they are derived from pyrolytic sources, and a ratio greater than 0.5 indicates that they are sourced from pyrogenic sources. The ratios plot of BaA/(BaA + Chr) against Flu/(Flu + Pyr) and ratios of BaA/(BaA + Chr) against IND/(IND + BghiP) are presented in Fig. 6. Generally, LMWPAHs originate from oil or fuel spills and have a short lifetime in the ecosystem, whereas HMWPAHs arise from combustion products, pyrolysis, or petrogenic origins [ 4 ]. As it is shown in Fig. 6, Flu/(Flu + Pyr) ratios are between 0.4–0.5 in all seasons except for summer for water samples, while the opposite was observed in sediment samples, indicating pyrolytic sources in both cases. Furthermore, these ratios were 0.5 were in autumn for sediment samples which explains that wood or coal, and grass combustion are the main PAHs origins. The BaA/(BaA + Chr) ratios for both water and sediment samples were more than 0.35, indicating pyrogenic sources, except for the summer season in sediments which are scattered and clustered between 0.2 and 0.35, indicating mixed sources (petrogenic/pyrogenic). The ratios of IND/(IND + BghiP) for water samples were more than 0.5 in the spring season only and for all four seasons, except for spring, for sediments samples, suggesting the contribution of combustion of solid fossil fuel-like biomass and coal in agricultural regions. The ratios of IND/(IND + BghiP) were between 0.2 and 0.5 in the winter, summer, and autumn seasons for water samples and in the spring season for sediments samples indicating pyrolytic sources. The BaP/BghiP ratio in both water and sediment sampling was more than 0.6 in all sampling seasons, indicating PAHs of petrogenic origin. Overall results indicate, the putative anthropogenic sources of PAHs were verified to be both pyrolytic (incomplete combustion of liquid fossil fuels and vehicle exhaust emissions) and pyrogenic (incomplete combustion of biomass and coal), with pyrogenic sources predominating over pyrolytic sources. 3.3. Principal Component Analysis (PCA) based on PAHs in water and sediment samples PCA was used to describe the individual loading of 16 PAHs variables in water and sediment samples from all six sites in the Danube River (Fig. 7). Bartlett's test revealed that the variables are substantially connected and appropriate for PCA analysis. The first two key components account for 88.67% of the overall variation in the set of findings related to water samples and 84.53% of the variance in the sediment samples. Generally, PCA1 was favourably dominated by high loadings of all examined PAHs in both water (Fig. 7, A) and sediments (Fig. 7, B). PCA analysis results corroborated with the previously observed distinction between sampling seasons, which were the concentrations of low molecular weight PAHs (Nap, Acy, Ace, Fl, Phe, and Ant) in water samples that were very high in cold seasons compared to the hot season. Specifically, from Fig. 7, it can be seen that according to PCA results, all the low molecular weight PAHs were characterized by cold seasons, which are winter, autumn, and spring. Contrarily, most of the high molecular weight PAHs occurred mainly in the summertime. As explained previously, this is attributed to poor water solubility and dissolution rate of HMWPAHs, which render them more resistant to decomposition, while LMWPAHs are more soluble and degradable during the hot season. It can be observed from Fig. 7 that the PCA results for sediment samples indicated that no different trends had been found for PAHs in seasonal sediment samples, demonstrating that sediments are independent of seasonal variations. 3.4. Eco-toxicological concerns and Incremental Lifetime Cancer Risk (ILCR) for sediment The sediment sample assessment for ecological risk followed the methodology outlined by [ 56 ]. PAHs levels in sediments were assessed according to sediment quality standards (SQGs) [ 57 ]. Compared with the concentration for each PAH to the Effect Range Low (ERL) and Effect Range Median (ERM) values, the ecological risk to aquatic species posed by contact with sediment-bound PAHs was ascertained. The SQGs involve three classifications of chemical concentrations that define the levels of adverse chemical effects on biology: 1) minimal effects range with rare biological effects (< ERL), 2) possible effects range with occasional biological effects (≥ ERL and < ERM), and 3) probable effects range with frequent biological effects (≥ ERM). Table S5 displayed the concentrations range and toxicity recommendations for 16 individual PAHs. The concentrations of all PAHs in sediments were less than ERL except for Acy and FI concentrations, indicating the possibility of rare biological effects. However, the concentrations of both Acy in winter and spring and FI in autumn and spring were above ERL and lower than ERM, suggesting occasional biological effects. Generally, except for the two mentioned PAHs concentrations, eco-toxicological concerns for the aquatic environment of the Danube River do not pose a significant hazard. The combined impact of the 16 PAHs pollutants in sediments suggests a low chance for negative biological impacts and a low ecological threat. To guarantee that the residual levels of PAHs in the sediments of the Danube River do not surpass the ecological quality criteria; routine monitoring of PAHs in sediments is required. In addition, initiatives for pollution control must be undertaken to avoid the spread of PAHs in the Danube River. For the purpose of comparing the carcinogenicity of PAHs to that of BaP, the toxic equivalency factor (TEF) approach was employed to determine the BaP equivalence (BaPeq) of PAHs [ 58 ]. Due to its high carcinogenicity, BaP has been chosen as a reference chemical in the TEF estimates and assigned a value of one so that the carcinogenicity of each PAH can be estimated relative to BaP. The recorded TEF values are shown in Table S1 . Based on their relative carcinogenicity to BaP, individual PAHs have unique TEF numbers. The formulas listed below are utilized to determine the toxic equivalent quotient (TEQ) for every location in the present investigation. $${\text{B}\text{a}\text{P}\text{e}\text{q}}_{i }= ({PAH}_{i }\times {TEF}_{i })$$ 1 $$\text{T}\text{E}\text{Q}={\sum }_{1}^{n}({PAH}_{i }\times {TEF}_{i })$$ 2 Where PAHi is the PAH concentration and TEFi is the toxic equivalency factor. The TEQ corresponds to sediment samples ranging from 29.88 in the winter season ng/g to 140.39 ng/g in the autumn season. According to the Canadian soil quality guidelines for the preservation of the ecosystem and human health, the threshold value of 600 ng/g is considered safe for humans [ 3 ]. In the present work, no value in any season exceeded the threshold value of TEQ. Using the USEPA's ILCR model, which examined the three main routes of exposure to contaminants (ingestion, dermal contact, and inhalation), a risk assessment to PAHs in river sediments was performed. This assessment was required because of people's daily reliance on the region's aquatic resources. ILCR is used to estimate the human cancer risk posed by exposure to environmental PAHs. The overall carcinogenic risk was determined by summing the hazards associated with the three routes of exposure. Table S6 and equations ( 3 , 4 , 5 , and 6 ) respectively explain the ILCR assessment parameters and model formulations [ 59 , 60 ]. $${\text{I}\text{L}\text{C}\text{R}}_{\text{i}\text{n}\text{g}\text{e}\text{s}\text{t}\text{i}\text{o}\text{n}}=\text{C}\text{S}\times {\text{I}\text{R}}_{\text{i}\text{n}\text{g}\text{e}\text{s}\text{t}\text{i}\text{o}\text{n}}\times \text{E}\text{F}\times \text{E}\text{D}\times \left({\text{C}\text{S}\text{F}}_{\text{i}\text{n}\text{g}\text{e}\text{s}\text{t}\text{i}\text{o}\text{n}}\times \sqrt[3]{\frac{\text{B}\text{W}}{70}}\right)\times {\left(\text{B}\text{W}\times \text{A}\text{T}\times {10}^{6}\right)}^{-1}$$ 3 $${\text{I}\text{L}\text{C}\text{R}}_{\text{i}\text{n}\text{h}\text{a}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}}=\text{C}\text{S}\times {\text{I}\text{R}}_{\text{i}\text{n}\text{h}\text{a}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}} \times \text{E}\text{F}\times \text{E}\text{D}\times \left({\text{C}\text{S}\text{F}}_{\text{i}\text{n}\text{h}\text{a}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}}\times \sqrt[3]{\frac{\text{B}\text{W}}{70}}\right)\times ({\text{B}\text{W}\times \text{A}\text{T}\times \text{P}\text{E}\text{F})}^{-1}$$ 4 $${\text{I}\text{L}\text{C}\text{R}}_{\text{d}\text{e}\text{r}\text{m}\text{a}\text{l} \text{c}\text{o}\text{n}\text{t}\text{a}\text{c}\text{t}}=\text{C}\text{S}\times \text{S}\text{A}\times \text{A}\text{F}\times \text{A}\text{B}\text{S}\times \text{E}\text{F}\times \text{E}\text{D}\times \left({\text{C}\text{S}\text{F}}_{\text{d}\text{e}\text{r}\text{m}\text{a}\text{l} \text{c}\text{o}\text{n}\text{t}\text{a}\text{c}\text{t}}\times \sqrt[3]{\frac{\text{B}\text{W}}{70}}\right)\times ({\text{B}\text{W}\times \text{A}\text{T}\times {10}^{6})}^{-1}$$ 5 $$\text{C}\text{a}\text{r}\text{c}\text{i}\text{n}\text{o}\text{g}\text{e}\text{n}\text{i}\text{c} \text{r}\text{i}\text{s}\text{k} = {{\text{I}\text{L}\text{C}\text{R}}_{\text{i}\text{n}\text{g}\text{e}\text{s}\text{t}\text{i}\text{o}\text{n}}+ \text{I}\text{L}\text{C}\text{R}}_{\text{d}\text{e}\text{r}\text{m}\text{a}\text{l} \text{c}\text{o}\text{n}\text{t}\text{a}\text{c}\text{t}}+ {\text{I}\text{L}\text{C}\text{R}}_{\text{i}\text{n}\text{h}\text{a}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}}$$ 6 CSF is the carcinogenic slope factor, which is represented in units of (mg kg-1day-1) −1 . According to the USEPA, the CSF concentrations of BaP for the three exposure pathways are 25, 7, 3 and 3.85 mg/kg/day − 1 [ 61 ]. CS is the total PAHs concentrations that transformed to hazardous equivalents of BaP using the Toxic Equivalence Factor (TEF) (in ng/g). Calculation of the ILCR relies heavily on the detection of PAHs as BaP-equivalent concentrations using the TEF of each PAHs relative to BaP. The total ILCR is equal to the sum of three routes: skin contact, oral consumption, and inhalation. If the ILCR is less than 1/10 6 , it is deemed inconsequential; if it is more than 1/10 4 , there is a reason for serious worry. For sediment samples, the inhalation component of the ILCR was shown to be negligible and was thus omitted. The ILCR values for adults and children detected in sediment samples from the Danube River are shown in Fig. 8 . The total ILCR in both children and adults are more than 1/10 4 in all seasons, with the highest values recorded in spring followed by winter seasons, which is really a matter of serious concern. In addition, these records are substantially larger than those reported in the Brisbane River in Australia [ 62 ]. When high amounts are found through long-term surveillance, the residents within the river basin area must be warned, and precautions must be taken to prevent human contact with sediments. 4. Conclusions The current work provides a detailed evaluation of PAHs concentrations, seasonal distribution, and ecological risk assessment in water and sediments gathered from six distinct sites along the Danube River in Hungary. Temporal and spatial variations of PAHs were investigated in both rivers' water and sediments, reflecting the anthropogenic sources along the Danube River. The findings highlighted a broad variance range of 16 PAHs contents in water with total concentrations of PAHs ranging from 283.10-541.91, 198.65-404.37, 160.03-324.63, and 228.26-378.19 ng/L for winter, spring, summer, and autumn, respectively. Sediment samples showed PAH ranging from 313.78-622.78, 312.75-595.41, 215.16-465.49, and 311.34-491.78 ng/g for winter, spring, summer, and autumn, respectively. The overall analysis of the results indicates that the putative anthropogenic sources of PAHs were verified to be both pyrogenic (incomplete combustion of biomass and coal) and pyrolytic (incomplete combustion of liquid fossil fuels and vehicle exhaust emissions); with pyrogenic origins predominating over pyrolytic sources. This might suggest that the industries essentially utilize fossil fuels, which would increase the PAHs emissions in the study area. Generally, except for Acy and FI concentrations, the eco-toxicological assessment of the Danube River environment showed no significant PAHs pollutants in sediments, suggesting a low chance for negative biological impacts and low ecological risk. The TEQ in sediment samples ranged from 29.88 ng/g in the winter season to 140.39 ng/g in the autumn season, which, in turn, is considered safe for humans. The total ILCR in both children and adults were calculated to be more than 1/10 4 in all seasons, with the highest values recorded in spring and followed by winter, which constitutes a concerning issue. Continuous monitoring of the PAHs would offer better insight into the scale of the pollution, which would help in devising effective mitigation strategies. Declarations Acknowledgements The Authors greatly appreciate the support of Mohammed A. Al-Seady and Ameer Abbas from the University of Debrecen and Budapest University of Technology and Economics Hungary in the sample collection. 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Ayoko, "Source apportionment and risk assessment of PAHs in Brisbane River sediment, Australia," Ecological Indicators, vol. 73, pp. 784-799, 2017. Additional Declarations No competing interests reported. Supplementary Files SupplementarymaterialDanubeRiver3032023.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Al-Juboori","email":"","orcid":"","institution":"New York University-Abu Dhabi Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Raed","middleName":"A.","lastName":"Al-Juboori","suffix":""},{"id":190806315,"identity":"226824c9-eb35-4f79-bd3b-2a60334cf811","order_by":4,"name":"Mónika Meiczinger","email":"","orcid":"","institution":"University of Pannonia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mónika","middleName":"","lastName":"Meiczinger","suffix":""},{"id":190806316,"identity":"12ad3fea-ce43-4ddc-b739-40f9389528ab","order_by":5,"name":"Manolia Andredaki","email":"","orcid":"","institution":"Liverpool John Moores University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manolia","middleName":"","lastName":"Andredaki","suffix":""},{"id":190806317,"identity":"8b028b43-505d-4304-8f6f-7c87b4df2276","order_by":6,"name":"Ibijoke A. Idowu","email":"","orcid":"","institution":"Liverpool John Moores University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ibijoke","middleName":"A.","lastName":"Idowu","suffix":""},{"id":190806318,"identity":"404746bf-2ea7-4434-b424-0f7666ad4fb4","order_by":7,"name":"Khalid S. Hashim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACCSBmBuIENgbmgwckoKKMDcRpYUsgUQsDA4/BAZgoXi2SDewPmAtq6vL4xM58OGCZc5iBv/0Am+QMPFqkgYYzzzh2uJhNOnfDAclthxkkziSwSW7Ao0WOgYeBmYftQGIbTAvDDQY2yQd4tQAdxvOvDqgl5wFYizwhLdIMDAbMvG3MIC0MYC0GIC34HCbZzGNwmLfvMFBLmgFQSzqP4ZnEZkt83pc43v7wMc+3usT5s5MfPpbcZi0nd/zwwZs9eLSAIuUAnA2MJR4CsYIGGD8Qr3YUjIJRMApGEAAAv0NIw+l9kB8AAAAASUVORK5CYII=","orcid":"","institution":"Liverpool John Moores University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Khalid","middleName":"S.","lastName":"Hashim","suffix":""}],"badges":[],"createdAt":"2023-04-01 16:14:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2765619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2765619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":35735334,"identity":"15a288d1-89bf-4402-8982-9b1db00dae1f","added_by":"auto","created_at":"2023-04-13 22:43:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":692302,"visible":true,"origin":"","legend":"\u003cp\u003eSampling points in the Danube River within the Hungarian region.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/8ee89bff611597931ea037a6.png"},{"id":35736450,"identity":"bf89cb96-f515-4d00-ac72-70428fe2ba82","added_by":"auto","created_at":"2023-04-13 22:51:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203298,"visible":true,"origin":"","legend":"\u003cp\u003ePAHs concentration in water in the six locations with respect to seasons.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/f81014b0471d6376d1f890fe.jpg"},{"id":35735335,"identity":"4582c7ac-9109-4ab4-a603-897cf10180f0","added_by":"auto","created_at":"2023-04-13 22:43:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":290664,"visible":true,"origin":"","legend":"\u003cp\u003ePAHs rings percentages in (A) water and (B) sediment among different sampling seasons: winter, spring, summer, and autumn.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/70688b6e6f504c0f63289a1a.jpg"},{"id":35736448,"identity":"e94cd8d7-f551-4705-b5ca-1bf055470756","added_by":"auto","created_at":"2023-04-13 22:51:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154130,"visible":true,"origin":"","legend":"\u003cp\u003ePAHs concentration in sediment samples among various sampling sites and seasons campaigns.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/e84304cf2c77446b263ab780.jpg"},{"id":35735340,"identity":"35f67a27-daef-48f2-a070-ab6cdc16871e","added_by":"auto","created_at":"2023-04-13 22:43:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1012128,"visible":true,"origin":"","legend":"\u003cp\u003e7CPAHs for each site for winter (A), spring (B), summer (C), and autumn (D).