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Dioxin-like and estrogenic activity screening in fractionated sediments from a German catchment after the 2021 extreme flood

Environmental Sciences Europe volume 36, Article number: 163 (2024) Cite this article

Abstract

Background

The flood in July 2021 is considered one of the largest flood disasters in Western Europe in decades, with massive socio-economic consequences. The potential emission and remobilization of anthropogenic pollutants can lead to additional environmental consequences, which need to be addressed in long-term mitigation strategies. The Inde River and its tributary, the Vichtbach River, form a catchment located at the transition from the low mountain ranges of the Eifel to the lowlands of the Lower Rhine Embayment in Germany. The area has been an industrial and mining hotspot for centuries, making it a high-risk area for flood sediment pollution. The present study provides an ecotoxicological screening of flood sediments of the Vicht–Inde catchment to gain an impression of the degree of contamination by organic pollutants by means of in vitro effect-based method. Sediment samples were collected within days after the flood and fractionated prior to biotesting, and supportive instrumental geochemical analysis was performed.

Results

Flood sediments did not reveal estrogenic potential, which was included in the testing strategy as a relevant endpoint for industrial chemicals and untreated wastewater. In contrast, moderate-to-high dioxin-like activity was observed in 70% of the sediment samples with a peak dioxin-like potential at the restored section of the Inde. Overall, four hotspot samples were identified as at risk, which aligned mostly with the high concentration of organic pollutants including PAHs and PCBs. The fractionation allowed the identification of PAHs and their derivates as the most likely toxicity drivers for dioxin-like activity in the sediments of the Vicht–Inde catchment.

Conclusion

The results provide first information on the prioritization of hotspot locations at risk for a detailed ecotoxicological profiling and a post-flood monitoring of organic contamination. The identified sinks of contamination in the floodplain areas can be considered a source for remobilization of pollution in future flood events, which is highly relevant for the receiving Rur River.

Background

In July 2021, substantial rainfalls occurred across western Germany and neighboring countries, including Belgium, France, Luxembourg, and the Netherlands. In Germany, the persistent rain caused extreme flood events in the regions Ahr, Erft, Inde and Rur, causing severe impact on human life and infrastructure that led to massive socio-economic consequences [1]. According to first estimations, the precipitation event had a return period of once in 400 years [2], however, the return period of the flood event varies spatially, and is not fully determined yet. The most significant damage occurred in the Ahr region, while the catchment of the Inde River, a tributary of the Rur River, was also dramatically affected. Though often neglected, the environmental consequences of the flood event need to be considered for long-term mitigation due to the potential emission and remobilization of anthropogenic pollutants [3]. In general, direct emissions from infrastructural damage in urban and industrial areas include, e.g., the release of industrial chemicals, biocides, fuel oils or untreated wastewater. Additionally, a flood remobilizes hazardous legacy substances, such as persistent organic pollutants (POPs), which are bonded to suspended particulates and sediments. As reviewed by Crawford et al. [4], this remobilization poses a significant risk to human and environmental health.

In the Inde catchment, many direct emissions of potentially hazardous compounds were observed during the flood event. The remobilization of POPs in flood-translocated suspended particles and sediments can be expected since the Inde River and its tributary systems [5, 6], notably the Vicht catchment, are impacted by centuries-long industrial and mining activities [1, 7, 8]. While the contamination with heavy metals due to the mining legacy is well known in the Vicht–Inde catchment [7, 9], and the impact of the 2021 extreme event on heavy metals is covered in detail in another article within this special issue [10], the relevance of (persistent) organic pollutants is poorly characterized. Nonetheless, a recent qualitative non-target screening for organic pollutants in the region indicated high contamination with organic pollutants during and shortly after the 2021 flood event [6]. Since POPs are being discussed as very relevant legacy pollutants in sediments [4], we see a high relevance to studying their toxicity in the Vicht–Inde catchment due to its history of industrialization. The detailed quantification of individual contaminants in target- or even non-target chemical analysis is rather time-consuming due to, e.g., extensive sample preparation (e.g., clean up). Additionally, instrumental chemical analysis alone cannot account for a complex mixture toxicity due to non-additive interactions of the variety of compounds. Thus, complementing bioassays shortly after a flood event can provide important information for a hazard and environmental risk assessment and guide intensive chemical screenings for risk driver identification [11, 12].

As one of the most sensitive biological endpoints after flood events we included the dioxin-like activity, accounting for different groups of organic substances such as polychlorinated dibenzo-p-dioxins and dibenzo-furans (PCDD/Fs), dioxin-like polychlorinated biphenyls (DL-PCBs), and polycyclic aromatic hydrocarbons (PAHs) [13]. Dioxin-like compounds typically activate the detoxification enzymes cytochrome P450 monooxygenases (CYPs) via binding the aryl hydrocarbon receptor (AhR) [14]. The upregulation of CYP enzymes in xenobiotic biotransformation can be quantified through bioanalytical tools such as the 7-ethoxyresorufin-O-deethylase (EROD) assay, accepted as a valuable biomarker in different matrices such as biological material or environmental samples (e.g., in fish: Whyte et al. [14], sediments, soil, water reviewed in Eichbaum et al. [15]). Previous research has concluded that the in vitro-based µEROD assay with H4IIE cells is an effective screening tool to detect dioxin-like compounds in sediments [15, 16], providing a time- and cost-efficient alternative to chemical analysis for the quantification of dioxin-like potential of samples [17]. We further included estrogenic activity measured through an effect-based method determining the potential to bind and activate the estrogen receptor alpha (ERα) relevant for different industrial chemicals and untreated wastewater since the Inde–Vicht catchment was affected by damaged sewage pipes and treatment plants [6]. Alkylphenols are one exemplary group of estrogenic chemicals that are widely used detergents released by industrial and municipal wastewater treatment plants tending to contribute to sediment contamination in many cases (e.g., [18]).

Since we expect the sediment samples from the Vicht–Inde catchment to contain a complex mixture of a variety of organic contaminants, which could also lead to masking effects in bioassays with the consequence of underestimating the toxicity, we fractionated the samples prior to biotesting. Fractionation allows the separation of contaminants, reducing the matrix interference, eliminating masking of biological responses, and supporting the identification of risk drivers (e.g. [19, 20]).

