3.1 Ecotoxicological status of the floodplain water bodies
To assess the current ecotoxicological status of the floodplain waterbodies, we tested water and surface sediment from the tributary (T), the ponds (P), and the flood depressions (F).
Figure 3 In vitro activities in water samples (w) from the tributary (T), the ponds (P), and the flood depressions (F) in the floodplain restoration site. (a) Baseline toxicity in the Microtox assay is expressed as the mean and standard error of the mean (SEM) of 50% effect concentration (EC50) based on the relative enrichment factor (REF) of the water samples. Non-toxic samples were set to 100 REF. (b) Mutagenicity in the Ames fluctuation assay is expressed as % revertants with > 20.8% revertants being considered mutagenic. Sample concentration in the test was 6.7 REF. The triangles represent the results for point mutations in th YG1042 strain, and the diamonds those for frameshift mutations in the YG1041 strain. Filled symbols indicates mutagenicity with metabolic activation (S9), and open symbols mutagenicity without S9. (c) Estrogenic activity in the Yeast Estrogen Screen (YES) expressed as mean and SEM in equivalents (EQ) of the positive substance 17β-estradiol (E2). (d) Activity in the Yeast Dioxin Screen (YDS) expressed as mean and SEM in EQ of the positive substance β-naphtoflavone (β-NF). (c, d) The dotted line represents the limit of quantification (LOQ).
In the water phase, we detected very weak baseline toxicity in only three of eight samples (T2w, P3w, F1w), all other samples were not active in this assay (Fig. 4a). Mutagenic potential (> 20.8% revertants) was observed exclusively in one sample from the tributary (T2w) and only in YG1041 strains after metabolic activitation with 36% revertants (Fig. 4b). The strongest estrogenic activities were found in the flood depressions with values of 1.14 ng E2-EQ/L in F1 and 0.56 ng E2-EQ/L in F2, which exceeded the EQS for E2 of 0.4 ng/L by a factor 2.85 and 1.4, respectively (Fig. 4c). The YDS showed weak effects above the LOQ (0.04 µg β-NF-EQ/L) in all water samples with the strongest activity of 0.29 µg β-NF-EQ/L in sample T2w (Fig. 4d). In this sample taken from the junction of both basins of the tributary, we found both, elevated dioxin-like activity and mutagnic potential in the YG1041 strain after metabolic activation. These results may indicate the remobilization of sediment-bound contaminants due to the higher flow velocity. Although the sediment sample at this site was not active in the respective assays, there may be contamination at other depths or areas of the water body. It is not known, whether and how the soil was redistributed during the construction work.
Figure 4 In vitro activities in surface sediment (ss) and soil (h) samples from the tributary (T), the ponds (P), the flood depressions (F), and the riparian area (R) in the floodplain restoration site. (a) Baseline toxicity in the Microtox assay is expressed as the mean and SEM of EC50 based on mg/sediment or soil equivalents (SEQ). Non-toxic samples were set to 100 mg SEQ. (b) Mutagenicity in the Ames fluctuation assay is expressed as % revertants with > 20.8% revertants being considered mutagenic. Sample concentration in the test was 66.7 mg SEQ/mL. The triangles represent the results for point mutations in th YG1042 strain, and the diamonds those for frameshift mutations in the YG1041 strain. Filled symbols indicate mutagenicity with S9, open symbols mutagenicity without S9. (c) Estrogenic activity in the YES is expressed as mean and SEM in EQ of the positive substance E2. (d) Activity in the YDS is expressed as mean and SEM in EQ of the positive substance β-NF. (c, d) The dotted line represents the LOQ. For abbreviations see Fig. 4.
In the respective surface sediment, we found moderate baseline toxicity with EC50 values below 21.4 mg SEQ in all water bodies except one groundwater-fed pond (P3) that showed only low activity of 54.8 mg SEQ (Fig. 5a). This is comparable with measured activities in other river sediments [51, 52]. The very low to absent activity in the associated water samples suggests that there is no remobilization of respective compounds. However, to assess potential harmful effects in organisms it may be useful to test parameters such as benthic community structure. In all other in vitro EMBs with sediments from the floodplain, we only found distinct activities in both flood depressions F1 and F2 (Fig. 5b-d). Including metabolic activation of the sample, F1ss and F2ss induced mutations in the YG1041 (35% and 26.6% revertants, respectively), F2ss also mutations in the YG1042 strain (33.6%). Also without metabolic activation, F2ss induced mutations in the YG1041 strain (29.8%) (Fig. 5b). The mutagenicity of both samples could be caused by legacy contaminants (see also 3.2).