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/0f6fe7c6ad49012b726045d0.jpg"},{"id":35735342,"identity":"19ba4802-f805-4fa6-8b8a-dbeaf2caa14c","added_by":"auto","created_at":"2023-04-13 22:43:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":331529,"visible":true,"origin":"","legend":"\u003cp\u003eCross plots of the diagnostic ratios of (A) BaA/(BaA+Chr) to IND/ (IND+BghiP) and (B) ratios BaA/(BaA+Chr) to Flu/(Flu+Pyr) for water and sediment samples in all seasons from the Danube River.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/9e831d6157164b3b2bdaeaf5.jpg"},{"id":35737432,"identity":"d13ce3d0-d278-468f-abe8-02cbe19b9b4e","added_by":"auto","created_at":"2023-04-13 22:59:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":299869,"visible":true,"origin":"","legend":"\u003cp\u003ePCA analysis for water (A) and sediment (B).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/eab403267f600527264ba4f2.jpg"},{"id":35735338,"identity":"dbfe68a5-edbc-42fa-a793-319e5e01039b","added_by":"auto","created_at":"2023-04-13 22:43:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2433014,"visible":true,"origin":"","legend":"\u003cp\u003eILCR levels in the Danube River sediments for adults and children. The red line is the Incremental Lifetime Cancer Risk (ILCR) (1/10\u003csup\u003e4\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/a3ac6cb2d8a3fe2e8af53ff8.jpg"},{"id":38862284,"identity":"0a9ee60b-dd47-448d-8796-dd4a40cf68e9","added_by":"auto","created_at":"2023-06-21 08:29:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2970910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/ec0acd2a-6328-4462-ab41-569770b7e8ae.pdf"},{"id":35736447,"identity":"af7684fa-6535-4ef9-84e9-90796baeaf12","added_by":"auto","created_at":"2023-04-13 22:51:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":76881,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialDanubeRiver3032023.docx","url":"https://assets-eu.researchsquare.com/files/rs-2765619/v1/ceae345ebc983224306809a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seasonal distribution, source identification, health and eco-toxicological risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in water and sediment from the Danube River in Hungary","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFreshwater is a valuable and scarce resource for both individuals and ecosystems. The protection and preservation of the freshwater are increasingly important with the drive to promote food security and sustainable development growth agenda for life below water. The majority of the world's biggest cities were constructed on or near regions of freshwater, mostly rivers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most research on aquatic systems is primarily concerned with the effects of anthropogenic activities and natural phenomena, such as volcanism and biological processes, on both human health and ecology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Population growth and corresponding increases in industrial, agricultural, and urban activities have increased the emission levels of different anthropogenic toxins into the ecosystem, among them Polycyclic Aromatic Hydrocarbons (PAHs) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to their toxicity, persistence, bioaccumulation, and possible detrimental health impacts on living organisms, these compounds have attracted considerable attention globally [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRivers, especially those in regions with heavy human activities, play a crucial role in transferring and transforming numerous contaminants and serve as an important connection between terrestrial and marine ecosystems [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rivers are significant conduits for the entry of land-based natural materials and pollutants directly into the seas; approximately 85% of the materials from land are transferred to the sea in several ways, such as municipal and industrial effluents, atmospheric deposition, riverine runoff, urban and agricultural runoff, sewage outfalls, and petrogenic sources [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these materials, PAHs constitute a pervasive class of chemicals generated by the fusion of two or more aromatic rings of hydrogen and carbon atoms in diverse configurations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Because PAHs possess concerning characteristics such as teratogenicity, carcinogenicity, and gene mutation, the United States Environmental Protection Agency (USEPA) and the European Union (EU) have identified 16 PAHs as priority pollutants among the hundreds of PAHs in the environment [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Due to their high octanol\u0026ndash;water partition coefficient and hydrophobic lipophilicity, PAHs entering the water get adsorbed on suspended particulate matter and ultimately settle into the sediment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, river sediments are susceptible to PAHs accumulation and release. Moreover, they are commonly used as an indicator for detecting probable emission sources and determining the exposure risk of PAHs to benthic biotas [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, the environmental fate and the possible ecological risk related to PAHs are serious matters of public concern [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As a result, PAHs have been the subject of environmental monitoring research for catchments worldwide. Numerous studies have employed water and sediment as important matrices for evaluating PAHs contamination in rivers [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], lakes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], dams [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] as well as seas [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Water Framework Directive (WFD) obligates the European Union member states to achieve a satisfactory quantitative and qualitative assessment of the status of all bodies of water [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Nonetheless, some bodies of water (for instance Danube River in Hungary) have not yet reached this objective, and emergent pollutants such as PAHs are not yet controlled [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies that have examined the PAHs level in the Danube River within the Hungarian regions can be categorized into two groups. The first group examined the upper area of the Danube River (i.e., the borders between Hungary and Slovakia) and the other group focused on the capital city (Budapest). Regarding the upper area of the Danube, Nagy, et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] investigated the PAHs concentrations and distribution in the surface water and bed sediments of the Hungarian upper section of the Danube River as well as in the Moson Danube branch during the period of 2001 to 2010. They found that the concentrations of 16 PAHs in water samples ranged from 25 to 1208 ng/L, which were dominated by 2\u0026ndash;3 rings of PAHs. The 16 PAHs concentrations in sediments ranged between 8.3 and 1202.5 ng/g in dry weight (dw). The same research group also continued the investigation within the upper part of the Danube River and its tributaries in the Autumn of 2012. The findings revealed that the total PAH concentrations in water samples ranged from 67 to 96 ng/L, and the PAHs concentrations ranged between 35.2 and 288.3 ng/g dw in sediments [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In another work, conducted in the upper part, the PAHs level in water from 2007 up to 2010 was reported to range between 25 and 357 ng/L [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The same group further investigated the PAHs concentration in water and sediments in the Moson Danube Arm and the upper area of the Danube River for the period 2014 to 2015 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The total concentration of PAHs in water was found to vary from 16 to 133 ng/L, and the concentration in sediments varied in the range of 118 and 283 ng/g. Comparing the findings from the long-term research by Nagy, et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] over the years, it can be noticed that concentrations of PAHs in water and sediment decreased dramatically. Moving to the second part of the PAHs level examination in the Danube River which was in the capital city, Visca, et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] evaluated the contamination in terms of PAHs level in the Danube River (only water samples) passing through Budapest with three locations inside the city. The samplings were performed in April 2017, November 2017, and October 2018. It should be mentioned; however, that information about the PAHs range is missing[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Caracciolo, et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] also conducted a study to monitor the PAHs level in Budapest at three different points without, however, revealing the coordinates of those sampling points. Their measurement was for PAHs level in water in April and November 2017. The finding revealed that total PAHs concentration ranged from 15.9 to 53.2 ng/L.