Hence the aim of the present study was to provide the first ecotoxicological screening in the Vicht–Inde catchment to gain an impression of the degree of contamination with organic pollutants in the flood sediments. In detail, we aimed to (a) quantify dioxin-like and estrogenic activity in sediment samples; (b) to identify hotspot sites for dioxin-like contamination; and (c) to identify hotspot fractions of dioxin-like activity to reveal the most relevant chemical groups as toxicity drivers.

The results will provide valuable information for a detailed ecotoxicological profiling of prioritized sites at risk and a post-flood monitoring of organic contamination. The findings can further support decision-making for future risk mitigation strategies. The study exemplifies further the importance of an extreme flood for the mass transport and relocalisation of dioxin-like and estrogenic particle-bound pollutants in a catchment.

Material and methods

Vicht–Inde catchment

The Inde is a river of 54 km length originating in the High Fens in eastern Belgium, which confluences with the river Rur around the city Jülich-Kirchberg (Fig. 1). The Vicht is the most significant tributary river to the Inde and is 23 km in length. It begins at the confluence of the Grölisbach and Dreilängerbach streams and flows into the Inde downstream of the city of Stolberg. It is characterized as a heavily modified water body due to anthropogenic impact flowing directly through the city of Stolberg [21]. The city of Stolberg has been an important industrial hotspot with a long tradition in mining and other industrial sectors such as glass production, pharmaceuticals, cosmetics, and household products settled in the last century. Downstream of the Vicht–Inde confluence, the river flows past a large number of contaminated sites before a restored section shapes the landscape.

Fig. 1
figure 1

Overview of the Inde–Vicht catchment in North Rhein-Westphalia (Germany) with sampling locations of flood sediments collected after the 2021 extreme flood event. Landuse based on Corine Land Cover 2018

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Sampling

Sediment samples in the Vicht–Inde catchment were collected following the flood event by an interdisciplinary research team of RWTH Aachen University and Goethe University Frankfurt (Fig. 1, exemplary pictures of sampled sites Figure SI1). The present study focused on ten selected surface sediment samples along the rivers Vicht and Inde (Table 1) collected within natural environmental settings in 1–3 cm depth in the range of 4 to 19 days after the heavy rainfalls in mid-July to ecotoxicologically characterize the degree of potential contamination with organic pollutants. Samples were prioritized according to documentation of infrastructural damage and fluvial-morphologic observations on site. The sampling sites covered one urban site allocated in the city center and vegetated floodplains, where flood deposits were especially clearly visible. All samples were collected in the receding flood wave.

Table 1 Prioritized sediment samples from the Vicht–Inde catchment
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Grain size composition of sediment samples

To assess the grain size composition of the fine sediment fraction, the routine for laser diffraction measurement, as established by Schulte et al. [22] was followed. The samples were dried at 35 °C, gently crushed in an apatite mortar, and sieved to < 2 mm. Organic matter was removed by treating the sediment repeatedly with 0.70 mL 30% H2O2 at 70 °C for several hours until the samples were bleached. To promote dispersion of particles, overnight treatment with 1.25 mL Na4P2O7 (0.1 mol L−1) in an overhead shaker followed [23, 24]. The laser diffraction measurements were performed with a Beckman Coulter LS 13 320. Grain size distribution was calculated following Mie theory (Fluid RI: 1.33; Sample RI: 1.55; Imaginary RI: 0.1) [22,23,24,25,26]. Due to their higher potential for absorbing pollutants to their surface, fine particles are particularly relevant when investigating fluvial sediments [27]. As many clay minerals are typically found up to the size fraction of fine silt [28] 6.3 µm was used to evaluate the grain size effect on toxicity of the sediments.

Sediment extraction and fractionation

The present study aimed to focus on the organic contamination, which led to the selection of accelerated solvent extraction (ASE) method. Heavy metals were not considered with this extraction method. Sediment samples were extracted and fractionated as detailed in Bellanova et al. [6]. Briefly, 15–20 g of sediment mixed with 2 g diatomaceous earth (Dionex ASE™ Prep DE) was extracted using accelerated solvent extraction (Dionex ASE 150, Thermo Fisher Scientific, Waltham, MA, USA). In addition to the ten sediment samples, one laboratory blank consisting of 1–2 g diatomaceous earth was extracted to exclude toxicity caused by sample preparation. Extraction conditions were set to 100 °C and 10 MPa for 5 min. Samples were extracted with 30 mL acetone, acetone/n-hexane (1:1), and n-hexane followed by drying and fractionating over microcolumn (conditioned at 200 °C) as described previously [29] and summarized in Table 2.

Table 2 Fractionation of prioritized sediment samples from the Vicht–Inde catchment
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50 µL of surrogate standard solution was added to each fraction of the samples. The surrogate standard consisted of 5.8 ng/µL fluoroacetophenone, 6.28 ng/µL d10-benzophenone and 6.03 ng/µL d34-hexadecane. For bioassays, the solvent was exchanged to dimethylsulfoxide (DMSO) by gently blowing down eluting solvents under a nitrogen stream and adding DMSO adjusting all extracts to a concentration of 20 g sediment equivalent (SEQ)/mL.

Solvent (DMSO) and surrogate standard controls were tested in addition to the individual fractions of laboratory blank and sediment extracts in the individual bioassays to exclude possible toxic effects caused by the solvent or surrogate standard.

Compound quantification in individual fractions

Gas chromatography–mass spectrometry (GC/MS) was applied for identification and quantification of individual compounds. GC/MS measurements were performed on a quadrupole Trace GC/MS (Thermo Finnigan LLC, USA) with helium as the carrier gas, equipped with a 30 m × 0.25 mm i.d. × 0.25 μm film ZB-5 fused silica capillary column (Zebron capillary GC column). Samples were measured with a temperature program starting at 60 °C (injector temperature 270 °C), splitless time of 60 s, a heating rate of 3 °C/min to 310 °C with an isothermal time of 20 min. Measurements were performed in full scan mode with a scan range from 35 to 650 m/z in positive electron impact ionization mode (EI +) with 7 eV electron energy.