Figure 5 Contamination in surface sediment (ss) and soil samples (h) from the tributary (T), the ponds (P), the flood depressions (F) and the riparian area (R) in the floodplain restoration site.
Polycyclic aromatic hydrocarbons (PAHs) that have accumulated in flood depressions (Fig. 6) are among the most well-known environmental mutagens [53, 54]. Estrogenic activities above the LOQ (0.7 ng E2-EQ/g SEQ) were exclusively found in the flood depressions with equivalent concentrations of 4.15 ng E2/g SEQ in F1 and 4.13 ng E2-EQ/g SEQ in F2 (Fig. 5c), which can be compared with the effect potentials found in Main river sediments and SPMs (Fig. 9c). We therefore assume that the activities in both flood depressions originate from the nearby Main river. Also possible, due to the proximity to the pedestrian path, would be another anthropogenic input, such as dog urine and feces.
Dioxin-like activities in the YDS were highest in both flood depressions with 78.2 µg β-NF/g SEQ in F1 and 164 µg β-NF-EQ/g SEQ in F2, corresponding to 283 and 414 pg TCDD-EQ/g SEQ, respectively. These are by far the highest dioxin-like activities found in this study. All other sediment samples showed weak activities below 5 µg β-NF-EQ/g SEQ.
As shown by the chemical analysis of the floodplain sediment samples (Fig. 6), both flood depressions are heavily contaminated with PAHs, with cumulative concentrations of 6.45 and 59.6 µg PAHs/g in F1ss and F2ss, respectively. These concentrations are considerably higher than in the tributary, the area of the future tributary and the ponds (< 0.5 µg/g) and the riparian area (< 0.9 µg/g) and correlate with activites found in the YDS. Particularly high are the concentrations of benzo[a]pyrene (28.3 µg/g) and benzo[e]pyrene (21.4 µg/g) in sample F2ss. Both PAHs are products of incomplete combustion of organic matter and are introduced into the aquatic environment inter alia by atmospheric deposition. However, the significant concentration differences in both flood depressions might also indicate additional sources. Since the flood depression F2 is located next to a popular fishing spot, the comparably high concentrations could also result from the improper disposal of barbecue charcoal and cigarettes. We also found persistent organic polutants (POPs) such as polychlorinated benzenes or biphenyls (PCBs) in the flood depressions, the soil samples from the floodplains, and also in the older Main river sediments (Fig. 10), suggesting that they are legacy contaminants.
Overall, sediment and water samples from the new tributary and the groundwater-fed ponds were unremarkable in the in vitro EBMs and showed a low chemical load compared to all other samples. In contrast, sediments from both flood depressions are active in all in vitro EMBs, and the EQS for E2 was exceeded in the water phase. The artificial flood depressions represent temporary water bodies that regularly fall dry and have no access to the Main river. Accordingly, they do not provide habitats for fish, but for insect larvae and amphibians, for example, which might be suitable organisms for an ERA that accounts for the bioavailability of detected contaminants.
3.2 Influence of potential legacy contamination
As a consequence of the major flood events in the last decades, pollutants from the Main river and the effluent of the former Cassella AG are hypothesized to have been deposited on the floodplain. We analyzed soil samples from upper horizons in the riparian zone (R) and the area of the future tributary (T4).
