Assessment of water pollution and environmental impacts in the Pineios river basin before and after extreme flood events in Thessaly, Greece

Prior to the flooding events caused by both storms Daniel and Elias during Sept 2023, water samples were collected along the Pineios river basin during May, June, and August 2023 as part of a preliminary monitoring effort (Fig.  2 ). The aim was to assess the presence of emerging and legacy pollutants with known endocrine-disrupting potential, as well as to evaluate microbial activity in the river water under baseline, pre-flood conditions. Following the extreme floods, additional sampling campaigns were conducted in the impacted estuarine areas of the Pineios river basin in November 2023, and again in February 2024. River water samples were collected from various depths across a grid of 24 predefined sampling locations (Fig.  2 ; Table S1 ), representing both upstream and downstream conditions within the basin. All samples were collected using 5-L Nalgene high-density polyethylene (HDPE) bottles (bisphenol and phthalate free; Tritan construction), equipped with polypropylene caps to avoid contamination. Immediately after collection, the sample bottles were wrapped in pre-combusted aluminum foil at 450 °C to minimize light exposure and prevent contamination, then transported to the laboratory in insulated bags using cooling gel packs. Microbiological analyses were performed within 12 h of sampling, while the remaining volume was stored at −20 °C until further chemical analysis. Fig. 2 Map showing the geographical distribution of the sampling sites along the Pineios river basin in the Thessaly region of central Greece Map showing the geographical distribution of the sampling sites along the Pineios river basin in the Thessaly region of central Greece A 200 mL aliquot of river surface water was subjected to solid phase extraction (SPE) using ISOLUTE C18 end-capped (EC) cartridges (500 mg/6 mL; Biotage, Sweden), following a modified version of U.S. Environmental Protection Agency ( 1995 ) Method 525.2. Prior to extraction, all samples were spiked with 200 ng of surrogate internal standards (IS): d 4 -dimethyl phthalate (d 4 -DMP), d 4 -DnBP, and d 4 -DEHP (Sigma-Aldrich). The SPE cartridges were pre-cleaned with 2 × 6 mL acetone and the sample flow was maintained at 2 drops per second. After loading, cartridges were eluted with 6 mL methyl tert-butyl ether (MTBE) followed by 2 × 6 mL of MTBE:n-hexane (1:1, v/v) into pre-cleaned 25-mL glass tubes. To remove residual water, approximately 0.5 g of Na 2 SO 4 was added to each extract. The tubes were vortexed for 30 s, centrifuged at 1800 rpm for 5 min, and the residual organic phase was transferred to new glass tubes. Solvent exchange was performed by evaporating the extracts under a gentle N 2 stream nearly to dryness, redissolving in 3 mL n-hexane, and re-evaporating to a final volume of 1 mL n-hexane. Finally, 100 ng of biphenyl was added to each sample as a volumetric pre-injection standard prior to instrumental analysis. Chemical analysis of phthalates and alternative plasticizers was conducted according to Giovanoulis et al. ( 2018 ) using a gas chromatography tandem mass spectrometry (GC-MS/MS) system (Agilent 7000; Agilent Technologies, Santa Clara, CA, USA) operated in electron impact (EI) ionization mode. Separation was achieved on a DB-5 column (30 m × 0.25 mm i.d., 0.25 µm film thickness). The instrument was equipped with an Agilent 7683B auto-injector, and injections were performed in pulsed splitless mode. Quantification was performed using MassHunter software (version B.04.00; Agilent Technologies, 2008). An 8-point internal standard calibration curve using high-purity reference standards (1–3000 ng/mL) was used, with 1/x weighting and a coefficient of determination r 2  > 0.992. All plasticizer analytical standards were purchased from Merck (Darmstadt, Germany). Method performance was assessed via spiked quality control (QC) samples, with recoveries ranging between 86 and 110%. Concentrations in water samples were blank corrected, if needed, using three procedural blanks and one field blank per batch. The limit of detection (LOD) for each compound was defined as three times the standard deviation of the blanks. If no signal was observed in the blanks, the LOD was based on a signal to noise ratio of three at the lowest calibration point. River water samples (200 mL) were extracted and analyzed following previously validated methodologies (Kärrman et al. 2021 ). Briefly, SPE cartridges EVOLUTE PFAS WAX (150 mg/6 mL; Biotage, Sweden) were conditioned sequentially with 4 mL 0.1% NH₄OH in methanol, 4 mL methanol, and 4 mL ultrapure water. Samples were acidified to pH 4 with glacial acetic acid and loaded at ~2 drops per second. Cartridges were then rinsed with 20 mL 0.01% NH₄OH, 10 mL ultrapure water, 4 mL pH 4 ammonium-acetate buffer, and 4 mL 20% methanol. After centrifugation (at 3000 rpm for 2 min), PFAS were eluted with 4 mL 0.1% NH 4 OH in methanol. Extracts were reduced under a gentle N₂ stream to 600 µL. Chromatographic separation was performed on a Nexera UPLC system coupled to a triple-quadrupole mass spectrometer LC-MS/MS 8060 NX, Shimadzu. A Shim-pack GIST-HP C18-AQ column (50 × 2.1 mm, 1.9 µm) was kept at 40 °C with a flow rate of 0.36–0.40 mL/min. The mobile phase consisted of (A) Milli-Q H 2 O with 2 mM ammonium acetate and (B) MeOH with 2 mM ammonium acetate. The gradient progressed from 95:5 (A:B) to 5:95 in 9 min, followed by a 2-min equilibration. The negative electrospray ionization (ESI⁻) source was operated with the following parameters: interface voltage −1.0 kV, interface temperature 300 °C, desolvation line temperature 150 °C, heat block temperature 350 °C, ion source temperature 600 °C, and heating gas flow 15 L/min. Quantification employed a 10-point internal standard calibration (0.01–20 ng/mL, 1/x weighting; r 2  > 0.99). The evaluation of possible cross contamination during sampling was conducted by using ultrapure water (Supelco) as a field blank. Moreover, three procedural blanks accompanied each batch to monitor laboratory background; all target PFAS were below detection limits in these blanks. QC samples spiked with 20 ng each of PFOA, perfluorooctanesulfonic acid (PFOS), and 6:2 fluorotelomersulfonic acid (6:2 FTS) were extracted with every batch to assess accuracy and precision (PFAS recoveries between 78 and 105%). Method detection limits were calculated as three times the standard deviation of blank signals; measurement uncertainty for individual PFAS averaged ~15%. Finally, the PFAS surrogate IS, and reference standards were obtained from Wellington Laboratories, while all organic solvents used in this study were of MS analytical grade and were purchased from Rathburn Chemicals Ltd (Walkerburn, Scotland, UK). The analysis of bacterial contamination in river water samples was conducted using the membrane filtration technique, following the standardized procedure described in International Organization for Standardization, ISO 7704:  2023 for membrane preparation and handling. For the detection and enumeration of Escherichia coli and total coliforms, the method prescribed in ISO 9308-1:  2014 was applied, while Enterococcus spp. was enumerated according to ISO 7899-2:  2000 . Water sampling was performed during early morning hours to minimize thermal degradation of microbial populations. Samples were collected in sterile Pyrex glass bottles (500 mL, blue screw cap), pre-sterilized via autoclaving at 121 °C for 30 min. Transportation to the laboratory was carried out in isothermal containers, maintaining a temperature of approximately 8 °C, and all microbiological analyses were initiated within 8 h of sample collection to preserve the integrity of microbial counts. Duplicate samples were collected at each site during all sampling occasions, covering riverine and coastal zones of the Pineios river basin in the Thessaly region, including areas near the cities of Larissa, Tyrnavos, and Elassona. For selective culturing, two growth media were employed. Slanetz-Bartley Medium (m- Enterococcus agar), as referenced in ISO 7899-2:  2001 , was prepared by dissolving 43.5 g/L and used for the enumeration of Enterococcus spp. For E. coli and coliforms, Harlequin® Chromogenic Coliform Agar (CCA) (Neogen, compliant with ISO 9308-1:  2014 ) was used at a concentration of 28.7 g/L. From each sample, 100 mL of water was filtered through sterile membrane filters, which were then placed on the appropriate selective agar medium: CCA for coliforms and E. coli , and Slanetz-Bartley Medium for Enterococcus spp., while incubation was performed at 37 °C for 24 ± 2 h in an inverted position. Colony morphology was used for preliminary identification: Enterococcus spp. colonies appeared dark red to brown, while total coliforms appeared pink to red, and E. coli showed a blue-violet coloration. Confirmatory testing for E. coli and coliforms included a two-step protocol: (i) suspect colonies were transferred to Tryptone Soy Agar (TSA) and incubated at 35–37 °C for 24 ± 2 h and (ii) oxidase testing using oxidase strips. Colonies showing no color change were classified as oxidase-negative (confirming coliform identity), whereas oxidase-positive colonies turned blue to light purple. For Enterococcus spp. confirmation, membranes were transferred from the Slanetz-Bartley Medium to Bile Esculin Agar plates and incubated at 44 ± 0.5 °C for 2 h, per ISO 7899-2:  2001 guidelines. According to ISO 9308-1:  2014 , total coliforms include all oxidase-negative colonies with pink to red coloration, as well as all dark blue-violet colonies observed on CCA plates. This methodological approach ensured both selectivity and specificity in the detection of key microbial water quality indicators under field-relevant conditions in a flood-affected Mediterranean watershed. Descriptive statistics, data visualization (including tables, charts and plots), and statistical significance testing between sampling dates and locations were conducted using GraphPad Prism 10.4.2, Microsoft Excel, and R (stats package). These tools were used to evaluate spatial and temporal trends in contaminant concentrations and microbial counts. From Table S2 , only compounds with a detection frequency (DF) ≥ 50% were included in the statistical analysis; non-detects were replaced by half the LOD to minimize bias. Prior to statistical analysis, all data were checked for normality using the Shapiro-Wilk test. As not all datasets met the assumption of normality, non-parametric tests were applied. The Mann-Whitney U test was used to compare independent groups, while the Wilcoxon matched-pairs signed rank test was employed to evaluate significant differences between paired samples from the same river estuary locations across different sampling occasions, before and after the flooding events in Thessaly. Statistical significance was considered at p  < 0.05 to determine whether emerging contaminant concentrations differed between time points.

Phthalate ester concentrations in the Pineios river basin exhibited substantial spatial and seasonal variation, with certain compounds reaching elevated levels. Total phthalate concentrations ranged from 0.1 to 39.7 µg/L, with median values between 1.1 and 5.3 µg/L across different sampling campaigns. Among individual phthalates, 7 out of 9 were nearly ubiquitous, while DEHP was consistently among the most abundant, with concentrations spanning 0.09–0.43 µg/L, and median values of 0.14–0.36 µg/L. DEHP contributed up to 33% of the total phthalate burden, and up to 23% of the overall plasticizer contamination (i.e., sum of phthalates and alternative plasticizers), reflecting its widespread use and persistence in aquatic environments. The other legacy phthalates DnBP, DiBP, and BBzP also featured prominently, with median concentrations of 0.16–1.89 µg/L, 0.017–0.1 µg/L, and 0.025–0.104 µg/L, respectively. Other high molecular weight phthalates were low (e.g., dipropylheptyl phthalate, DPHP median 0.008–0.011 µg/L) or even not detected in any of the surface water samples collected throughout the monitoring campaign (Table S3 ; Table S4 ). The absence of high molecular weight phthalates such as diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP) in the surface waters of the Pineios river basin likely reflects their physicochemical properties, particularly their hydrophobic nature and strong affinity for sediments. These compounds possess high octanol-water partition coefficients (logK ow ), indicating a strong tendency to adsorb to organic matter and particulate materials in aquatic environments. Consequently, they preferentially partition into sediments rather than remaining dissolved in the water column. This behavior is consistent with findings from other studies, which have reported that high molecular weight phthalates are more prone to accumulate in sediments due to their low water solubility and high hydrophobicity. Therefore, their absence in surface water samples does not necessarily indicate a lack of contamination but rather suggests a predominant association with sediment compartments (Zhang et al. 2018 ). In contrast, low molecular weight phthalates such as DMP and diethyl phthalate (DEP) showed considerable concentrations, with DMP median values 0.1–3.5 µg/L and DEP 0.11–0.22 µg/L, indicating widespread use and diffuse sources throughout the catchment. Compared to the Venta river in Lithuania, where median concentrations of DnBP and DEHP reached 1.83 µg/L and 1.17 µg/L respectively, the corresponding phthalate levels in the Pineios river were generally lower (0.702 µg/L and 0.223 µg/L respectively), although still reflecting diffuse sources of contamination in the basin (Kerine et al. 2011 ). Phthalate levels in the Pineios river basin are comparable to those reported in central Spain, where the Manzanares river, flowing through the city of Madrid, showed the highest contamination with an average of 0.724 ± 0.783 µg/L, followed by the Jarama river at 0.667 ± 0.359 µg/L, reflecting the influence of nearby urban and industrial activities, particularly plastics manufacturing (Domínguez-Morueco et al. 2014 ). Alternative plasticizers, recognized as emerging contaminants, were detected across the Pineios river basin with a concentration range of 0.02–11.4 µg/L and median values between 0.06 and 0.149 µg/L. Among these compounds, 3 out of 5 were omnipresent, and di(2-ethylhexyl) terephthalate (DEHT) clearly dominated, with median concentrations spanning 0.048–0.147 µg/L across all sampling sites. On average, DEHT