Compound identification was achieved via the mass spectra NIST MS database (National Institute of Standards and Technology (NIST), U.S. Department of Commerce, USA). Compound verification based on mass spectral parameters was performed with standard reference materials. Quantification of compound concentrations was accomplished by integrating specific ion chromatograms in combination with external four-point calibration of the reference material. Since fractions B2, 3 and 4 were most relevant for the biologically derived dioxin-like activity (see “Results” section), 19 PCBs and 16 US EPA priority PAHs were quantified. Concentrations of chemicals were provided per sediment dry weight (dw).

Cultivation of H4IIE and U2-OS cell

Rat hepatoma H4IIE cells (purchased from ATCC, American Type Culture Collection, USA) were cultivated in 75-cm2 flasks in Dulbecco’s modified Eagle medium with phenol red (DMEM, low glucose, pyruvate, no glutamine, 2% GlutaMAX, Gibco, United Kingdom) supplemented with 10% fetal calf serum (FCS, Gibco Mexico) according to Schiwy et al. [30]. Double-transfected human osteosarcoma U2-OS cells from Biodetection Systems (BDS, The Netherlands) were cultured in DMEM (with phenol red) and F12 medium (1:1), supplemented with 7.5% FCS, non-essential amino acids, and a penicillin–streptomycin solution (10,000 units, 10 mg/mL) according to Legler et al. [31] and Sonneveld et al. [32]. Both cell types were incubated at 37 °C, 5% CO2, and 95% humidity up to an overall cell density of 80–90% before passaging. All bioassays were performed with cell passages reaching 80% confluency.

Neutral red retention assay

To exclude false-negative results in mechanism-specific bioassays related to cytotoxicity, the neutral red retention (NR) assay was performed according to Repetto et al. [33]. The assay was performed with H4IIe cells and overall procedures, including seeding, exposure and incubation time, were maintained identical to the corresponding mechanistic bioassay (detailed below in µEROD). Cytotoxic effects to U2-OS cells were extrapolated from the results obtained from H4IIe cells due to limited sample volume (150 µL). Uptake and retention of the NR dye in viable cells was quantified by fluorescent measurement (excitation: 530 nm, emission: 645 nm) after incubating cell layer with NR solution (0.05 mg/mL) for 3 h followed by twice washing with PBS and cell lysis (1% glacial acetic acid, 50% ethanol in water). Cytotoxicity of exposed cells was normalized to the unexposed control cells. If cell viability was affected, a concentration–response curve was fitted using a 4-parameter non-linear regression model with variable slope (top, bottom fixed to 100, 0) and 20% effect concentration (EC20) was calculated as a threshold for an acceptable cytotoxic range in the mechanistic assays (highest test concentration at EC20).

µEROD assay

µEROD exposure procedure

The µEROD assay was performed according to Schiwy et al. [30] to study the dioxin-like potential. 50 µL of a cell suspension at a density of 200,000 cells/mL in DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, supplemented with 10% FCS and 2% GlutaMAX, Gibco, United Kingdom) was seeded in each well of a 96-well plate. Well-plates were incubated first for 1 h at room temperature and then for 2 h at 37 °C, 95% humidity and 5% CO2. In the meantime, serial dilutions (1:2) of sample extract concentrations, which have been excluded for cytotoxic effects in the NR Assay, and of the reference substance 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were prepared in 1% DMSO medium. For exposure, 50 µL of the exposure dilutions were transferred to the cells in four technical replicates resulting in an overall exposure concentration range of 0.4–100 mg SEQ/mL and 0.15–6 pg/mL for samples and TCDD, respectively. Each well-plate contained the calibration series of TCDD and two sediment extracts (tested in 6 concentrations). Resulting solvent content was kept constant at 0.5% DMSO. Cells were incubated at identical conditions as described previously for 72 h. Afterwards, exposure medium was replaced by 100 µL 100-fold ethoxyresorufin solution (49 mL PBS with MgCl2 and CaCl2, 500 μL of 8 μM ethoxyresorufin, and 500 μL of 1 mM dicoumarol) in the dark. Well-plates were incubated for another 30 min followed by addition of 75 µL MeOH and incubation for 20 min to stop the reaction. Fluorescence was measured in a multiplate reader (Tecan Sparks, Tecan GmbH, Germany) at 585 nm excitation and 630 nm emission.

µEROD activity quantification and statistical analysis

µEROD data were analyzed using Microsoft Excel (2016) and plotted in Prism 9 (GraphPad, San Diego, USA). Due to sample volume limitations, 1 or 2 biological replicates with 4 technical replicates per plate were available per fraction and sample. The range of the EC50 (TCDD) and the z-factor were used as validity criteria.

Concentration–response curves were established for EROD induction normalized to maximum EROD induction of TCDD. For this, relative fluorescence unit (RLU) were corrected for corresponding blank value and the mean of the technical replicates was normalized to the maximum RLU of TCDD on the well-plate. Afterwards concentration–response curves of normalized RLU values were plotted in Prism 9 using the logistic non-linear regression model with variable slope and top and bottom fixed to 1 and 0, respectively (Eq. 1):

$$y=\text{bottom}+\frac{(\text{top}-\text{bottom})}{1+{10}^{(\text{logEC}50-x)*\text{hillslope}}}.$$
(1)

Based on the concentration–response curves bioassay-derived TCDD equivalents (bio-TEQ10s) were calculated from the EROD induction as described in [17] by using a non-linear regression GRC model in Microsoft Excel with top and bottom in the range of maximum TCDD fluorescence and minimum solvent control fluorescence, respectively. Bio-TEQs at 10% effect concentrations (TEQ10) were calculated according to Eq. (2) and plotted in Prism 9. All calculations were based on sediment dry weight (dw):

$${\text{Bio-TEQ}}_{10} \left( {\frac{{{\text{pg}}}}{{{\text{g}},{\text{dw}}}}} \right) = \frac{{{\text{TCDD}}\;{\text{EC}}_{10} \left( {\frac{{{\text{pg}}}}{{{\text{mL}}}}} \right)}}{{{\text{sample}}\;{\text{EC}}_{10} \left( {{\text{gSEQ}},\frac{{{\text{dw}}}}{{{\text{mL}}}}} \right)}}.$$
(2)