None of the soil samples showed estrogenic activities (Fig. 5c). Also, baseline toxicity and dioxin-like activities were low with EC50 values of 50.6 to 76.6 mg SEQ and 2.13 to 4.72 µg β-NF-EQ/g SEQ, respectively (Fig. 5a, d). However, we found strong mutagenic potential in all samples (T4h1, T4h2, R1h1, R1h2) in both strains, as well as with and without metabolic activation (Fig. 5b). In the riparian zone, we found up to 91.2% revertants and in the area of the future tributary up to 75.6%. Since both sampling sites are affected by at least 5-year floods, we assume that the observed mutagenicity is related to the hypothesized legacy contamination, which is further supported by the sensitivity of the used Ames strains to nitrated aromatic hydrocarbons. For over 150 years, the local industry has been producing and discharging mutagenic tar dyes without an operating WWTP [55, 56]. Concerning the development potential of the restoration site and remobilization of potential legacy contamination, the decisive factor will be whether the contaminated soil has direct contact with the water body. Fortunately, apart from the two flood depressions F1 and F2, none of the existing water bodies showed mutagenic activities, which can possibly be explained by their water depth. Soil samples were only taken from the upper two horizons with a depth of up to 44.5 cm, which is similar to the depth of the flood depressions. The sediment samples from the tributary and the ponds were taken from much deeper layers. This may indicate that the potential legacy contamination affects only the upper soil layers. Apart from this, the ponds are located in less heavily flooded areas and are therefore less contaminated with potential legacy contamination. However, we recommend considering the high mutagenicity in the tested soil layers when planning and implementing the future tributary.
3.3 Influence of the current industrial effluent on Main water and SPM quality
To assess the current impact of the on-site industrial plant, we analyzed a 24 h composite sample from the WWTP effluent (E), water samples from six sites along the Main river (M) (Fig. 7), and SPM samples collected over 35 days at three sampling sites (Fig. 9).
Figure 7 In vitro activities in water samples (w) from the Main river (M) and a 24 h composite sample of the wastewater treatment plant effluent (E) of the on-site industrial facility. (a) Baseline toxicity in the Microtox assay is expressed as mean and SEM of EC50 based on the REF of the samples. Non-toxic samples were set to 100 mg REF. (b) Mutagenicity in the Ames fluctuation test is expressed as % revertants with > 20.8% revertants being considered mutagenic. Sample concentration in the test was 6.7 REF. Due to cytotoxicity, sample E was tested at 16.7 REF (*). The triangles represent the results for point mutations in th YG1042 strain, and the diamonds those for frameshift mutations in the YG1041 strain. Filled symbols indicate mutagenicity with S9, open symbols mutagenicity without S9. Samples were tested in a 16.7-fold, sample E in 6.67-fold (*) enrichment. (c) Estrogenic activity in the YES is expressed as mean and SEM in EQ of the positive substance E2. (d) Activity in the YDS is expressed as mean and SEM in EQ of the positive substance β-NF. (c, d) The dotted line represents the LOQ. Statistical significances are provided in Table S1. For abbreviations see Fig. 4.
In the effluent sample, we found high baseline toxicity with an EC50 of 2.88 REF. This activity was still measurable directly at the discharge point (M2) with an EC50 value of 42.1 REF, which equals a 14.3-fold dilution. In the following transects, along with the reference site M1, we found no baseline toxicity (Fig. 7a). The effect potentials found in this study are common for industrial effluents, which are often more potent in the Microtox assay than municipal effluents [57, 58]. This might be relevant for the functionality of the biological treatment stage, as high toxicity to bacteria may impact the purification process [59]. All SPM samples showed high baseline toxicity, both upstream and downstream of the WWTP, with EC50 values of 3.34, 3.42, and 6.02 mg SEQ, respectively (Fig. 9a). We further found mutagenic potential in the effluent sample in three of the four tested Ames assays: 47.8% revertants in the YG1042 and 50.6% in the YG1041 strain without metabolic activation, as well as 49.5% in the YG1041 strain after metabolical activation of the sample. Again, the effect potential was still measurable at the discharge site M2 with 