constituted 92.8% of the total alternative plasticizer load, underscoring its widespread occurrence and environmental relevance (Fig. S2 ). DEHT alone contributed up to 8% of the overall plasticizer burden, underscoring its increasing environmental relevance and supporting its classification as an emerging contaminant. Other alternative plasticizers such as di(2-ethylhexyl) adipate (DEHA) and acetyl tributyl citrate (ATBC) were also consistently detected, though at lower concentrations, with median levels below 0.011 µg/L, corresponding to 4.3% and 2.5% of the total alternative plasticizer concentrations, respectively. In contrast, tris(2-ethylhexyl) trimellitate (TOTM, 0–29% DF) and diisononyl cyclohexane-1,2-dicarboxylate (DINCH, 0–14% DF) were rarely detected or entirely absent in surface river waters, mirroring the patterns observed for other high molecular weight phthalate esters (Fig. S2 ; Table S5 ). Alternative plasticizers are significantly less studied in river waters than in sediments, despite their increasing use and potential environmental impact. DEHA concentrations in coastal waters of northern Spain were low, averaging 0.012 µg/L (Sánchez-Avila et al. 2013 ). In the Pineios river basin, DEHA concentrations were comparable, with median values ranging from 0.085 to 0.011 µg/L (mean 0.094 µg/L). DEHT, although rarely investigated, showed exceptionally high concentrations in coastal waters from the south Mediterranean (634 µg/L) (Jebara et al. 2021 ), significantly higher than the Pineios estuarine levels, where a mean of 0.511 µg/L was observed. ATBC has only been reported in a single study with a concentration around 0.05 µg/L (Smith et al. 2015 ), while in Pineios, levels ranged lower at 0.003–0.007 µg/L. Currently, there are no published data on DINCH or TOTM in estuarine waters (Billings et al. 2024 ), consistent with findings from Pineios where DINCH remained undetected and TOTM was only identified at 3 out of 24 locations, ranging from < LOD to 0.009 µg/L. These results underscore the complexity of monitoring emerging plasticizers in aquatic environments and emphasize the need for expanded, targeted surveillance in estuarine and riverine systems. Thirteen out of 63 PFAS were consistently detected across the Pineios river basin and sampling periods. Compounds such as perfluoropropionic acid (PFPrA, 13–86% detection frequency, DF), perfluorobutanoic acid (PFBA, 75–86% DF), perfluoropentanoic acid (PFPeA, 50–86% DF), perfluorohexanoic acid (PFHxA, 38–80% DF), perfluoroheptanoic acid (PFHpA, 60–100% DF), PFOA (70–88% DF), perfluorodecanoic acid (PFDA, 50–86% DF), perfluoroundecanoic acid (PFUnDA, 20–43% DF), and perfluorotridecanoic acid (PFTrDA, 13–38% DF) were among the most frequently observed carboxylic acids. Among sulfonic acids, perfluorobutane sulfonic acid (PFBS, 63–88% DF), perfluorohexane sulfonic acid (PFHxS, 0–30% DF), PFOS (14–63% DF), and 6:2 FTS (14–40% DF) were detected. Total median PFAS concentrations were 2.26–5.77 ng/L, with individual concentrations spanning 0.05 to 11.9 ng/L (Table S6 ). The presence of long-chain PFAS and fluorotelomer compounds underscores the risks of persistence and potential bioaccumulation associated with this chemical group in aquatic systems (Wang et al. 2024 ). Furthermore, next-generation dodecafluoro-3H-4,8-dioxanonanoic acid (ADONA) was detected twice at 0.05 ng/L at estuarine samples (i.e., locations S4 and S7; Fig.  2 ), aligning with omnipresent detections in nearby seawater where the Pineios river discharges (Lougkovois et al. 2025 ). In comparison to the Danube river, where average surface water concentrations of PFOA, PFOS, and PFHxA reached 12.1 ng/L, 6.11 ng/L, and 10.0 ng/L respectively (Buljovcic et al. 2022 ), and compared to the average ground water levels of PFOA (3 ng/L), PFOS (4 ng/L), perfluorononanoic acid (PFNA, 0 ng/L), and PFHpA (1 ng/L) reported in a broader European survey (Loos et al. 2010 ), PFAS levels detected in the Pineios river basin were generally lower. However, they frequently exceeded the proposed 4.4 ng/L limit for PFAS-24, expressed as PFOA equivalents (SCHEER  2025 ), and were often close to and sometimes above the EU environmental quality standard (EQS-AA) threshold of 0.65 ng/L for PFOS (EU 2020 ). This suggests comparable environmental concern despite the Pineios region’s more limited industrial activity. PFAS concentrations in the Fyris river near Stockholm Arlanda airport, Sweden, were substantially higher, with ΣPFAS averaging 300 ± 120 ng/L at sites impacted by historic use of PFAS containing firefighting foams and peaking at 620 ng/L. In contrast, river segments in the Uppsala area, primarily influenced by municipal wastewater discharges, showed lower average concentrations of 19 ± 13 ng/L, while a relatively pristine upstream reference site recorded 6.4 ± 8.7 ng/L (Nguyen et al. 2022 ). Treated effluents from nine Swedish wastewater treatment plants (WWTPs) showed average PFOS and PFOA levels of 2.8 ng/L and 3.93 ng/L, respectively (Swedish EPA  2025 ). Also, the poorly studied 6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB) was identified as the predominant PFAS in a river in northern France receiving wastewater from an industrial WWTP (Boiteux et al. 2017 ), whereas PFAS were not measured in 24-h composite influent and effluent samples from a municipal WWTP in Thessaloniki, Greece (Ofrydopoulou et al. 2021 ). Furthermore, a recent UK-wide survey reported the first systematic investigation of trifluoroacetic acid (TFA) pollution in rivers, detecting TFA in 31 out of 32 sites with a national average concentration of 2.21 µg/L (Fidra 2025 ). This confirms previous findings that TFA levels are orders of magnitude higher than those of other PFAS (Arp et al. 2024 ), underscoring its widespread occurrence and origins from both direct applications and degradation of precursor PFAS and fluorinated greenhouse gases (F-gases). In this context, although PFAS concentrations in the Pineios river appear modest, they still frequently exceed key regulatory thresholds, reinforcing the need for comprehensive monitoring in catchments affected by both industrial and non-industrial pollution sources. In the estuarine zone of the Pineios river, sampling points S1 to S13 (excluding S10) cover approximately 5000 hectares shaped by intensive agriculture and seasonal tourism (Fig. S3 ). Agricultural practices, such as kiwifruit cultivation involving heavy fertilizer application, may contribute to nutrient and organic pollution downstream. Additionally, the widespread use of plastic films, irrigation tubing, greenhouse covers, and agrochemical packaging likely introduces phthalates and alternative plasticizers into the environment via soil leaching and runoff. Also, tourism-related wastewater and personal care products further contribute to plasticizer loads during the summer months. Sampling points S10 and S19 lie in the Tempi Valley, a narrow corridor through which the full flow of the Pineios river passes through the highly active Thessalian plain, an area characterized by dense agricultural and livestock operations. Further upstream, sampling points S20, S22, and S24 are located along the Titarisios tributary of the Pineios river, in a region predominantly characterized by agricultural and agri-food