Besides bio-TEQ values, chemically derived TCDD equivalents (chem-TEQ) were calculated for the fractions B2, B3 and B4 (mostly contributing to EROD activity) according to Eq. (3). The relative potency factors were derived from literature. The World Health Organization (WHO) toxicity equivalent factors (TEF) for PCBs [34] and the relative potency (REP) for in vitro H4IIE cells for PAHs according to Willet et al. [35] and Machala et al. [36] were used:

$$\text{Chem-TEQ}(\frac{\text{pg}}{g, dw})=\sum \left(\text{conci}*\text{TEFi}\right)(\text{with conci}=\text{concentration of compound }i).$$
(3)

ERα-CALUX assay

ERα-CALUX exposure procedure

The ERα chemical-activated luciferase reporter gene expression (ERα-CALUX) assay was performed according to Legler et al. [31] and Sonneveld et al. [32] to investigate the xenobiotic activation of the human estrogen receptor α (ER α). Cells were seeded at a density of 100,000 cells/mL in 100 µL assay medium (DMEM/F12 (1:1), with stripped FCS (7.5%), penicillin–streptomycin (10,000 units, 10 mg/mL), and without phenol red) in a 96-well plate and incubated for 24 h as described above. Serial dilutions (1:2) of sediment extracts and the reference compound 17-ß estradiol (E2) were prepared in 1 mL assay medium at 0.1% DMSO shortly prior to application. After removing the medium, cells were exposed to 200 µL of the sediment extracts (1.25–20 mgSEQ/mL) and E2 (0.1 pM to 1 nM) in triplicates. As for µEROD, cytotoxic exposure concentrations were excluded. One 96-well plate contained the calibration series of E2 and two sediment extracts (tested in 5 concentrations). After 24 h of exposure, the medium was removed, cells were lysed (30 µL lysis mix: 25 mM Tris, 2 mM DTT, 2 mM CDTA, 10% glycerol, 1% TritonX®-100) for 15 min and luminescence was measured in a multiplate reader (100 µL luciferin substrate mix: 20 mM Tricine, 1.07 mM (MgCO3)4 Mg(OH)2, 2.67 mM MgSO4·7H2O, 0.1 mM EDTA, 1.5 mM DTT, 539 μM d-luciferin, 5.49 mM ATP; reaction stop: 100 µL 0.2 M NaOH).

ERα activity quantification and statistical analysis

ERα CALUX data were analyzed using Microsoft Excel and plotted in Prism 9. Test validity criteria for E2 calibration series were curve fit (R2 > 0.98), minimum induction factor (> 5), quality of technical replicates (Z-factor > 0.6). Three biological replicates were performed in total.

Concentration–response curves for E2 and sample dilution series were prepared as described for the µEROD assay with ER induction normalized to maximum induction of E2. E2 equivalents (EEQ) were not calculated, since no sample reached activity above the limit of quantification (LOQ).

Results

Cytotoxicity

Cell viability of H4IIE cells exposed to the sediment fractions up to 100 mgSEQ/mL was quantified using NR retention assay. Laboratory blank and the internal standard did not alter cell viability (Table SI1). Cytotoxicity was less than 5% at all concentrations for most sites and fractions, except for two samples from the Inde (I5, I6, Fig. 2). The strongest impact on cell viability was observed for sample I5 with a concentration > 25 mgSEQ/mL showing a concentration-dependent increase in cytotoxicity up to almost 100% for the fraction B6. A mean 20% effect concentration (EC20) of 20.2 mgSEQ/mL was calculated (Table SI1). Less toxic but still with concentration-related increase in cytotoxicity was sample I6. All fractions induced cytotoxicity > 5%, but always < 25% resulting in no mean EC20 value being calculated. EC20 was used to derive maximum exposure concentrations for mechanism-specific assays on estrogenic and dioxin-like activity.

Fig. 2
figure 2

Cytotoxicity of two fractionated sediment extracts (I5, I6) from the Vicht–Inde catchment collected after the flood event 2021. Cytotoxicity in H4IIE cells was normalized to the negative control, and the mean and standard deviation were plotted (n = 2)

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Estrogenic activity

The estrogenicity of the sediment fractions was studied using the ERα-CALUX assay. Neither controls (laboratory blank and internal standard) nor any sediment fractions at any site showed an estrogenic potential in the studied exposure concentrations (max. 100 mgSEQ/mL) as shown by relative ERα induction normalized to maximum E2 induction (Fig. 3). Since all responses were below the limits of quantification, no EEQ values were calculated.

Fig. 3
figure 3

Relative ERα induction of fractionated sediment extracts from the Vicht–Inde catchment collected after the flood event 2021 in the ER-CALUX assay with U2-OS cells. Data were normalized to maximum induction of the reference compound estradiol (E2). Data points and error bars indicate mean and standard deviation of the replicates (n = 3). For E2 a four-parameter concentration–response curve (non-linear regression model with variable slope, bottom and top fixed to 0 and 1) was fitted

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Dioxin-like activity

The dioxin-like activity of chemical fractions from sediment extracts of the Vicht–Inde catchment was investigated by means of the µEROD assay. H4IIE cells were exposed to concentrations up to a maximum of 100 mgSEQ/mL depending on the viability range determined in the NR retention assay. EROD induction was observed for different fractions of different sampling sites. Neither sample preparation procedure (extraction, fractionation) nor internal standard application contributed to the dioxin-like potential of sediment extracts, since both the laboratory blank and internal standard did not induce a response within the µEROD assay (Fig. 4).