44.7%, 62.1%, and 80.8% revertants, respectively. Even 700 m downstream the WWTP discharge at site M3, we found 35% revertants in the YG1041 strain after metabolic activation. All other transect samples were not active in the Ames tests (Fig. 7b). Due to cytotoxicity, the effluent sample E was tested at a lower concentration (6.67 REF) than the other water extracts (16.7 REF), which could explain the comparably high activity in the Main river samples. The SPM samples showed no mutagenic potential (Fig. 9b). In all water samples from the Main river, estrogenic activities were below the LOQ (0.41 ng E2-EQ/L). In the effluent sample, we found no measurable estrogenic activity (Fig. 7c). In contrast, all SPM samples were active in the YES, with activities decreasing in the direction of flow from 5.61 ng E2-EQ/g SEQ at the reference site to 1.81 and 1.16 ng E2-EQ/g SEQ at sites M5 and M6, respectively (Fig. 9c). Other studies on SPM from the Rhine (Germany) and Meuse river (Netherlands) reported similar or lower E2-EQs of up to 2.35 [60] and 0.7 ng/g SEQ [61], respectively. The YDS showed significant to highly significant activities above the LOQ (0.01 µg β-NF-EQ/L) in all Main water samples compared to the reference site M1 (0.03 µg β-NF-EQ/L) (Fig. 7d; statistical data: Tab. S1). The highest activity was 6.22 µg β-NF-EQ/L in the effluent sample, followed by 0.35 µg β-NF-EQ/L at the discharge point, which equals an 18-fold dilution. The remaining downstream transects (M3-M6), showed weak dioxin-like activities of 0.04 to 0.06 µg β-NF-EQ/L, corresponding to a 1.3- to 2-fold increase compared to the reference site (Fig. 7d). We also found dioxin-like activities in all SPM samples. As observed in the YES and contrary to our expectation that the effluent causes to a higher dioxin-like activity in SPM samples downstream of the discharge, the activities decreased in the direction of flow from 14.8 µg β-NF-EQ/g SEQ at the reference site to 4.72 µg β-NF-EQ/g SEQ at site M5, and to 2.9 µg β-NF-EQ/g SEQ at site M6 (Fig. 9d; statistical data: Tab. S1), corresponding to 121, 67.5 and 52.7 pg TCDD-EQs/g SEQ, respectively. As the activities refer to dry weight, these observations could be explained by varying levels of organic matter, which would affect the pollutant load. However, the activities were lower than reported for SPM samples from the Rhine and the Neckar river with TCDD-EQ values ranging from 1160 pg/g to peak concentrations of 6640 pg/g during flood events [62–65].
Figure 8 Contamination in water samples (w) from the Main river (M) and the WWTP effluent sample (E). M1 represents the reference site. (a) Cumulative concentrations of all categories without the main contaminants hexamethoxymethylmelamine (HMMM) (b), benzotriazole (BT) and 5-methyl-1-H-benzo-triazole (5M-1H-BT; hatched part) (c).
Chemical analyses of the water samples from the Main river and the effluent sample show the same tendency as the in vitro EBMs (Fig. 8). The cumulative concentration of all contaminants is about 4.2 mg/L in the effluent and 0.6 mg/L at the discharge point M2, which corresponds to a 7-fold dilution. It then decreases greatly, so that the chemical profile of the Main transects downstream of the discharge is similar to the one at the reference site. Particularly striking is the main contaminant hexamethoxymethylmelamine (HMMM; category: polymer additives), which accounts for approximately 95% of the total chemical load in the effluent sample E (3.9 mg/L). It is then strongly diluted downstream, but stabilizes at about 6 µg/L at sites M5 and M6. HMMM is a cross-linker of melamine resins that are mainly used in the automotive industry. Not much is known about the toxic potential of HMMM, but over 21 persistent and mobile transformation products (TPs) have been found recently [66]. We assume that the TPs of HMMM also occur in high concentrations in the Main river. However, the TPs were not analysed in the target screening due to the lack of corresponding standards. After cleavage of all side chains, melamine remains. Its high persistence, mobility and toxicity make it important on a global scale. Lütjens et al. (2023) [67] detected melamine in about 90 % of th European surface waters studied, with a concentration of 1.4 µg/L measured in the Main river in Frankfurt. They further identified the production of melamine-containing products as a major pathway for the presence of melamine in surface waters. The second most common pollutant in the effluent sample E is benzotriazole (BT; category: personal care and household) with