activities. Point source pollution from domestic and industrial effluents can have a well-defined and localized impact on water quality, while non-point source pollution from intensively cultivated land is more diffuse and difficult to quantify (Fytianos et al. 2002 ; Giakoumatos et al. 2024 ). The presence of dairies, wineries, and livestock farms in this area raises concerns about potential organic pollution, particularly in cases where wastewater treatment infrastructure may be inadequate, thereby contributing to the degradation of surface water quality. Recent findings of elevated trifluoroacetic acid (TFA), which is a persistent PFAS, in European wines suggest agro-industrial processes as emerging pathways for PFAS contamination (GLOBAL 2000,  2025 ). Facilities such as dairies and wineries also use plasticized materials (e.g., in hoses, tanks, packaging), which can release plasticizers into the aquatic environment when improperly managed. Moreover, total PFAS concentrations of 4.3 and 3.6 ng/L were consistently detected at sampling points S14 and S16, respectively, both situated near the city of Larissa (~164,095 inhabitants) and its industrial zone (Hellenic Statistical Authority  2024 ). This area is characterized by diverse industrial and municipal activities, which are known contributors to PFAS emissions. Industrial operations can leave persistent PFAS residues in soil and groundwater, which may migrate and impact adjacent water bodies (Schroeder et al. 2021 ). These overlapping agricultural and industrial sources in the Pineios basin may explain the co-occurrence of PFAS and plasticizers, underscoring the need for targeted monitoring and improved regulatory control in these vulnerable catchment zones. Among phthalate esters, DEHP is currently regulated under the EU Water Framework Directive (WFD) as a priority substance, with an environmental quality standard (EQS) of 1.3 µg/L for inland surface waters (EU 2020 ). However, most other legacy phthalates and emerging alternative plasticizers remain unregulated, despite their frequent detection in the environment and growing evidence of endocrine disruption and ecotoxicological effects. This highlights a critical gap in current regulatory frameworks. In the present study, all DEHP concentrations remained below the EQS threshold, suggesting limited risk from this compound alone. Nevertheless, the high variability in total plasticizer concentrations, coupled with the presence of numerous other plasticizers (both regulated and unregulated), raises concerns regarding the cumulative impact of these substances. This cumulative plasticizer burden introduces considerable uncertainty regarding the ecological safety of surface waters in the Pineios river and poses potential risks to downstream and connected aquatic ecosystems, particularly in the context of extreme weather events and contaminant mobilization. For PFAS, the EU WFD (EU 2020 ) introduces a health-based guideline value of 4 ng/L that has been established for PFAS-4 (i.e. PFOA, PFOS, PFNA, PFHxS), a threshold for drinking water sources of 100 ng/L for the sum of 20 PFAS of most concern, together with 6:2 FTS consisting of the broader PFAS-21 group (Table S2 ), and a benchmark value of 500 ng/L for total PFAS increasingly used in risk assessments, acknowledging cumulative exposure and mixture toxicity. Additionally, the extended PFAS-24 group (Table S2 ), which includes emerging substances such as perfluoro-2-propoxypropanoic acid (GenX), ADONA, perfluoroacetic acid, 2-[(5-methoxy-1,3-dioxolan-4-yl)oxy], ammonium salt (C6O4), and fluorotelomer alcohols, is proposed to be regulated at 4.4 ng/L expressed as PFOA equivalents, under upcoming EU legislation set to take effect in January 2026 (SCHEER  2025 ). Furthermore, PFOS remains a priority hazardous substance under the WFD with an EQS of 0.65 ng/L (annual average) for surface waters. In this study, a comprehensive panel of 63 PFAS compounds was analyzed in surface water samples from the Pineios river basin. Concentrations were evaluated against thresholds for PFAS-4, PFAS-21, PFAS-24, and total PFAS, to assess potential human health risks and the environmental quality status of the river system (Table S7 ). These evolving EU regulatory frameworks are particularly relevant for Greece, especially in flood-prone regions like Thessaly, where extreme weather events have increased the likelihood of pollutant mobilization. As illustrated in Fig.  3 , the measured median concentrations approached established or proposed limits, and in several instances (especially for PFAS-24) exceeded the proposed threshold of 4.4 ng/L (expressed as PFOA equivalents). These findings underscore the need for targeted monitoring and risk management, particularly in the context of climate-driven hydrological disturbances, which may amplify the mobilization and dispersion of PFAS. Fig. 3 Median-max-min concentrations of A DEHP, the sum of phthalate esters, alternative plasticizers, and total plasticizers; and B PFOS, the sum of PFAS-4, PFAS-21, PFAS-24, and total PFAS in water samples across the Pineios river basin, Thessaly, Greece, collected before and after the extreme flood events in September 2023. Red lines indicate applicable EU regulatory water limit values Median-max-min concentrations of A DEHP, the sum of phthalate esters, alternative plasticizers, and total plasticizers; and B PFOS, the sum of PFAS-4, PFAS-21, PFAS-24, and total PFAS in water samples across the Pineios river basin, Thessaly, Greece, collected before and after the extreme flood events in September 2023. Red lines indicate applicable EU regulatory water limit values Moreover, certain PFAS contamination pathways may have been overlooked in the present assessment. Several pesticide active ingredients, particularly insecticides, acaricides, and fungicides, contain or degrade into PFAS (Donley et al. 2024 ). These substances are commonly used in crop protection across Greece, and the intensive agricultural activities in the Pineios basin make it plausible that such compounds enter surface waters via runoff, drainage, or leaching. Importantly, these PFAS-based pesticides or their degradation products can give rise to highly persistent substances such as TFA, a short-chain PFAS. TFA may also form through metabolic processes in humans and animals exposed to fluorinated pesticides classified as PFAS, thus contributing to the overall PFAS burden in aquatic environments (PAN Europe 2024 ). However, the analytical method employed in this study was not designed to detect TFA or fluorinated pesticide residues in surface water. Including such analytes in future monitoring efforts would offer a more complete picture of PFAS pollution in one of Greece’s most agriculturally intensive regions. Moreover, a broader evaluation of the contamination profile of the Pineios river is essential to safeguard the touristic bathing areas near its estuary in accordance with the standards set by the EU Bathing Water Directive (EEA 2025 ). It is important to note that significant pollution of surface and groundwater remains outside the scope of Bathing Water Directive assessments. Such pollution may be further exacerbated by the effects of climate change. Therefore, enhancing the resilience of water systems is of paramount importance