Fig. 4
figure 4

Relative EROD induction of fractionated sediment extracts from the Vicht–Inde catchment collected after the 2021 flood event in the μEROD assay with H4IIE cells. Data were normalized to maximum induction of the reference compound TCDD. Data points and error bars denote mean and standard deviation of replicates (n = 1–2, no error bars: n = 1). Data were fitted to a four-parameter concentration–response curve (non-linear regression model with variable slope, bottom and top fixed to 0 and 1)

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Fraction B1, containing the lowest polarity of organic contaminants did not induce any dioxin-like response across all samples, followed by fraction B6 containing the highest polarity of compounds and reaching a maximum of 50% induction of the TCDD reference compound in the highest exposure concentration for only three sediment samples at the Inde (I3, I4 and I7, Fig. 4). The remaining fractions B2 to B5 led to increasing µEROD activity with increasing exposure concentrations reaching the maximum response of the reference compound TCDD for several sampling sites (Fig. 4).

Based on the concentration–response curves, the bio-TEQ10s were calculated to quantify the dioxin-like activity allowing a direct comparison to in vitro-based dioxin-like activity of sediment samples from literature. For fraction B1 no bio-TEQ10s were derived, followed by fraction B6 with very low bio-TEQ10s for some sites (Fig. 5). The by far highest µEROD induction caused fraction B4 containing mainly PAHs and corresponding more polar derivates with 7 out of 10 sampling sites leading to bio-TEQ10 values ≥ 400 pg/gSEQ (Fig. 5). Within this, the flood sediment I6 induced the highest response with a bio-TEQ10 of 1900 pg/gSEQ followed by sample I4 with a bio-TEQ10 of 1040 pg/gSEQ, while in contrast the samples I2, I7, and V1 led to comparably low EROD induction of 55, 110 and 290 pg/gSEQ, respectively.

Fig. 5
figure 5

The Vicht–Inde streams with dioxin-like activity and organic contaminants of sediment samples after the 2021 flood event. Dioxin-like activity was quantified with µEROD assay in H4IIE cells and shown as bioassay-derived TCDD equivalents (bio-TEQ10). Concentrations of individual PCBs/PAHs were quantified in fractions B2 and B3/4. Sediments were sampled within 4–19 days after the heavy rainfalls in July

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The fractions of less polar compounds (fractions B2 and B3), as well as more polar compounds (fraction B5) compared to fraction B4 showed dioxin-like activities in the range of 50–410 pg/gSEQ for 50% of the sites (B2), 11–70 pg/gSEQ (B3) and 112–440 pg/gSEQ for 80% of the samples (B5).

Organic chemical and granulometric analysis

GC/MS analysis revealed concentrations of 3000–34,000 ng/gdw ∑PAHs (fractions B3,4), with sample V1 showing the highest concentration followed by I6 and I3 containing 27,000 and 25,000 ng/gdw, respectively (Fig. 5). PCBs (fraction B2) were detected in constant concentrations for almost all sites in the range of 1000–2000 ng/gdw (∑PCBs) with the exception of V3 reaching a tenfold higher concentration (10,000 ng/gdw) (Fig. 5).

Based on the concentrations of PCBs and PAHs in the fractions B2–B4, the chem-TEQ values were derived. Chem-TEQ values for total PCBs ranged between 6 and 100 pg/gdw, with sediment from site I3 showing the highest toxicity equivalent (Fig. 6). Chem-TEQs calculated for PAH fractions were below 10,000 pg/gdw for the three sites I7 (2400 pg/gdw), I1 (8700 pg/gdw), and V2 (7400 pg/gdw), while remaining sediments from the catchment were in the range of 11,000–24,000 pg/gdw with V1 showing the highest chem-TEQ. Based on both TEQ values, a moderate correlation between bio- and chem-TEQ was observed (Fig. 7, R2 = 0.56), with the overall trend of increasing bio-TEQ with increasing chem-TEQ.

Fig. 6
figure 6

Calculated chemically derived TCDD equivalents (chem-TEQs) based on quantified PCBs and PAHs of sediment samples from the Vicht–Inde catchment after the 2021 flood event. Chem-TEQs were reported for fractions B2 (containing PCBs) and B3 with B4 (containing PAHs)

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Fig. 7
figure 7

Correlation of bioassay-derived TCDD equivalents (bio-TEQ10s) with chemically derived TCDD equivalents (chem-TEQs) for dioxin-like activity in sediment samples from the Vicht–Inde catchment. Dots represent bio-/chem-TEQ values of fractions B2, B3/4 of 10 sediment extracts. Bio-TEQs were derived from µEROD assay, while chem-TEQ values were calculated from chemical analysis and corresponding REPs. Equation: y = 0.05*x + 115.2, R2 = 0.56

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The grain size of sediment samples varied across the river courses with more sites of fine than coarse grain sizes (Fig. 8, median grain size see Table SI2). The finest sediment was sample I6 followed by V3 and I1, respectively. In contrast, three sediments (I7, V1, V2) were characterized by a rather coarse grain size.

Fig. 8
figure 8

Grain size distributions of the < 2 mm fraction of the samples collected along the Vicht (upper panel) and the Inde River (lower panel). To assess the grain size composition of the fine sediment fraction, the routine for laser diffraction measurement was applied

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Discussion

Estrogenic and dioxin-like hazard of sediments from the Vicht–Inde catchment after the 2021 flood event

Previous research has shown that in addition to the water phase containing many estrogenic compounds originating from industrial and urban wastewater also the particulate phase (suspended particulate matter and river sediments) can accumulate xenoestrogenic organic pollutants, highlighting the relevance of monitoring estrogenicity after flood events [37]. Furthermore, it has been demonstrated that endocrine disruptors can be remobilized from sediments during flood events resulting in readily bioavailable and ecotoxicologically relevant concentrations [38, 39]. As important organic contaminants for the present study, PAHs and their derivates have been identified as ER-agonists in, e.g., crude oils as well as refined fossil fuels [40] or when tested as single substances [41, 42], while PCBs were reported to induce rather weak ER-activation [43]. Overall, the contribution of PAHs to estrogenicity in river sediments is reported contradictory. Certain PAHs and their metabolites have been identified to likely contribute to a detected estrogenic activity in river sediments from the Czech Republic [44], while another study could not correlate PAHs with particle-bound estrogenicity [45]. In the present study, it is possible that the main estrogenicity drivers were associated with the water phase, which, however, was not investigated.