\u003c/p\u003e \u003cp\u003eThe overall conclusion regarding the studies conducted for measuring the PAHs level in the Danube River within the framework frame of Hungarian borders is that most of these investigations were conducted in the upper part of the river, and with the latest sampling campaign in the period of 2014\u0026ndash;2015 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Other studies have reported PAHs level in the area of Budapest, however, with unclear conclusions due to missing information such as the PAHs ranges, sampling locations, and overlooking the sediments part, which is considered an important PAHs sink [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. With a view to narrowing the gap in knowledge regarding PAHs concentration in the Danube River, the present investigation was designed to adequately investigate the spatial and temporal PAHs contamination of the river basin in Hungary. The main objectives are: 1) The spatial and temporal assessment of PAHs contamination in the Danube River; 2) The identification of the primary sources of PAHs contamination; 3) The evaluation of the eco-toxicological risk assessment of PAHs and; 4) The identification and qualification of the Incremental Lifetime Cancer Risk (ILCR), for both adults and children.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Chemicals\u003c/h2\u003e\n\u003cp\u003eAll solvents used were High-performance liquid chromatography (HPLC)-grade and acquired from Fisher Chemical Co. (USA), with a minimum purity of 99%. Supelco (Bellefonte, PA, USA) supplied the reference standards (QTM PAH-Mix, 2000 \u0026micro;g/mL) of the 16 PAHs. Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e shows the abbreviation and chemical structure information of the considered 16 prioritized PAHs. Sigma-Aldrich, USA, supplied solid-phase extraction membranes (ENVI\u0026trade;-18 DSK SPE Disk, diameter 47 mm), Sodium sulfate anhydrous, Silica gel (desiccant\u0026thinsp;~\u0026thinsp;2\u0026ndash;5 mm), as well as PAH recovery standards. Sodium Sulfate Anhydrous and Silica gel were put in a furnace (FI 600\u0026thinsp;\u0026minus;\u0026thinsp;60, Borel) at a temperature of 500\u0026deg;C for 4 h to remove any moisture or organics before being transferred to a desiccator for storage until usage. In the experiments, Milli-Q water with a resistivity of 18.2 MΩ\u0026bull;cm at room temperature and a total organic carbon value of less than 5 ppb was utilized. Prior to each measurement, glassware was first cleaned using ultrasonic cleaners (Heidolph\u0026trade;, Fisher Scientific), washed afterwards with acetone, n-hexane, methanol, and dichloromethane to eliminate background pollutions and dried at 105\u0026deg;C before use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Study area\u003c/h2\u003e\n\u003cp\u003eWith a length of 2,780 km, the Danube is the second-longest river in Europe; its catchment area is 801,500 km\u003csup\u003e2\u003c/sup\u003e. The water of the Danube River Basin is used for a variety of reasons, including the production of drinking water, industrial and agricultural activity, leisure, hydroelectric power generation, and transportation. The length of the Hungarian Danube segment is 417 km (1850\u0026ndash;1433 river kilometres (rkm)). Six locations were investigated along the proposed segment. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the sampling locations along the Danube River. Both S1 and S2 are representatives of the northern sites in our campaign in an area affected by both agricultural and industrial activities. Site 3 was taken at the heart of Budapest city, which happens to be situated downstream of a wastewater treatment plant, the second largest plant in the city (processing 180,000-200,000 m\u003csup\u003e3\u003c/sup\u003e/day of wastewater). Site 4 is located downstream of a polystyrene factory which is impact-resistant polystyrene and expandable polystyrene. Site 5 is located downstream of different industries, such as paper mill companies and electricity suppliers. Site 6 is located furthest south with respect to the other samples. This site was selected to be representative of the collective anthropogenic activities upstream. Table S2 shows the coordinates of the sampling sites.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Samples collection and pretreatment\u003c/h2\u003e\n\u003cp\u003eThroughout the study, the water and sediment samples were collected on a monthly basis, from February 2022 to February 2023, over a 12-calendar month period. Water samples were obtained at depths from 5 to 30 cm in 1 L brown glass containers that had been previously cleaned and then transported to the laboratory for analysis. Sediment samples were collected from the river bed at a depth 0\u0026ndash;10 cm and were then placed in clean polyethylene bags and transferred to the laboratory for further processing. Sediment samples were dried at 25 \u003csup\u003eo\u003c/sup\u003eC and were ground and sieved through a 2 mm mesh to remove any roots, debris, or large particles. Finally, they were dry-frozen until the time of further use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4. Extraction procedures of PAHs\u003c/h2\u003e\n\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.1 Water sample extraction procedure\u003c/h2\u003e\n\u003cp\u003eSolid phase extraction (SPE) was used to extract one litter of filtered water samples for the purpose of water sample extraction. The water sample extraction followed the previously reported procedures [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. An SPE membrane was pre-washed using six milliliter of dichloromethane (DCM) before conditioning (activation) with six milliliter of methanol and six milliliter of ultrapure water, and ten milliliter of methanol. A ten microliter of surrogate standard mixture solution was added to one litter of the water sample. This step was to enrich the sample, which was then passed through the SPE at a flow rate of 3 mL/min. After the extraction was finished, a vacuum pump was utilized to dry the column, and six milliliter of dichloromethane was added to soak it for 5 minutes before elution into a clean glass test tube. By using nitrogen, the eluate was concentrated to 0.5 mL prior to the addition of ten microliter of standard mixture solution. Then the samples were analyzed using gas chromatography-mass spectrometry (GC\u0026ndash;MS) analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.2 Sediment sample extraction procedure\u003c/h2\u003e\n\u003cp\u003eThe freeze-dried sediment samples were pulverized using a mortar and pestle and were sieved using a 100-mesh to remove large particles. Sediment samples dried and homogenized were put into brown glass vials for further laboratory analysis. The sediment sample extraction followed the previously reported procedures [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. A sample of 2 grams was precisely weighed out. Five milliliters of acetone/n-hexane (1:1, v/v) and a standard surrogate solution were added to the test tube. Afterwards, they were vortexed for 60 seconds, followed by 15 minutes of ultrasonic extraction in a water bath. The test tubes were then centrifuged for 20 minutes at 2000 rpm to separate the solid and liquid phases. Using a Pasteur pipette, the supernatant was transferred into another clean test tube. Then, five milliliters of a 1:1 mixture of acetone and n-hexane was added to each sample. In one test tube, the extracts were mixed, and activated copper was added for desulfurization. Then, sodium sulfate anhydrous was applied to eliminate water, followed by a concentration step to 0.5 milliliters using a nitrogen-blowing concentrator. Finally, an internal standard solution was added for the GC/MS analysis. As described in previous work by the present authors [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], PAHs