a concentration of 134 µg/L. BT is completely diluted in the Main river, so that background concentrations of about 1 µg/L at the reference site are immediately found again downstream of the discharge. Including 5-methyl-1-H-benzotriazole (5M-1H-BT), the background levels in the Main river were about 1.5 µg/L. BTs are corrosion inhibitors and are widely used in industry and households, e.g., in dishwashing agents. They are high-volume production chemicals that are regularly detected in rivers around the world, with reported concentrations up to 7 µg/L [68]. They are highly soluble in water and can be toxic to aquatic organisms [69]. Excluding the main contaminants HMMM and BT with 5M-1H-BT, the effluent sample consisted mainly of components of the categories personal care and household as well as biocides. Besides BT and 5M-1H-BT, the most common contaminant in the Main river (M1, M3-M6) was the sweetener sucralose with constant concentrations of around 1 µg/L. Regardless of the industrial effluent, all SPM samples revealed a cumulative concentration of up to 8 µg/g, which are among the highest compared to the other sediment and soil samples (Fig. 10). The detected compounds in the SPM samples were mainly PAHs (up to 2.7 µg/g) and polymer additives (up to 1.8 µg/g). A decrease in intermediates is accompanied by an increase in POPs (mainly 1,2,4-trichlorobenzene). The main contaminant HMMM was found in the SPM samples in comparably low but increasing concentrations after the WWTP reference site (M1p: 0.23 µg/L, M5p: 1.5 µg/L, and M6p: 1.4 µg/L). Detailed information on the results of the chemical analysis is provided in the supplementary material.
In conclusion, based on the results of the water analysis, we found that the local industrial plant represents a point source for pollutants that, at the time of sampling, caused baseline toxicity, mutagenic and dioxin-like activites. However, the dilution effect in the Main river is much greater than in smaller rivers [57] so environmentally relevant activities in the water phase can only be observed at the discharge point and, in case of mutagenicity, additionally 700 m downstream. We found no critical in vitro activities in the water samples at the inflow level of the future tributary. Further, the effluent does not seem to have an enhancing effect on the measured in vitro activities in the SPM samples. In particular, estrogenic and dioxin-like activities do not increase but decrease downstream of the treatment plant. This could be due to exchange processes, as the chemical analysis indicates a shifting contamination profile as well. Assuming that SPM pollution levels are similarly high over time, their sedimentation may have a negative impact on floodplain development. This was shown, for example, by Schulze et al. [62] who chemically and biologically analyzed SPMs and compared frequently versus infrequently inundated floodplain soils. However, measurements of the present study depend on single grab samples and SPM samples covering 35 days, so that a more comprehensive approach with repeated sampling campaigns would be nessecary for general statements.
3.4 Influence of former industrial effluents on Main river sediments
To also reflect the influence of the local industrial plant over the past years and to identify potential legacy contamination in the Main river (M), we tested surface sediment and additional core sediment from the restored bank flattening (M6) (Fig. 9).
Figure 96 In vitro activities in suspended particulate matter (SPM; p), surface (ss) and core sediment (sc) samples from the Main river (M). (a) Baseline toxicity in the Microtox assay is expressed as mean and SEM of the EC50 based on mg sediment or SPM equivalents (SEQ). Non-toxic samples were set to 100 mg SEQ. (b) Mutagenicity in the Ames fluctuation assay is expressed as % revertants with > 20.8% is considered mutagenic. Sample concentration in the test was 67.7 mg SEQ/mL. Due to cytotoxicity, sample M6p was tested at 26.7 mg SEQ/mL (*). The triangles represent the results for point mutations in th YG1042 strain, and the diamonds those for frameshift mutations in the YG1041 strain. Filled symbols indicate mutagenicity with S9, open symbols mutagenicity without S9. (c) Estrogenic activity in the YES is expressed as mean and SEM in EQ of the positive substance E2. (d) Activity in the YDS is expressed as mean and SEM in EQ of the positive substance β-NF. (c, d) The dotted line represents the LOQ. Statistical significances are provided in Table S1. For abbreviations see Fig. 4.