for both human and environmental health. Beyond the chemical contamination patterns described above, the floods also produced pronounced microbiological impacts. Assessing fecal indicator bacteria provides complementary insight into the immediate public health dimension of the event and the effectiveness of post-flood water quality recovery. Comparisons between sampling campaigns revealed significant increases in fecal indicator bacteria following the September 2023 flood events. Mean E. coli concentrations rose from 208.4 cfu/100 mL prior to the floods to 242.5 cfu/100 mL in the post-flood samples, while Enterococcus spp. showed a more pronounced increase from 136.8 cfu/100 mL to 326.2 cfu/100 mL ( p  < 0.05) after the floods. Similarly, total coliform levels increased from 774.8 cfu/mL before the floods to 986.2 cfu/mL afterward. The most affected sites were those located downstream and in the estuarine zone (S1–S13), particularly near wastewater discharge points (S7) and flooded agricultural areas (S3, S4). Upstream stations showed comparatively lower bacterial loads and faster recovery by February 2024, consistent with reduced anthropogenic influence. These patterns indicate that flooding mobilizes large volumes of fecal material and microbial contaminants from urban and rural sources into the lower river system. High levels of total coliforms were consistently detected across all sampling periods in the Pineios river, with median concentrations spanning 165–1428 cfu/100 mL (Fig.  4 ), indicating widespread microbial contamination. Their persistent presence alongside established fecal indicators raises serious concerns for both environmental and public health, as elevated E. coli and Enterococcus spp. levels are strongly associated with increased risks of gastrointestinal and dermatological infections in recreational and agricultural water users (Wyer et al. 2018 ; Odonkor and Ampofo 2013 ). Seasonal variations in E. coli , Enterococcus spp., and total coliform concentrations are documented across European inland waters, influenced by factors such as precipitation, ambient temperature, and surrounding land use (Stallard et al. 2016 ). Similar to the trends observed in the Pineios river (Fig.  4 ), these fluctuations often result in exceedances of established microbial quality thresholds, posing considerable risks to both environmental and public health. For instance, studies in the Rhine and Moselle rivers in Germany have shown that rainfall events and increased river discharge significantly elevate levels of fecal indicator bacteria, primarily due to runoff from agricultural areas and urban surfaces transporting fecal contaminants into adjacent water bodies (Herrig et al. 2019 ). In the Göta älv river (Sweden), E. coli concentrations exhibited seasonal patterns similar to those in the Pineios river, with higher levels and greater variability during colder months; median concentrations in February reached 180 cfu/100 mL (Göta älv) vs 309 cfu/100 mL (Pineios), and maximum values of 1300 and 1010 cfu/100 mL, respectively. Key factors influencing E. coli levels at water intake points included water temperature, upstream microbial loads, and precipitation (Sokolova et al.  2022 ). In general, inland bathing waters, such as rivers and lakes, are typically more vulnerable to microbiological quality deterioration than coastal sites, largely due to their closer proximity to agricultural runoff and untreated or partially treated sewage discharges (UK Environment Agency 2023 ). Fig. 4 Median-max-min total concentrations of bacterial indicators ( Escherichia coli , enterococci, and total coliforms) across the Pineios river basin, Thessaly, Greece, collected before and after the extreme flood events in September 2023. Red lines indicate applicable EU regulatory water limit values Median-max-min total concentrations of bacterial indicators ( Escherichia coli , enterococci, and total coliforms) across the Pineios river basin, Thessaly, Greece, collected before and after the extreme flood events in September 2023. Red lines indicate applicable EU regulatory water limit values Before the flood events in Thessaly, E. coli concentrations at most sites remained below or near the EU bathing water threshold of 250 cfu/100 mL (Wyer et al. 2018 ), with exceedances observed only at S7 and S9 near wastewater outfalls. After the floods, however, exceedances occurred at over 70% of sites, with peak values up to 1010 cfu/100 mL recorded at S4 (estuarine zone) and 860 cfu/100 mL at S12 (flooded farmland). Enterococcus spp . levels also rose markedly from typical pre-flood values of 45–80 cfu/100 mL (EU Directive 2006 /7/EC) to as high as 413 cfu/100 mL post-flood, indicating substantial fecal contamination across the basin. These increases correspond temporally with reported wastewater infrastructure failures and livestock mortality following the September 2023 floods. The elevated bacterial loads are attributed to decaying animal carcasses, the failure of wastewater infrastructure, and runoff from affected agricultural zones, consistent with post-flood contamination mechanisms documented in Greece and other Mediterranean catchments (Diakakis et al. 2025 ; Balta et al. 2024 ; Herrig et al. 2019 ). These findings underscore the urgent need for targeted remediation measures, effective carcass disposal strategies, and sustained microbiological monitoring throughout the Pineios river basin. In addition to consistently elevated values, fecal indicator bacteria levels displayed marked seasonal variation. Exceedances of water quality thresholds occurred both during the summer months, a pattern likely driven by warmer temperatures that enhances bacterial persistence and proliferation, and during the winter after the flood events. Seasonal peaks are further influenced by agricultural runoff and episodic wastewater treatment failures. Heavy precipitation events mobilize pathogens from soils and sediments into the water column (Herrig et al. 2019 ), while flood-related infrastructure damage amplifies contamination sources (Diakakis et al. 2025 ). Moreover, clusters of gastroenteritis and respiratory infections were reported in the flood-affected region, indicating a rising public health burden (Diakakis et al. 2025 ; Mas-Coma et al. 2025 ). Post-event analyses by Diakakis et al. ( 2025 ) described reports from local health authorities of elevated gastrointestinal and respiratory symptoms in Thessaly communities during the weeks following the September 2023 floods, concurrent with widespread wastewater infrastructure failure and surface water contamination. Similar associations between exceedances of E. coli and Enterococcus spp. and outbreaks of gastroenteritis after flood events have been documented in Mediterranean and international studies (Mas-Coma et al. 2025 ; Perez Arredondo et al. 2021 ). These published findings strengthen the interpretation that the microbial exceedances observed in the Pineios basin represent not only environmental deterioration but also a tangible risk to human health. Overall, these dynamics highlight the persistent microbiological risks in the region’s surface waters and reinforce the importance of year-round bacterial monitoring, robust wastewater management systems, and integrated public health strategies