were measured using GC\u0026ndash;MS (gas chromatography mass spectrometry) type Agilent 6890 N 5975C mass selective detector, USA system. Helium was utilized as a carrier gas at 1.5 ml/min using an HP-5MS gas chromatography column (30 m 0.32 mm 0.25 \u0026micro;m). The selective ion scanning (SIM) mode was utilized for quantitative analysis. The injector temperature was set to 300 \u003csup\u003eo\u003c/sup\u003eC. The oven temperature was programmed as follows: the initial temperature was set to 100\u0026deg;C for one minute, after which it increased to 300\u0026deg;C at a rate of 8\u0026deg;C per minute and remained at 300\u0026deg;C for 39 minutes. Triplicate measurements were recorded for each sample resulting in a relative standard deviation of less than 10.2%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.3 QC/QA\u003c/h2\u003e\n\u003cp\u003eIn this study, the quality controls included triplicate samples, matrix spike standards, calibration standards, a procedural blank, and detection limits. Prior to each measurement, the glassware was first cleaned using ultrasonic cleaners, washed afterwards with acetone, n-hexane, methanol, and dichloromethane to eliminate background pollution and dried at 105\u0026deg;C before use. Utilizing the dry weight approach, the concentration of 16 PAHs in sediment samples was determined. The lowest detection limits (LOD) were determined using analyte concentration and a 3-fold signal-to-noise ratio [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. The range of LOD for water was between 0.02 and 0.59 ng/L, while for sediment was between 0.37 and 0.96 ng/g in dw. PAHs recovery was determined by spiking water and sediment samples with standard solutions. A procedure blank (solvent), a spiked blank (standards added to solvent), and sample triplicates were conducted for each sample. Analysis of method blanks proved the absence of detectable PAH contamination. The recovery ranges for 16 PAHs in water, and sediment samples were 91.5%\u0026plusmn; 4.3\u0026ndash;104.1% \u0026plusmn; 7.6% and 86.8%\u0026plusmn; 5.5\u0026ndash;99.2% \u0026plusmn; 6.2%, respectively. For water samples, the recovery ranges of the spiking standards were 92.8% \u0026plusmn; 4.5\u0026ndash;109.7% \u0026plusmn; 6.9%, and for sediment samples, they were 90.5% \u0026plusmn; 8.3\u0026ndash;97.4%\u0026plusmn; 4.4%. For recovery, the concentrations of 16 PAHs were adjusted. Reference and blank samples were measured to confirm the accuracy of the analysis. Moreover, each sample was measured three times, resulting in a relative standard deviation between 1.7% and 10.4%, which is within the acceptable limit (\u0026lt;\u0026thinsp;25%). Mean values are presented for the measurements. The data in this study were tested to the Kolmogorov-Smirnov normality at a significance level of 0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. PAHs spatial distribution and seasonal variation in water and sediment\u003c/h2\u003e\n\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.1. Water\u003c/h2\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the 16 identified PAHs concentrations in the water of the Danube River. The total PAHs contents in water for all seasons were ranging from 224.85 (in summer) to 365.87 ng/L (in winter). The seasonal concentration variation of PAHs was 283.10-541.91 ng/L in winter, 198.65-404.37 ng/L in spring, 160.03-324.63 ng/L in summer, and 228.26-378.19 ng/L in autumn. The concentrations of LMWPAHs (low molecular weight PAHs, 2\u0026ndash;3 rings): Nap, Acy, Ace, Fl, Phe, and Ant) in water samples of the Danube River were greater than the concentrations of HMWPAHs (high molecular weight PAHs 4\u0026ndash;6 rings): Flu, Pyr, BaA, Chr, BaP, DBA, BkF, BbF, IND and BghiP, and in all four seasons. The HMWPAHs recorded the highest values during the summer season (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), being congruent with literature studies from other countries [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In Fig.\u0026nbsp;3, the PAHs profile in water and sediment is presented. Ace was found to be the most predominant component, with levels between 16 and 47.86 ng/L, followed by Nap, Acy, FI, Ant, and Phe with values between 11.9 and 45 ng/L, 15.5 and 44 ng/L, 7.17 and 40.9 ng/L, 10.5 and 40.2 ng/L, and 11 and 36 ng/L, respectively. The highest HMWPAHs in water samples were Bghip (11.4\u0026ndash;28.3 ng/L), IND (8.25\u0026ndash;17.99 ng/L), DBA (9.66\u0026ndash;17.88 ng/L), BaP (9.13\u0026ndash;17.5 ng/L), BkF (7.29\u0026ndash;17.2 ng/L), BbF (9.56\u0026ndash;17.1 ng/L), Pyr (7.81\u0026ndash;14.99 ng/L), Chr (10.16\u0026ndash;14.70 ng/L), Flu (5.99\u0026ndash;11.05 ng/L), and BaA (8.2-10.13 ng/L). The concentrations of \u0026sum;PAHs in the water of the Danube River were greater in the winter, spring, and autumn seasons compared to the summer season, which was attributed to the lower temperatures during these sampling times. The variation in the patterns of PAHs in water throughout various sample periods (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) is likely related to connected with the PAHs molecular weight and degradation, which is more evident during warm seasons [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. In contrast to HMWPAHs (4\u0026ndash;6 rings) which have a poor water solubility and dissolution rate and are, hence, more resistant to decomposition, LMWPAHs (2\u0026ndash;3 ring PAHs) are more degradable and soluble during warm seasons. The 2\u0026ndash;3 rings PAHs are higher in cold seasons, and 4\u0026ndash;6 rings PAHs are higher in the hot season (Fig.\u0026nbsp;3); this is because of the reduction in the content of LMWPAHs in water. Furthermore, the current data reveal that PAHs concentration in water also varies across the sampling sites. The highest concentrations of PAHs in water were found at the S3 sampling location during cold seasons (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), in conjunction with high amounts of LMWPAHs, indicating a local origin of recently generated LMWPAHs resulting from atmospheric deposition caused by vehicular and industrial emissions from various plants.\u003c/p\u003e\n\u003cp\u003eThe \u0026sum;PAHs concentrations in the water samples in all seasons (ranges from the lowest of 224.85 in summer to the highest of 365.87 ng/L in winter) are higher than those found in the Danube River and tributaries (67\u0026ndash;96 ng/L) from the territory of Hungary [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], Danube River (16\u0026ndash;133 ng/L) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e], Danube River measurements ( 15.9 to 53.2 ng/L) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Contrarily, these concentrations are lower than those found in the Raba River (the largest Danube tributary in Hungary), with a range between 41\u0026ndash;437 ng/L [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. These variations are likely due to the difference in the selected sampling sites. This study deliberately selected sites where the potential source of contamination lies ahead. Generally, these concentrations are also lower than those found in other countries such as the Tiber River in Italy, Humen River, Bai Chao and Chaobai Rivers in China, with PAHs range of 10.3\u0026ndash;951.6 ng/L, 311.13\u0026ndash;1012.80 ng/L, and 55\u0026ndash;882 ng/L, respectively [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, it was similar to those reported in China by Chen, et al. [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e] in the Yinma River (23.2-386.9 ng/L).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003e3.1.2 Sediment\u003c/h2\u003e\n\u003cp\u003eThe concentrations of PAHs in sediments are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. PAHs concentrations in all four seasons for the sediment samples were ranging from 316.72 in summer to 422.98 ng/g dw in winter. The concentration variation of PAHs within seasons is as follows: 313.78-622.78 ng/g in winter, 312.75-595.41 ng/g in spring, 215.16-465.49 ng/g in summer, and 311.34-491.78 ng/g in autumn. The variance of PAHs in the sediments of the Danube River indicates that PAHs levels vary with sampling sites, indicating the anthropogenic sources along the Danube River, as well as with seasons, given that the samples were gathered at various periods and places. Contrarily to the reported concentrations of PAHs in water, HMWPAHs are more prevalent than LMWPAHs in sediments of the Danube River (Fig.