In contrast to estrogenicity, sediments from the Vicht–Inde catchment showed clear dioxin-like activity that mostly correlated with the quantified organic pollutants in the individual fractions (risk driver identification see below). Concentrations of PAHs observed in the present study can be considered high in comparison to values reported for fluvial sediments in other European regions; In extremely contaminated areas such as a small harbor basin of the Elbe River (Germany) or a highly petrochemical-polluted canal in Serbia total PAHs in sediments reached concentration of 509 µg/g and 120 µg/g, respectively, which is approximately tenfold higher than the present observations [46, 47]. Even higher PAH concentrations were reported for sediments from one of the most contaminated coastal and estuarine systems around the world, the Elizabeth River (USA). This catchment has been impacted by military and industrial activities (wood treatment plant) until the 1990s and subsurface sediments contained a total PAH concentration of 2,500 µg/g [48]. Compared to previous flood events, the concentrations of organic contaminants in sediments are in the range of catchments with large anthropogenic impacts. Following the Elbe flood in 2002, sediment samples were analyzed along the river course and its major tributaries, which had been exposed to excessive contaminant emission until 1990 due to industrial activities. At specific hot spot locations, very high concentrations of PAHs were detected within the first weeks after the flood event such as 45, 14 and 9.8 µg/g for fluoranthene, benzo(b)fluoranthene, and benzo(a)pyrene, respectively [49]. Additionally, surface sediments collected after a flood event in 1997 contained total PAHs up to 40 µg/g [50]. The sediments from the Vicht–Inde catchment aligned mostly with those reported post-flood PAH concentrations. However, PCBs were detected in roughly tenfold higher concentrations in the Vicht–Inde catchment compared to previous studies [50].

High concentrations of the organic contaminants PCBs and PAHs likely induced the high dioxin-like activities determined by the bioassay. According to a classification of in vitro EROD activity in river sediments by Keiter et al. [51] the sediments I1 (urban), I3, I4 and I6 (rural) are considered “strongly toxic”, followed by three out of six remaining sediment samples to be categorized “moderately toxic” (V1, V3, I5). The dioxin equivalents (bio-TEQs) were comparable to different sites reported in numerous previous studies. Research on surface sediments from the rivers Danube (Germany) or Hyeongsan (Korea) has derived bio-TEQs of up to 5,000 pg/g, and 1,520 pg/g [52, 53] in crude sediment extracts by means of in vitro EROD with RTL-W1 or H4IIE luc assay. Those sediments were also claimed to be highly contaminated which aligns with the present findings. The moderately contaminated sediments from the present study (V1, V3, I5) were in a comparable range of the likewise moderately contaminated sediments along the Elbe estuary with bio-TEQs of 15 – 322 pg/gdw [46]. Considering EDA approaches and hence testing of individual fractions instead of a complex mixture, the present results were also in the range of earlier studies on contaminated sites. Eichbaum et al. [16] revealed PCB-associated bio-TEQs up to 76 pg/gdw in sediments from the Zollelbe (Germany) in H4IIE cells, which aligns with the range of 5—400 pg/gSEQ for PCB fractions (B2) observed in the Vicht–Inde catchment.

However, even 10- to 100-fold higher toxicity equivalents have been reported in highly contaminated sediment samples from a river in the Czech Republic, an industrial wastewater channel in Serbia and an Elbe River harbor with bio-TEQs up to 23,000, 34,600 and 200,000 pg/gdw by means of the H4IIE luc assay [46, 47, 50]. It has to be considered that the dioxin equivalents of the present study were mainly compared to those obtained from EROD activities in other cell lines such as RTL-W1 or other assay procedures such as H4IIE luc. Though a direct comparison is limited due to, e.g., metabolic capacities of different cell lines, many previous studies directly comparing those methods showed bio-TEQs mostly in the same orders of magnitude [46, 50, 54].

Linking the dioxin-like hazard of sediments from the Vicht–Inde catchment to ecological consequences, especially the hotspot sediment samples have the potential to cause negative effects on ecosystem and human health (details on hotspot location see below). Some of the dioxin-like compounds are considered carcinogenic, teratogenic, and immune system modulating (e.g., [34]), which is relevant for human and environmental health especially when considering the persistence resulting in significant bioaccumulation and biomagnification of some organic xenobiotics. Especially for PCB congeners, it has been well documented that they can be found in higher trophic organisms such as fish [55] and humans (e.g., in tissues, blood, or milk [56]) due to biomagnification. Ecological consequences of AhR-mediated toxicity were for example reviewed by Di Giulio and Clark [57], showing among others elevated rates of liver cancer or even microevolutionary adaptation in fish populations in the enormously contaminated Elizabeth River.

Identification of groups of chemicals driving dioxin-like activity

The fractionation applied in this study allowed the identification of PAHs and their derivates (e.g., alkylated congeners, fraction B4) as the main toxicity drivers for dioxin-like activity in the sediments of the Vicht–Inde catchment. This was shown by both biologically (bio-TEQ) as well as chemically derived toxicity equivalents (chem-TEQ). Though PCBs (fraction B2) and PCDD/DFs (not quantified, part of fraction B2) are strong AhR-agonists in H4IIE cells [58, 59], the corresponding fraction B2 induced much lower dioxin equivalents than fraction B4 containing the PAHs. In detail, mainly the higher molecular weight PAHs, such as benzo(k)-fluoranthene, benzo(b)fluoranthene, dibenz(a,h)anthracene, indeno(1,2,3-c,d) pyrene, benzo(a)pyrene, chrysene, and benz(a)anthracene did likely contribute to the observed high EROD activity since they are known to bind to the AhR and activate cyp1a-related transcription in H4IIE cells [35] in contrast to non-binding low molecular weight PAHs. This trend of high PAH and low PCB contribution to the overall dioxin-like potential aligns with various aforementioned studies using fractionation and biotesting approaches [46, 47, 50].