to prevent waterborne disease transmission. The September 2023 floods profoundly altered the chemical and microbiological quality of the Pineios river system, exposing its high vulnerability to contaminant mobilization during extreme hydrological stress. A comparative assessment of water quality parameters before and after the flood events demonstrated substantial increases in pollutant levels, highlighting the multi-dimensional nature of flood-induced contamination. Specifically, median total plasticizer concentrations increased fourfold (1.3 → 5.4 µg/L), with significant spikes in legacy plasticizers such as DnBP and DEHP. These increases were most pronounced at estuarine sampling points near flood-affected urban and agricultural zones, suggesting strong links to runoff and compromised infrastructure. Simultaneously, fecal indicator bacteria levels, despite some variability, also increased markedly, signaling microbial contamination and elevated public health risks. This includes observed post-flood increases in E. coli (1.2-fold), Enterococcus spp. (2.4-fold, p  < 0.05), and total coliforms (1.3-fold). These trends likely reflect surface runoff following the floods, animal carcass decomposition, and overwhelmed or damaged wastewater treatment facilities. While PFAS exhibited more stable concentrations, their ubiquitous detection across all sampling campaigns, including long-chain and fluorotelomer variants, highlights their persistence in the aquatic environment. Although post-flood PFAS concentrations did not exhibit dramatic increases, possibly due to their lower water-phase concentrations compared to plasticizers and tendency to partition to sediments, their persistent detection across all campaigns, even at low ng/L levels, and in some cases approaching or exceeding regulatory thresholds, remains a concern. This raises questions about sediment-bound reservoirs and long-term exposure risks within the river system. Triggered by Storm Daniel and subsequently intensified by Storm Elias, the September 2023 flooding severely disrupted municipal infrastructure across the Thessaly region, leading to the temporary shutdown of major WWTPs in Larissa, Giannouli, and Mandra (Diakakis et al. 2025 ; Lekkas et al. 2024 ). The uncontrolled discharge of untreated urban wastewater into the Pineios river, as reported by regional environmental authorities, likely introduced high loads of emerging contaminants into the aquatic environment (Lougkovois et al. 2025 ). Among these, plasticizers, as well as persistent compounds like PFAS, have been detected at elevated concentrations, underscoring the vulnerability of freshwater systems during extreme weather events. The overflow of WWTPs may have also released pharmaceuticals, surfactants, and even illicit drug residues into the river, while the destruction of industrial and transport infrastructure may have contributed to the spread of industrial chemicals, including long-chain PFAS (Lougkovois et al. 2025 ). Another documented incident involved central heating oil tanks, commonly located in the basements of urban apartment buildings in Greece, which were submerged during the flood (Lekkas et al.  2024 ). As a result, significant volumes of heating oil were released, contaminating the stormwater system of the city of Larissa with a complex mixture of hydrocarbons and other organic pollutants. These observations are in accordance with findings from Strandberg et al. ( 2024 ), who characterized fuel-induced water contamination and demonstrated that such incidents not only alter chemical composition and odor thresholds but also pose ecotoxicological risks to aquatic ecosystems and drinking water resources. Figure  5 presents the temporal trends of phthalate esters and PFAS concentrations in surface water samples collected at the Pineios river estuary, following the influx of large volumes of floodwaters from upstream areas (Fig.  1 ). Following the extreme flooding events in September 2023, an increase in phthalate ester concentrations was observed, particularly in the November 2023 sampling campaign. Legacy compounds such as DEHP and DnBP showed statistically significant elevations ( p  < 0.05) compared to pre-flood levels, likely reflecting contaminant mobilization from urban, industrial, or sediment sources during the flood. By February 2024, 5 months post-flood, concentrations of most phthalates exhibited a declining trend, approaching values measured prior to the flooding. This pattern suggests a potential return to baseline conditions, although seasonal variability, particularly differences between summer, autumn, and winter, must also be considered when interpreting the observed trends. A similar post-flood temporal pattern was observed for other phthalates (e.g., DMP, DiBP, BBzP, and DPHP), as well as several alternative plasticizers, although these changes were not statistically significant. In contrast, as PFAS concentrations were consistently much lower than those of plasticizers, they did not exhibit major fluctuations across the different sampling periods. Nonetheless, their continuous detection across all time points underscores their persistence in the aquatic environment and raises concerns about long-term accumulation in sediment matrices, which were not evaluated in this study but warrant future investigation. By February 2024, concentrations of most phthalates had declined toward baseline, whereas PFAS and fecal indicators remained elevated, demonstrating slower attenuation and greater persistence. This contrast indicates that plasticizer inputs were largely episodic, whereas PFAS and microbial contamination sustained longer-term impacts on water quality and ecosystem health. Fig. 5 Bar charts showing median concentrations and ranges of A individual phthalate esters (DMP, DEP, DiBP, DnBP, BBzP, DEHP, DPHP) and phthalates sum and B individual compounds within the PFAS-4 group (PFOA, PFNA, PFOS, PFHxS), along with the summed concentrations of PFAS-4, PFAS-21, PFAS-24, and sum of all PFAS. Data were collected at the Pineios river estuary 1 month before, 1 month after, and 5 months after the flood events in Thessaly, Greece (September 2023). Asterisks (*) indicate statistically significant differences between sampling dates ( p  < 0.05) Bar charts showing median concentrations and ranges of A individual phthalate esters (DMP, DEP, DiBP, DnBP, BBzP, DEHP, DPHP) and phthalates sum and B individual compounds within the PFAS-4 group (PFOA, PFNA, PFOS, PFHxS), along with the summed concentrations of PFAS-4, PFAS-21, PFAS-24, and sum of all PFAS. Data were collected at the Pineios river estuary 1 month before, 1 month after, and 5 months after the flood events in Thessaly, Greece (September 2023). Asterisks (*) indicate statistically significant differences between sampling dates ( p  < 0.05) The health impacts associated with floods are multifaceted and evolve over time, encompassing both immediate and delayed consequences. While acute outcomes such as injuries, bacterial infections, and disrupted access to healthcare are frequently observed, increasing scientific evidence underscores the importance of long-term and cumulative health burdens. Floods often intensify pre-existing vulnerabilities, disproportionately affecting children, the elderly, and individuals with