\u0026nbsp;3), which is in accordance with other studies [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. LMWPAHs had greater water solubility owing to lower octanol-water coefficients and were more volatile than HMWPAHs, which makes them record the lowest concentrations values in sediment samples [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. In contrast, HMWPAHs have low water solubility, greater partitioning coefficients, and high hydrophobicity in aqueous conditions [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. BaP, Chr, BbF, and BkF are the most predominant PAHs in sediments samples, with records ranging between 28.79 and 52.47 ng/g, 16.16 and 50.82 ng/g, 22.94 and 42.62 ng/g, as well as 14.97 and 42.42 ng/g, respectively-followed by BkF, Pyr, Bghip, and BaA with values between 26.32\u0026ndash;36.85 ng/g, 11.41-36.00 ng/g 13.34\u0026ndash;33.81 ng/g, and 10.17\u0026ndash;32.88 ng/g, respectively. Consistent with water samples, S3 recorded the highest PAH concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). IND and Flu had the lowest concentrations with values ranging from 17.01\u0026ndash;29.38 ng/g and 9.00-21.45 ng/g, respectively. LMWPAHs that had the highest concentrations are Ant (13.21\u0026ndash;24.86 ng/g), FI (11.90\u0026ndash;23.30 ng/g), and Phe (14.59\u0026ndash;23.21 ng/g), while Acy, Nap, and Ace scored the lowest concentration ranges of 11.67\u0026ndash;18.46 ng/g, 14.01\u0026ndash;19.23 ng/g, and 11.20\u0026ndash;22.00 ng/g, respectively. No distinctive patterns were identified for PAHs in sediments samples during the different seasons indicating the sediments independence of temporal variations. Sediment pollution assessed by total PAHs concentrations may be categorized as follows: (A) low polluted (less than100 ng/ g), (B) moderately polluted (between101 and 1000 ng/g), (C) highly polluted (between 1001 and 5000 ng/g), and (D) very polluted (more than 5000 ng/g) [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consequently, the pollution levels of the sediments from the Danube River can be categorized as low pollution.\u003c/p\u003e\n\u003cp\u003eThe seven carcinogenic PAHs (CPAHs) account for most of the total PAHs ranging from 181.09 in summer to 240.02 ng/g in winter, with the CPAHs exhibiting the same spatial distribution as \u0026Sigma;PAHs in S3. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the 7CPAHs distribution in water and sediment along the Danube River in all seasons. Variations in the spatial distribution of PAH concentrations in sediments can be attributed to various factors [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], such as (1) discharge of untreated municipal wastewater, traffic emissions, industrial activities, and fuel consumption; (2) various hydrodynamic systems related to meteorological conditions that can stimulate resuspension and re-deposition of the sediments; (3) alteration in sediment textural characteristics based on spatial properties of sampling sites and (4) the presence of redox conditions in sediments and PAH biodegradation. The PAHs maximum concentration in site 3 sampling locations can be due to the position of S3 inside Budapest and downstream of the wastewater treatment plant. The PAHs concentrations in wastewater are a significant cause of concern for the industry. PAHs are regarded as hazardous components because of their highly toxic and polluting possibilities, which can persist for many decades in the environment, and their carcinogenic, genotoxic, and mutagenic effects, which can cause irreparable harm to individuals' health [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. PAHs pollution can occur in wastewater effluent when they are not eliminated, and as a result, they can enter river water from these sources [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe concentrations of PAHs in sediment samples from the Danube River (316.72 in summer \u0026minus;\u0026thinsp;422.98 ng/g, dw in winter) were higher than those detected in the Hungarian upper section of the Danube River and its tributaries (35.2 to 288.3 ng/g) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] and Danube River and Moson Danube Arm (Hungary) (118\u0026ndash;283 ng/g ) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] but lower than those reported in Hungarian upper section of the Danube River and the Moson Danube branch (8.3 to1,202.5 ng/g) [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Generally, the measured PAHs concentrations in the present study were comparable to those detected in Soltan Abad River, Iran (180.3\u0026ndash;504.0 ng/g) as well as Ovia River, Nigeria (5.25-573.33 ng/g) [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. Followed by the lower comparison to the concentrations that were reported by Liu, et al. [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e] in Rivers in Shanghai, China (248.89\u0026ndash;36198.23 ng/g).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Sources identification ratios of PAHs in water and sediment samples\u003c/h2\u003e\n\u003cp\u003eBased on the average concentrations of individual PAHs in water and sediments for each sampling season (winter, spring, summer, and autumn), the following diagnostic ratios were determined to identify the dominant sources and the related emission routes. The precise meaning of each ratio related to Flu/(Flu\u0026thinsp;+\u0026thinsp;Pyr), LMW/HMW, Flu/Pyr, BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr), IND/(IND\u0026thinsp;+\u0026thinsp;BghiP), IND/BghiP, and BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr) with the determined ratios are given in Table S3 and Table S4 for water and sediments, respectively. According to the literature, a ratio of BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr) less than 0.2 suggests that PAHs are mostly generated from petrogenic inputs (liquid fuel discharges), a ratio between 0.2 and 0.35 shows that PAHs are sourced from mixed sources (petrogenic/pyrogenic), and a ratio greater than 0.35 indicates that PAHs are primarily formed from pyrogenic - combustion of solid fuel - natural sources such as biomass and coal. A ratio of Flu/ (Flu\u0026thinsp;+\u0026thinsp;Pyr) less than 0.4 indicates that PAHs originate from petrogenic inputs, a ratio between 0.4 and 0.5 indicates that they are derived from pyrolytic (burning of liquid fossil fuels and crude oil, vehicles), and a ratio greater than 0.5 indicates that they are sourced from pyrogenic - combustion of solid fuel. A ratio of IND/ (IND\u0026thinsp;+\u0026thinsp;BghiP) lower than 0.2 indicates that PAHs originate from petrogenic inputs, a ratio between 0.2 and 0.5 indicates that they are derived from pyrolytic sources, and a ratio greater than 0.5 indicates that they are sourced from pyrogenic sources.\u003c/p\u003e\n\u003cp\u003eThe ratios plot of BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr) against Flu/(Flu\u0026thinsp;+\u0026thinsp;Pyr) and ratios of BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr) against IND/(IND\u0026thinsp;+\u0026thinsp;BghiP) are presented in Fig.\u0026nbsp;6. Generally, LMWPAHs originate from oil or fuel spills and have a short lifetime in the ecosystem, whereas HMWPAHs arise from combustion products, pyrolysis, or petrogenic origins [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. As it is shown in Fig.\u0026nbsp;6, Flu/(Flu\u0026thinsp;+\u0026thinsp;Pyr) ratios are between 0.4\u0026ndash;0.5 in all seasons except for summer for water samples, while the opposite was observed in sediment samples, indicating pyrolytic sources in both cases. Furthermore, these ratios were \u0026lt;\u0026thinsp;0.4 in the summer season for water samples and in both the winter and spring seasons for sediment samples indicating petrogenic inputs. The only time these ratios\u0026thinsp;\u0026gt;\u0026thinsp;0.5 were in autumn for sediment samples which explains that wood or coal, and grass combustion are the main PAHs origins.\u003c/p\u003e\n\u003cp\u003eThe BaA/(BaA\u0026thinsp;+\u0026thinsp;Chr) ratios for both water and sediment samples were more than 0.35, indicating pyrogenic sources, except for the summer season in sediments which are scattered and clustered between 0.2 and 0.35, indicating mixed sources (petrogenic/pyrogenic). The ratios of IND/(IND\u0026thinsp;+\u0026thinsp;BghiP) for water samples were more than 0.5 in the spring season only and for all four seasons, except for spring, for sediments samples, suggesting the contribution of combustion of solid fossil fuel-like biomass and coal in agricultural regions. The ratios of IND/(IND\u0026thinsp;+\u0026thinsp;BghiP) were between 0.2 and 0.5 in the winter, summer, and autumn seasons for water samples and in the spring season for sediments samples indicating pyrolytic sources. The BaP/BghiP ratio in both water and sediment sampling was more than 0.6 in all sampling seasons, indicating PAHs of petrogenic origin. Overall results indicate, the putative anthropogenic sources of PAHs were verified to be both pyrolytic (incomplete combustion of liquid fossil fuels and vehicle exhaust emissions) and pyrogenic (incomplete combustion of biomass and coal), with pyrogenic sources predominating over pyrolytic sources.