Overall, the present study revealed a moderate correlation between bio- and chem-TEQ, indicating that the quantified chemicals contributed to the observed AhR-mediated EROD activation with some limitations. Mainly for PAHs (fractions B3,4) bio- and chem-TEQ differed for some sampling sites by a factor of 10 to 50 with higher chem-TEQs than bio-TEQs. The highest discrepancy for bio and chem-TEQ correlation was observed for the samples V1 and I1. Sediment V1 contained the highest PAH concentration across all samples, while the biological response was moderate. In contrast, sediment I1 had a strong toxic response in the bioassay, while the PAH concentration was rather low. A tenfold difference as well as a bio-/chem-TEQ correlation of R2 = 0.5 have been reported in many earlier studies but mainly with higher biologically derived values indicating that other compounds which have not been chemically quantified contributed to the biological response [17, 20, 46, 54, 60]. Especially for PAHs it is known that derivates such as hydroxylated, alkylated, or heterocyclic PAHs are more potent AhR-agonists than their parent compounds in different in vitro assays (RTL-W1 and H4IIE cells, [61, 62]). Hence, the moderate correlation observed in the present study might be related to the contribution of non-quantified compounds (e.g., PCDDs/Fs, other organic compounds), a mixture toxicity with antagonistic interactions of individual compounds, or overestimation of toxicity (chem-TEQ) due to unprecise REP values. However, the latter might be less likely since REP values specifically derived for H4IIE cells were used for the present calculation.

It cannot be neglected that fraction B5, typically containing fatty acids and phthalate, did induce EROD with dioxin equivalents in the range of some samples of fraction B2. To our knowledge, potential xenobiotics from this fraction do rather not induce the AhR-mediated EROD. Nonetheless, the biological activity in fraction B5 can probably be explained by a process-related shift in the elution of more polar and simultaneously highly potent PAH congeners for AhR-mediated toxicity, such as heterocyclic PAHs, from fraction B4 to fraction B5. Fractionation by hand is a continuous process and the shifted elution of potent substances can be caused by environmental influences such as external temperature. The quantification of potential dioxin-like compounds in fraction B5 could further optimize the hazard assessment of sediment-bond dioxin-like activity. Overall, we would like to highlight that fractionation can increase the sensitivity of a biological response through reducing masking effects but simultaneously it can dilute the activity if compounds are eluted into adjacent fractions. As reviewed in detail in Alverez-Mora et al. [63], fractionation requires a fine interplay between the sensitivity of the bioassay and the number of fractions provided.

Longitudinal assessment of anthropogenic pollutant emission—hotspot identification

Except for V2, I2, and I7, all sediment samples caused considerable dioxin-like activity. Those effects and corresponding high concentrations of AhR-agonists are likely attributed to a combination of direct emission of pollutants during flooding and remobilization of legacy pollutants. PAHs are ubiquitous compounds that could have been released from damaged infrastructure (fuel and petroleum oil from heating), road runoff, or overflown industry [6]. Besides this acute release, PAHs could also originate from remobilized legacy pollution. In that context, for example, Hudjetz et al. [64] demonstrated that firmly bonded PAHs from aged sediments were remobilized under flood simulating conditions in an annular flume experiment and thus became bioavailable to exposed fish. In contrast to PAHs, PCBs can be considered good indicators for the remobilization of legacy pollutants since they have been banned worldwide for many years but still accumulate due to their high persistence. The PCB contamination of the Vicht–Inde catchment could originate from former mining, since those substances have been used in underground mining processes as discussed recently [5].

The present study did not identify increasing dioxin-like activity with the longitudinal course of the water flow from the rivers Vicht and Inde. Instead, four hotspot samples of dioxin-like activity were identified at different locations in the catchment. In this respect, the dioxin-like potential correlated well with the proportion of fine sediment grain size but less with the total organic content (TOC, see Figure SI2). Sediments containing high proportions of fine sediments (< 6.3 µm) were those with high dioxin-like activities, as indicated by the correlation of ∑bio-TEQ with fine sediment grain size. Overall, the accumulation of organic pollutants in the floodplain areas is not surprising, since they are prone to be important sinks for deposition of fine suspended organic matter and remobilized sediments as demonstrated in many previous studies on sediments after flood events (e.g., [5, 65,66,67]).

The strongest dioxin-like activity was observed for site I6, which is located at a restored section of the Inde River close to an open-pit lignite mining area [68]. The peak dioxin-like activity can likely be explained by a high deposition of fine sediments with high concentration of organic pollutants in this area. The strong sedimentation of fine particulate matter in this section is caused by a strongly decreased water flow velocity due to the increased structural diversity (restoration to more natural conditions). The sedimentation of particulate matter in this lowland section is even more enhanced by a lower gradient compared to upstream parts of the Inde River. For future risk assessments, it would be desirable to consider the flow velocity as well, and utilize hydrological and hydraulic models to locate potential pollutant sinks and sources, as for example sinks were evaluated in the city of Eschweiler [10]. However, modeling high-energy events, such as the flood of mid-July 2021, poses additional challenges. Gauges were destroyed or no longer provided reliable values, and existing rainfall-runoff models reached their limit of calibration (e.g., [69]). Further research is needed here, to provide additional hydrologic and hydraulic data to support future evaluations.

The high dioxin-like activity of sediment I1 was likely caused by an acute input of organic pollutants following the flooding. This site is fully urban and was impacted by a direct discharge of the small river Itterbach, which contained untreated urban water discharges from cesspits of closely located houses during the flood event (see Blümel et al. [70] who provide evidence for this pollution by intensive sampling approach). To some extent the strong response might additionally originate from fuel oils or exhaust particulates, residues of tire wear, road dust, from the huge parking area (sampling site of I1) or destroyed industrial infrastructure, all known for their dioxin-like potential [71, 72]. The latter of potential contamination due to destroyed infrastructure and industrial sectors from the upstream city of Stolberg or Eschweiler (e.g., former Leblanc soda plant and industry for paper packing material) might rather likely explain also the two remaining hotspot samples I3 and I4.