In the transect samples downstream of the reference site, we found varying baseline toxicities that correlate with the respective sediment types we found at each side (Fig. S1). The sandy sediments M4ss and M6ss showed high baseline toxicities with EC50 of 7.89 and 7.20 mg SEQ, respectively. The core sediment from the restored site was less active than the surface sediment (EC50 of 24.7 mg SEQ). Since the reference site with comparable sediment characteristics showed low baseline toxicity (EC50 of 63.6 mg SEQ), we suspect an influence of the WWTP effluent. Clayey sediments showed no (M2ss) or weak (M3ss and M5ss) activities (Fig. 12a). All samples showed dioxin-like activities above the LOQ (0.17 µg β-NF/g SEQ). Compared to the reference site, it was increased at all sites downstream of the WWTP discharge. We observed the highest activities in the sandy river sediments at sites M4 (8.23 µg β-NF-EQ/g SEQ, corresonding to 89.7 pg TCDD-EQ/g SEQ) and M6, with a decreasing activity from the surface (18.3 µg β-NF-EQ/g SEQ, corresponding to 135 pg TCDD-EQ/g SEQ) towards the core sediment (4.26 µg β-NF-EQ/g SEQ, corresponding to 64.1 pg TCDD-EQ/g SEQ) of the restored site (Fig. 9d). These results are consistent with the expected higher sedimentation rate in a bay located on a sliding slope. The dioxin-like activities found in this work are similar to those reported by Otte et al. for the Elbe river [70], and about 10- to 50-fold lower than measured at “highly cotaminated” sites along the Elbe and Danube river [71, 72]. However, they are orders of magnitude higher than results from less polluted rivers such as the Nidda or the Horloff [18]. Mutagenic potential was found only in the YG1041 strain (31.8% revertants) after metabolic activation in clayey sediments directly at the discharge site (M2) (Fig. 9b). Elevated estrogenic activities above the LOQ (0.70 ng E2-EQ/g SEQ) were detected in almost all samples downstream of the reference site (0.06 ng E2-EQ/g SEQ), with some being statistically significant to very significant (Fig. 10c; statistical data: Tab. S1). The highest activity with 4.91 ng E2-EQ/g SEQ was detected in clayey sediment from site M5. In general, also with respect to the mutagenicity at site M2, it can be assumed that the remobilization rate from clayey sediments is low, which minimizes their influence on the development of the floodplain. However, the restored site M6 showed comparably high estrogenic activities as well, with 2.9 ng E2-EQ/g SEQ in the surface and 2.31 ng E2-EQ/g SEQ in the core sediment (Fig. 9c). These values are slightly higher than reported for other European rivers using similar assays and extraction methods [73, 74].
Figure 10 Contamination in suspended particulate matter (p; SPM), surface (ss), and core sediment (sc) from the Main (M) river. M1 represents the reference.
Sediments act as a sink for hydrophobic and persistent compounds and can therefore reflect a long period of pollution. Accordingly, the contamination of the Main river sediments (Fig. 10) indicated that the effluent of the local industrial plant was an important point source for such pollutants in the past. For example, PAH contamination is higher in the sandy sediments downstream of the WWTP. Compared to sediment samples that showed similar dioxin-like activities, the cumulative PAH concentrations found in this work are significantly higher with up to 5.1 µg/g [70]. In addition to atmospheric deposition, which is the typical origin, PAHs can also enter the environment through wastewater discharges, as they also have industrial uses [75, 76]. Phenanthrene, for example, shows the highest concentration of all PAHs in Main river sediment with 2.5 µg/g at site M4. It is used for the synthesis of dyes [77]. The second most common PAH in the river sediments is fluoranthene with up to 0.4 µg/g, which is used for the synthesis of pharmaceuticals [78]. However, combustion and athmospheric deposition or unburned fossil fuels are typically the main source of PAHs in the environment. Like most PAHs, both phenanthrene and fluoranthene are rated as very persistent and very bioaccumulative and as Substances of Very High Concern.
Ultimately, sediments from the restored bank were particularly conspicuous in the in vitro EBMs and the chemical analysis. We detected decreasing effects from the surface to the core sediment for baseline toxicity, as well as estrogenic and dioxin-like activity. As the boulders were replaced with sand in 2014, there has been considerable contamination of the new river sediments within the last six years. We therefore hypothesize long term accumulation of contaminants from the Main river in the tributaries as well. The good chemical status of sediments from the tributary may consequently reflect its short existence.