chronic diseases or socioeconomic disadvantages (Valavani et al. 2024 ). Of particular concern is prolonged exposure to environments contaminated by floodwaters carrying hazardous chemicals. The concurrent mobilization of phthalates and PFAS can result in sustained health risks that extend well beyond the immediate disaster phase. To more clearly understand the long-term health risks associated with flood-related contamination, it is essential to consider the biological mechanisms through which key flood-borne chemicals, such as PFAS, phthalates, and toxic metals, affect human physiology. PFAS, such as PFOS and PFOA, have been shown to activate peroxisome proliferator-activated receptors, generate mitochondrial reactive oxygen species, inhibit nuclear factor kappa B, and interfere with thyroid and sex hormone signaling. These disruptions have been linked to kidney and testicular cancers, reduced vaccine efficacy, endocrine dysfunction, and impaired neurodevelopment (DeWitt et al. 2012 ; Grandjean and Budtz-Jørgensen 2013 ; Sunderland et al. 2019 ; ATSDR 2021 ; Steenland and Winquist 2021 ). Phthalates, such as DEHP and DnBP, can bind to androgen and estrogen receptors, inhibit steroidogenic enzymes, and induce oxidative stress through cytochrome P450 1A1 upregulation. These mechanisms have been implicated in adverse reproductive and endocrine outcomes, including infertility, endometriosis, polycystic ovary syndrome, altered timing of puberty, and metabolic syndrome (Hauser and Calafat 2005 ; Stahlhut et al. 2007 ; EFSA 2019 ). Similarly, metals such as lead generate oxidative stress and displace essential metal ions like calcium, zinc, and iron. This leads to inhibition of heme synthesis, renal tubular damage, and dysregulation of vascular signaling. Clinically, such effects manifest as anemia, chronic kidney disease, hypertension, cardiovascular morbidity, neurodevelopmental delays, and adverse pregnancy outcomes (Lanphear et al. 2005 ; ATSDR 2020 ; Dang et al. 2024 ). Together, these mechanistic insights emphasize that the chemical pollutants identified in the Pineios river during and after the floods are not only environmental hazards but also potential drivers of chronic disease burden within exposed populations. The Pineios flood exemplifies a One Health challenge in which human well-being, livestock productivity, and ecosystem integrity are deeply interconnected. Following extreme flooding, pollutants can accumulate on agricultural land and be transferred to livestock via contaminated soil, forage, or water, ultimately entering the human food chain through milk and meat products (Lake et al. 2005 , 2015 ). Simultaneously, floodwaters mobilize manure and fecal pathogens, including antimicrobial-resistant Enterobacteriaceae, from inundated farms into aquatic ecosystems, where they may colonize wildlife and facilitate re-entry into human populations through recreational contact, water use, or food contamination (Perez Arredondo et al. 2021 ). Additionally, habitat degradation caused by flooding reduces aquatic biodiversity, disrupting natural ecological buffers and elevating the risk of zoonotic disease emergence (Balta et al. 2024 ). These dynamics reflect a complex One Health scenario, in which environmental pollution, food safety, and public health intersect. These dynamics underline the need for coordinated environmental, veterinary, and public health surveillance supported by early-warning systems and post-flood response planning to detect and mitigate risks promptly (Mas-Coma et al.  2025 ). Establishing cross-sectoral monitoring and targeted remediation programs, particularly in estuarine and agricultural zones where pollutants concentrate, is essential to restore environmental quality and strengthen the resilience of water resources in flood-prone regions. While this study provides valuable insight into the presence and distribution of emerging contaminants and bacterial indicators in the Pineios river basin following extreme flooding events, several limitations must be acknowledged. Firstly, the current analysis focused solely on surface water samples. The inclusion of sediment samples would offer a more comprehensive understanding of contaminant dynamics, particularly for hydrophobic compounds such as plasticizers and PFAS, which are known to accumulate in sediment matrices. Sediment analysis could provide additional evidence of long-term pollution trends, contaminant persistence, and potential remobilization during high-flow events. Secondly, the temporal resolution of sampling was limited to specific dates across a span of several months. A higher sampling frequency, especially immediately before and after flood events, would enhance our ability to capture short-term fluctuations and episodic peaks in contaminant levels. This would improve the interpretation of contaminant fate and transport mechanisms and inform more timely mitigation strategies. Furthermore, the spatial distribution of sampling points, while representative of the basin, may not have fully captured local-scale variations or directly pinpointed contamination hotspots. Incorporating a greater number of sampling locations, particularly near known or suspected point sources, such as wastewater treatment plant outlets, industrial zones, and agricultural discharge areas, would enable better source attribution. Such spatial refinement would strengthen risk assessments and support more targeted and effective remediation or management interventions. To ensure these improvements are implemented efficiently, an integrated coordination mechanism is required. Implementation of these measures should follow a One Health framework that connects environmental and health data streams. Coordinated epidemiological surveillance and environmental biomonitoring, covering microbial, chemical, and antimicrobial-resistance indicators, are needed to track disease trends and exposure-related morbidity in affected communities (Mas-Coma et al.  2025 ; Balta et al. 2024 ). Establishing basin-level response protocols, including post-flood rapid assessment and remediation of wastewater and livestock-contaminated areas, would enhance preparedness for future extreme events. Furthermore, building analytical capacity and methodological coverage is critical to complement these management actions. Analytical methods should be expanded and improved to capture a broader range of PFAS compounds, including TFA, which may arise from the degradation or metabolic transformation of PFAS-based pesticides. Since many PFAS remain undetected using standard targeted methods, the integration of non-targeted screening (NTS) and high-resolution MS can significantly enhance the scope of detection, thereby improving risk assessment accuracy (Megson et al. 2024 ). Given the continuous emergence of new PFAS and their potential environmental and health impacts, such methodological advancements are crucial for informed regulation and comprehensive environmental monitoring (Androulakakis et al.  2022 ). Addressing these limitations in future studies would provide a more holistic and granular understanding of contaminant behavior in the Pineios river and, more broadly, support the development of science-based policies for managing emerging pollutants in Mediterranean river basins.