Consequences and future mitigation strategies for the Vicht–Inde catchment

The present study was able to identify AhR-mediated effects, but cannot predict the long-term consequences for ecosystem health. Nonetheless, adverse effects of dioxin-like contamination after flood events have been described to aquatic species such as fish and cattle grazing on floodplains, which is of relevance for human exposure via food chain [73]. Overall, it is known that the natural recovery of contaminated sites is limited to biodegradable and bioavailable substances. Further it depends on the local environmental conditions and is very slow. In that respect, previous post-flood monitoring still detected PAHs and PCBs around 6–13 µg/g and 0.06–0.09 µg/g without huge changes within 4 years after the severe flood at the Elbe River [74]. However, sedimentary PAHs from an enormous oil spill were reported to be reduced to regional background levels in the environment within a 7.5-year period after the spill [75]. Hence, it is recommended to monitor the degree of organic contamination in the region in future research, especially considering the persistence of some organic pollutants detected in the Vicht–Inde catchment.

The Vicht–Inde catchment is expected to be impacted by more frequent extreme floods in future according to climate change projections in Europe [76]. Therefore, it should be highlighted that the sinks of contamination in the floodplain areas are a source for remobilization of pollution in future flood events with a high relevance for the downstream Rur catchment. Since the Inde catchment was described to contain a low level of protection measures [5], new approaches should be considered for future mitigation strategies.

Conclusion

Considering previous European flood events, the present concentrations of organic contaminants in surface sediments of the Vicht–Inde catchment can be considered comparably high and are of environmental concern. Corresponding to quantified organic contaminants, moderate-to-high dioxin-like activity was observed in 70% of the sediments. No sediment-associated estrogenicity in any organic fraction from the catchment was identified, though estrogenicity can be a relevant toxicity pathway in flood sediments. The present study furthermore revealed that the fractions containing mainly PAHs contributed most to the overall dioxin-like potential. Nonetheless, it was not possible to differentiate if the analyzed priority PAHs or the more potent derivate such as alkylated or heterocyclic PAHs were responsible for the biological activity. Additionally, other organic contaminants not quantified within the present analysis could have caused the AhR activation. To elaborate more details about toxicity drivers, it is recommended to consider other organic pollutants than priority PAHs and PCBs present in the fractions. It is important to consider that the present study focused on organic compounds in the Inde–Vicht catchment, while heavy metals are another group of contaminants of high relevance due to the long mining tradition. Hence, the present study provided a first ecotoxicological screening, but for comprehensive risk assessment the mixture toxic pressure needs to be examined in future research.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

AhR:

Aryl-hydrocarbon receptor

ASE:

Accelerated solvent extraction (ASE)

bio-TEQs:

Bioassay derived TCDD equivalents

chem-TEQs:

Chemically derived TCDD equivalents

DL-PCBs:

Dioxin-like polychlorinated biphenyls

DDX:

p,pʹ-DDT and metabolites

EROD:

7-Ethoxyresorufin-O-deethylase

ERα-CALUX:

ERα chemical-activated luciferase reporter gene expression

GC/MS:

Gas chromatography–mass spectrometry

LABs:

Linear alkylbenzene

NR:

Neutral red retention

POPs:

Persistent organic pollutants

PAHs:

Polycyclic aromatic hydrocarbons

PCDD/Fs:

Polychlorinated dibenzo-p-dioxins and dibenzo furans

REP:

Relative potency

TCDD:

Tetrachlorodibenzo-p-dioxin

SEQ:

Sediment equivalent

TEF:

Toxicity equivalent factors

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Acknowledgements

The authors thank Simone Wollenweber, head of laboratory at the Department Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, for supporting the practical work by performing the neutral red retention assay with the fractionated sediment samples. The authors further thank Yvonne Esser and Annette Schneiderwind for their support during laboratory analysis. We thank the sampling teams from RWTH Aachen University and Goethe University (Verena Esser, Elena Klopries, Janine Freyer, Lennart Schelter, Carole Detampel, Fabian Weichert, Andreas Schiwy, Markus Schmitz).

Funding

Open Access funding enabled and organized by Projekt DEAL. This work received funding from the Robust-Nature Excellence Network provided by the Goethe University Frankfurt, Germany and the German Research Foundation (DFG—Project number 497800446).

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Authors and Affiliations

  1. Department Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Frankfurt Am Main, Germany

    Sarah Johann, Moritz Düster & Henner Hollert

  2. Department Environmental Media Related Ecotoxicology, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany

    Sarah Johann & Henner Hollert

  3. Institute of Neotectonics and Natural Hazards, RWTH Aachen University, Aachen, Germany

    Piero Bellanova

  4. Institute of Organic Biochemistry in Geo-Systems, RWTH Aachen University, Aachen, Germany

    Jan Schwarzbauer

  5. Department of Geography, RWTH Aachen University, Aachen, Germany

    Alexandra Weber & Frank Lehmkuhl

  6. Institute of Hydraulic Engineering and Water Resources Management, RWTH Aachen University, Aachen, Germany

    Stefanie Wolf & Holger Schüttrumpf

  7. LOEWE Centre for Translational Biodiversity Genomics (LOEWE‐TBG), Frankfurt Am Main, Germany

    Henner Hollert

  8. Competence Centre Water Hesse, Frankfurt Am Main, Germany

    Henner Hollert

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  1. Sarah Johann
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Contributions

Conceptualization: HH, FL, HS, SW, SJ; Investigation/Methodology: MD, AW, PB; Data analysis: MD, SJ, AW, PB; Supervision: SJ, HH; Visualization: MD, AW, SJ; Writing—Original draft preparation: SJ; Writing—Review and Editing: MD, PB, JS, AW, SW, HS, FL, HH. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sarah Johann.

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Competing interests

The authors declare that they have no competing interests. Henner Hollert is Editor-in-Chief of the Journal Environmental Sciences Europe and was not involved in the review process of this manuscript. He was completely blinded for this submission.

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Johann, S., Düster, M., Bellanova, P. et al. Dioxin-like and estrogenic activity screening in fractionated sediments from a German catchment after the 2021 extreme flood. Environ Sci Eur 36, 163 (2024). https://doi.org/10.1186/s12302-024-00989-4

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