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Principal Component Analysis (PCA) based on PAHs in water and sediment samples\u003c/h2\u003e\n\u003cp\u003ePCA was used to describe the individual loading of 16 PAHs variables in water and sediment samples from all six sites in the Danube River (Fig.\u0026nbsp;7). Bartlett's test revealed that the variables are substantially connected and appropriate for PCA analysis. The first two key components account for 88.67% of the overall variation in the set of findings related to water samples and 84.53% of the variance in the sediment samples. Generally, PCA1 was favourably dominated by high loadings of all examined PAHs in both water (Fig.\u0026nbsp;7, A) and sediments (Fig.\u0026nbsp;7, B). PCA analysis results corroborated with the previously observed distinction between sampling seasons, which were the concentrations of low molecular weight PAHs (Nap, Acy, Ace, Fl, Phe, and Ant) in water samples that were very high in cold seasons compared to the hot season. Specifically, from Fig.\u0026nbsp;7, it can be seen that according to PCA results, all the low molecular weight PAHs were characterized by cold seasons, which are winter, autumn, and spring. Contrarily, most of the high molecular weight PAHs occurred mainly in the summertime. As explained previously, this is attributed to poor water solubility and dissolution rate of HMWPAHs, which render them more resistant to decomposition, while LMWPAHs are more soluble and degradable during the hot season. It can be observed from Fig.\u0026nbsp;7 that the PCA results for sediment samples indicated that no different trends had been found for PAHs in seasonal sediment samples, demonstrating that sediments are independent of seasonal variations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Eco-toxicological concerns and Incremental Lifetime Cancer Risk (ILCR) for sediment\u003c/h2\u003e\n\u003cp\u003eThe sediment sample assessment for ecological risk followed the methodology outlined by [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. PAHs levels in sediments were assessed according to sediment quality standards (SQGs) [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. Compared with the concentration for each PAH to the Effect Range Low (ERL) and Effect Range Median (ERM) values, the ecological risk to aquatic species posed by contact with sediment-bound PAHs was ascertained. The SQGs involve three classifications of chemical concentrations that define the levels of adverse chemical effects on biology: 1) minimal effects range with rare biological effects (\u0026lt;\u0026thinsp;ERL), 2) possible effects range with occasional biological effects (\u0026ge;\u0026thinsp;ERL and \u0026lt;\u0026thinsp;ERM), and 3) probable effects range with frequent biological effects (\u0026ge;\u0026thinsp;ERM). Table S5 displayed the concentrations range and toxicity recommendations for 16 individual PAHs. The concentrations of all PAHs in sediments were less than ERL except for Acy and FI concentrations, indicating the possibility of rare biological effects. However, the concentrations of both Acy in winter and spring and FI in autumn and spring were above ERL and lower than ERM, suggesting occasional biological effects. Generally, except for the two mentioned PAHs concentrations, eco-toxicological concerns for the aquatic environment of the Danube River do not pose a significant hazard. The combined impact of the 16 PAHs pollutants in sediments suggests a low chance for negative biological impacts and a low ecological threat. To guarantee that the residual levels of PAHs in the sediments of the Danube River do not surpass the ecological quality criteria; routine monitoring of PAHs in sediments is required. In addition, initiatives for pollution control must be undertaken to avoid the spread of PAHs in the Danube River.\u003c/p\u003e\n\u003cp\u003eFor the purpose of comparing the carcinogenicity of PAHs to that of BaP, the toxic equivalency factor (TEF) approach was employed to determine the BaP equivalence (BaPeq) of PAHs [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. Due to its high carcinogenicity, BaP has been chosen as a reference chemical in the TEF estimates and assigned a value of one so that the carcinogenicity of each PAH can be estimated relative to BaP. The recorded TEF values are shown in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. Based on their relative carcinogenicity to BaP, individual PAHs have unique TEF numbers. The formulas listed below are utilized to determine the toxic equivalent quotient (TEQ) for every location in the present investigation.\u003c/p\u003e\n\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ1\" class=\"mathdisplay\"\u003e$${\\text{B}\\text{a}\\text{P}\\text{e}\\text{q}}_{i }= ({PAH}_{i }\\times {TEF}_{i })$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ2\" class=\"mathdisplay\"\u003e$$\\text{T}\\text{E}\\text{Q}={\\sum }_{1}^{n}({PAH}_{i }\\times {TEF}_{i })$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere PAHi is the PAH concentration and TEFi is the toxic equivalency factor.\u003c/p\u003e\n\u003cp\u003eThe TEQ corresponds to sediment samples ranging from 29.88 in the winter season ng/g to 140.39 ng/g in the autumn season. According to the Canadian soil quality guidelines for the preservation of the ecosystem and human health, the threshold value of 600 ng/g is considered safe for humans [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the present work, no value in any season exceeded the threshold value of TEQ.\u003c/p\u003e\n\u003cp\u003eUsing the USEPA's ILCR model, which examined the three main routes of exposure to contaminants (ingestion, dermal contact, and inhalation), a risk assessment to PAHs in river sediments was performed. This assessment was required because of people's daily reliance on the region's aquatic resources. ILCR is used to estimate the human cancer risk posed by exposure to environmental PAHs. The overall carcinogenic risk was determined by summing the hazards associated with the three routes of exposure. Table S6 and equations (\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) respectively explain the ILCR assessment parameters and model formulations [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ3\" class=\"mathdisplay\"\u003e$${\\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{i}\\text{n}\\text{g}\\text{e}\\text{s}\\text{t}\\text{i}\\text{o}\\text{n}}=\\text{C}\\text{S}\\times {\\text{I}\\text{R}}_{\\text{i}\\text{n}\\text{g}\\text{e}\\text{s}\\text{t}\\text{i}\\text{o}\\text{n}}\\times \\text{E}\\text{F}\\times \\text{E}\\text{D}\\times \\left({\\text{C}\\text{S}\\text{F}}_{\\text{i}\\text{n}\\text{g}\\text{e}\\text{s}\\text{t}\\text{i}\\text{o}\\text{n}}\\times \\sqrt[3]{\\frac{\\text{B}\\text{W}}{70}}\\right)\\times {\\left(\\text{B}\\text{W}\\times \\text{A}\\text{T}\\times {10}^{6}\\right)}^{-1}$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ4\" class=\"mathdisplay\"\u003e$${\\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{i}\\text{n}\\text{h}\\text{a}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}=\\text{C}\\text{S}\\times {\\text{I}\\text{R}}_{\\text{i}\\text{n}\\text{h}\\text{a}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}} \\times \\text{E}\\text{F}\\times \\text{E}\\text{D}\\times \\left({\\text{C}\\text{S}\\text{F}}_{\\text{i}\\text{n}\\text{h}\\text{a}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}\\times \\sqrt[3]{\\frac{\\text{B}\\text{W}}{70}}\\right)\\times ({\\text{B}\\text{W}\\times \\text{A}\\text{T}\\times \\text{P}\\text{E}\\text{F})}^{-1}$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ5\" class=\"mathdisplay\"\u003e$${\\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{d}\\text{e}\\text{r}\\text{m}\\text{a}\\text{l} \\text{c}\\text{o}\\text{n}\\text{t}\\text{a}\\text{c}\\text{t}}=\\text{C}\\text{S}\\times \\text{S}\\text{A}\\times \\text{A}\\text{F}\\times \\text{A}\\text{B}\\text{S}\\times \\text{E}\\text{F}\\times \\text{E}\\text{D}\\times \\left({\\text{C}\\text{S}\\text{F}}_{\\text{d}\\text{e}\\text{r}\\text{m}\\text{a}\\text{l} \\text{c}\\text{o}\\text{n}\\text{t}\\text{a}\\text{c}\\text{t}}\\times \\sqrt[3]{\\frac{\\text{B}\\text{W}}{70}}\\right)\\times ({\\text{B}\\text{W}\\times \\text{A}\\text{T}\\times {10}^{6})}^{-1}$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equ6\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equ6\" class=\"mathdisplay\"\u003e$$\\text{C}\\text{a}\\text{r}\\text{c}\\text{i}\\text{n}\\text{o}\\text{g}\\text{e}\\text{n}\\text{i}\\text{c} \\text{r}\\text{i}\\text{s}\\text{k} = {{\\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{i}\\text{n}\\text{g}\\text{e}\\text{s}\\text{t}\\text{i}\\text{o}\\text{n}}+ \\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{d}\\text{e}\\text{r}\\text{m}\\text{a}\\text{l} \\text{c}\\text{o}\\text{n}\\text{t}\\text{a}\\text{c}\\text{t}}+ {\\text{I}\\text{L}\\text{C}\\text{R}}_{\\text{i}\\text{n}\\text{h}\\text{a}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}$$\u003c/div\u003e\n\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eCSF is the carcinogenic slope factor, which is represented in units of (mg kg-1day-1)\u003csup\u003e\u0026minus;1\u003c/sup\u003e. According to the USEPA, the CSF concentrations of BaP for the three exposure pathways are 25, 7, 3 and 3.85 mg/kg/day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. CS is the total PAHs concentrations that transformed to hazardous equivalents of BaP using the Toxic Equivalence Factor (TEF) (in ng/g). Calculation of the ILCR relies heavily on the detection of PAHs as BaP-equivalent concentrations using the TEF of each PAHs relative to BaP. The total ILCR is equal to the sum of three routes: skin contact, oral consumption, and inhalation. If the ILCR is less than 1/10\u003csup\u003e6\u003c/sup\u003e, it is deemed inconsequential; if it is more than 1/10\u003csup\u003e4\u003c/sup\u003e, there is a reason for serious worry.\u003c/p\u003e\n\u003cp\u003eFor sediment samples, the inhalation component of the ILCR was shown to be negligible and was thus omitted. The ILCR values for adults and children detected in sediment samples from the Danube River are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. The total ILCR in both children and adults are more than 1/10\u003csup\u003e4\u003c/sup\u003e in all seasons, with the highest values recorded in spring followed by winter seasons, which is really a matter of serious concern. In addition, these records are substantially larger than those reported in the Brisbane River in Australia [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. When high amounts are found through long-term surveillance, the residents within the river basin area must be warned, and precautions must be taken to prevent human contact with sediments.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe current work provides a detailed evaluation of PAHs concentrations, seasonal distribution, and ecological risk assessment in water and sediments gathered from six distinct sites along the Danube River in Hungary. Temporal and spatial variations of PAHs were investigated in both rivers' water and sediments, reflecting the anthropogenic sources along the Danube River. The findings highlighted a broad variance range of 16 PAHs contents in water with total concentrations of PAHs ranging from 283.10-541.91, 198.65-404.37, 160.03-324.63, and 228.26-378.19 ng/L for winter, spring, summer, and autumn, respectively. Sediment samples showed PAH ranging from 313.78-622.78, 312.75-595.41, 215.16-465.49, and 311.34-491.78 ng/g for winter, spring, summer, and autumn, respectively. The overall analysis of the results indicates that the putative anthropogenic sources of PAHs were verified to be both pyrogenic (incomplete combustion of biomass and coal) and pyrolytic (incomplete combustion of liquid fossil fuels and vehicle exhaust emissions); with pyrogenic origins predominating over pyrolytic sources. This might suggest that the industries essentially utilize fossil fuels, which would increase the PAHs emissions in the study area. Generally, except for Acy and FI concentrations, the eco-toxicological assessment of the Danube River environment showed no significant PAHs pollutants in sediments, suggesting a low chance for negative biological impacts and low ecological risk. The TEQ in sediment samples ranged from 29.88 ng/g in the winter season to 140.39 ng/g in the autumn season, which, in turn, is considered safe for humans. The total ILCR in both children and adults were calculated to be more than 1/10\u003csup\u003e4\u003c/sup\u003e in all seasons, with the highest values recorded in spring and followed by winter, which constitutes a concerning issue. Continuous monitoring of the PAHs would offer better insight into the scale of the pollution, which would help in devising effective mitigation strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors greatly appreciate the support of Mohammed A. Al-Seady and Ameer Abbas from the University of Debrecen and Budapest University of Technology and Economics Hungary in the sample collection. The authors also thank Mr Mostafa Mahmood from the Budapest University of Technology and Economics for his logistic support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available upon a reasonable request by contacting the corresponding author via [email protected]; [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFund\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026Uacute;NKP-22-3-I-PE-5 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund supported this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. 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Ayoko, \u0026quot;Source apportionment and risk assessment of PAHs in Brisbane River sediment, Australia,\u0026quot; \u003cem\u003eEcological Indicators, \u003c/em\u003evol. 73, pp. 784-799, 2017.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Danube River, Water, Diagnostic ratios, Polycyclic aromatic hydrocarbons, Sediment, Risk assessment","lastPublishedDoi":"10.21203/rs.3.rs-2765619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2765619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Danube is one of the largest transboundary rivers word-wide, having several tributaries. The discharges from industrial activities and wastewater treatment facilities affect the river's aquatic environment. These discharges pose a serious risk to aquatic life by degrading the water and sediment quality. Therefore, 16 Polycyclic Aromatic Hydrocarbons (PAHs) compounds in six different locations were examined along the river over 12 months to investigate the temporal and special variations of the compounds in water and sediment. The findings highlighted a broad variance range in PAHs concentration in water within a year, ranging from 224.85 ng/L in summer to 365.87 ng/L in winter, whereas PAHs in sediment samples recorded values ranging from 316.72 ng/g in dry weight in summer to 422.98 ng/g in dry weight in winter. The overall results indicate that the putative anthropogenic sources of PAHs were of pyrolytic and pyrogenic origin, with pyrogenic sources being more prominent. Generally, except for Acenaphthylene and Fluorene concentrations, the eco-toxicological concerns for the aquatic environment of the Danube River do not pose a significant threat. In addition, the combined impact of the 16 PAHs pollutants in sediments suggests a low chance for negative biological impacts and low ecological risk. The total ILCR for both children and adults is more than 1/10\u003csup\u003e4\u003c/sup\u003e in all seasons, with the highest values recorded in spring followed by winter time, which becomes a matter of urgency.\u003c/p\u003e","manuscriptTitle":"Seasonal distribution, source identification, health and eco-toxicological risk assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in water and sediment from the Danube River in Hungary","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-13 22:42:57","doi":"10.21203/rs.3.rs-2765619/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfdbb1ef-85b6-448c-966f-7c64e6d1ea87","owner":[],"postedDate":"April 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":20610839,"name":"Earth and environmental sciences/Environmental sciences/Environmental chemistry"},{"id":20610840,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"}],"tags":[],"updatedAt":"2023-06-21T08:29:27+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-13 22:42:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2765619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2765619","identity":"rs-2765619","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}