Safeguarding freshwater systems such as the Pineios river is essential for the environmental sustainability and socioeconomic resilience of Greece’s most agriculturally productive region. River water serves not only as a vital resource for irrigation and food production but also plays a key role in public health protection and ecological integrity (Ingrao et al. 2023 ). However, the growing impacts of climate change, extreme weather events, and diffuse anthropogenic pollution are placing unprecedented strain on freshwater quality and availability. The severe flooding events that struck the Pineios basin in September 2023 clearly demonstrated the vulnerability of river systems to complex mixtures of chemical and microbiological contaminants. These floods disrupted wastewater infrastructure, mobilized pollutants from agricultural and industrial sources, and introduced significant public and ecological health risks. Plasticizer concentrations showed a marked increase immediately after the flooding, while fecal indicator bacteria and several PFAS, especially compounds within the PFAS-4 and PFAS-24 groups, frequently approached or exceeded proposed EU safety thresholds. Furthermore, multiple PFAS compounds were omnipresent in surface water samples, reinforcing concerns about their environmental persistence and potential bioaccumulation. As the frequency and intensity of climate-related hazards continue to rise, the likelihood of such contamination events will only increase. Ensuring the resilience of river basins like Pineios will therefore require sustained investment in climate-resilient water and sanitation infrastructure, strengthened monitoring capacity, and coordinated action under a One Health framework. The findings illustrate that both emerging and legacy contaminants, including plasticizers, PFAS, and microbial pathogens, can persist in the environment and enter complex exposure pathways that threaten human and ecosystem health. Their continued release through industrial discharges, agricultural runoff, and poor waste management highlights the need for comprehensive, preventive pollution-control strategies. Additionally, the potential for long-range transport and transformation into unmonitored byproducts presents further regulatory challenges. PFAS pose enduring concerns due to their extreme persistence, broad use, and resistance to removal, while effective risk assessment is hampered by their vast numbers, unknown mixture effects, and lack of effective mitigation technologies (Wang et al. 2017 ). Ultimately, protecting Mediterranean river systems demands a proactive, science-informed policy framework that links monitoring data with risk management, encourages regulatory innovation, and promotes coordinated preparedness for climate-driven contamination events. This integrative approach will be key to maintaining the resilience of freshwater resources in vulnerable regions.

The Pineios river basin, situated in the Thessaly region of central Greece, is a critical hydrological system that supports Greece’s largest and most important agriculturally productive plain. Originating in the Pindos mountains, the river flows for approximately 262 km, crossing an extensive network of tributaries before passing through the Tempi valley and eventually discharging into the Aegean Sea. Encompassing a drainage area of about 10,700 km 2 , the basin plays a vital role in providing essential services for irrigation, drinking water supply, fisheries, and hydropower for the region (Papaioannou et al. 2007 ). However, this region is increasingly vulnerable to the intensifying effects of global warming and climate change, which manifest in more frequent and extreme weather events such as droughts, heatwaves, wildfires, and catastrophic flooding. Storm Daniel, which occurred between September 4 and 8, 2023, brought extreme rainfall and catastrophic flooding to the Thessaly region of central Greece, severely impacting both urban and rural areas. The Pineios river basin received rainfall equivalent to nearly half its average annual total rainfall over just a few days, as it was one of the most intense precipitation events ever recorded in Greece (Dimitriou et al. 2024 ). The event caused unprecedented damage to infrastructure, displaced communities, and mobilized vast volumes of sediments and pollutants throughout the Pineios river basin. The evolution of the flooded areas during Storm Daniel is illustrated in Fig.  1 . Substantial volumes of water moved east from the western Thessaly region, following the course of the Pineios river toward its estuary. Several locations, particularly those near the estuarine zone, were directly impacted by the flooding. This resulted in the mobilization of soil and sediments from previously contaminated hotspot areas, with the floodwaters transporting these materials throughout the river basin and reshaping the contamination profile of the Pineios river. Also, thousands of hectares of farmland remained submerged for days, resulting in sediment deposition that significantly altered the chemical properties of the soil (Iatrou et al. 2024 ). Just weeks later, the region experienced further disruption from Storm Elias (September 25–28, 2023), which affected parts of the already heavily flooded landscape, compounding the environmental and societal consequences. The spatial distribution of rainfall during September 2023, associated with both storms Daniel and Elias, highlights the extent and intensity of precipitation across Thessaly (Fig. S1 ). These climate-driven disturbances can generate both acute and chronic chemical burdens in freshwater systems, reinforcing the urgency to improve our understanding of emerging threats and to develop strategies that enhance societal resilience. Therefore, rivers like Pineios are critical zones for evaluating not only ecological impacts but also human health risks posed by complex mixtures of pollutants following extreme hydrometeorological events. Fig. 1 Progression and extent of flooding across the Thessaly region during Storm Daniel (September 2023), highlighting severely affected zones, including the Pineios river basin, where surface overflow led to environmental and infrastructural impacts Progression and extent of flooding across the Thessaly region during Storm Daniel (September 2023), highlighting severely affected zones, including the Pineios river basin, where surface overflow led to environmental and infrastructural impacts Among the emerging and legacy contaminants of concern are plasticizers, such as phthalate esters and their main alternatives, as well as the per- and polyfluoroalkyl substances (PFAS), also known as “forever chemicals” (Ateia and Scheringer 2024 ). All of them have wide industrial use and growing toxicological scrutiny. Several phthalates, such as di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), benzyl butyl phthalate (BBzP), and di(2-ethylhexyl) phthalate (DEHP), have long been used as plasticizers and are now classified as Substances of Very High Concern (SVHCs) under EU REACH due to their endocrine-disrupting properties. Also, despite replacing alternative plasticizers claiming to have an improved toxicological profile with less harmful effects on living organisms and the environment, the phenomenon of regrettable substitution remains a concern, as these replacements may also be widely dispersed and poorly characterized in the environment so far (Qadeer et al. 2024 ). PFAS, known for their exceptional chemical stability and ubiquity in consumer goods, from cosmetics (e.g., mascara, foundation) to water-repellent fabrics (e.g., school uniforms, carpets), lubricants, and firefighting foams, are especially persistent and have been linked to adverse health effects including immunotoxicity, metabolic disruption, and carcinogenicity (e.g., perfluorooctanoic acid, PFOA) (Grandjean and Clapp 2015 ). Overall, human exposure to endocrine-disrupting chemicals such as plasticizers and PFAS primarily occurs through diet and drinking water (Giovanoulis et al. 2018 ; Sunderland et al. 2019 ). In addition, bacterial indicators, frequently used in water quality assessments, may signal fecal contamination or infrastructure failure, posing direct risks to public health and aquatic ecosystems (Odonkor and Ampofo 2013 ). This study investigates the concentrations and distribution of phthalates, alternative plasticizers, PFAS, and bacterial indicators in the Pineios river basin (Thessaly, Greece) before and after the extreme flood events of September 2023. As climate change drives more frequent and intense weather events, such as those triggered by storms Daniel and Elias, understanding the environmental consequences becomes increasingly urgent. The aim is to assess exceedances of environmental quality standards and evaluate pollutant mobilization patterns to inform future risk management in flood-prone areas. With limited national data available, particularly for PFAS and plasticizers, this work provides crucial measurements to support the implementation of EU regulations and guide local authorities in identifying contamination hotspots and prioritizing remediation and monitoring strategies.

Below is the link to the electronic supplementary material. ESM 1 (DOCX 2.76 MB) (DOCX 2.76 MB)