3.1 Metabolites detected in river waters
Peak picking followed by annotation (based on MS1 fullscan) resulted in 145 target peaks with m/z and retention time matching to the target metabolite with a tolerance of 5 ppm and +/- 0.1 min, respectively. Some target metabolites were annotated multiple times due to picking multiple peaks at a single precursor ion mass with given retention time tolerance. Removal of false positives and peak filtering using intensity and resolution reduced the target list to 106 peaks. Based on additional MS/MS fragment comparison with reference standards, we confirmed the presence of 12 target metabolites in the river waters. The identified compounds belong to different classes of natural compounds including coumarins, alkaloids, isoflavone and others. In general, the identified metabolites contain one or more phenolic groups representing a class of compounds found most abundantly in vegetation (Puri et al. 1998; Afendi et al. 2011). The names and chemical structures of the identified metabolites are given in Fig. 2.
3.2 Distribution of measured metabolites in river waters
In 18 out of 38 rain event samples PSMs were detected (9 samples from each location – Leipzig and Bode catchment), while in none of the dry weather control samples any of the target PSMs were found. In the Bode catchment, larger numbers of metabolites were detected in rivers impacted by agricultural than natural vegetation (Fig. 1 and Table S4 in SI).
Among the identified 12 metabolites, 11 were detected in samples from the ELP catchment and 10 were found in the Bode catchment. In both catchments, the coumarin derivatives scopoletin and fraxidin were detected with the highest frequency with 9 samples from each catchments (Figs. 3 and 4). Esculetin, another coumarin derivative was the third most frequently detected PSM in the Bode catchment with 20% while it was detected in 9% of the samples from the ELP catchment. The high detection frequency of fraxidin and esculetin is in good agreement with its formation by Fraxinus excelsior, a frequent tree in central European floodplains including the ones under investigation here. Scopoletin is produced by Scopolia species, but also the very frequently occurring stinging nettle Urtica dioica. However, all three compounds are present in a wide range of plants, which might contribute to emissions (Whang et al. 2005). The isoflavone formononetin is the third most frequently occurring PSM in the ELP catchment with 22%, while it was found in 7% of the Bode catchment samples. Other compounds were detected only in specific samples from a specific season such as lycorine and narciclasine occurring in Amaryllidacea, which show a high abundance within short growth periods such as Galanthus species in early spring. Although coniferyl aldehyde is a lignin component of many plants, it could be detected only in the EPL catchment and the coumarin psoralen only in Bode catchment. Natural compounds stemming likely from human consumption such as the piperidin alkaloid piperine as a component of pepper and nicotine from tobacco could be detected in both catchments.
3.3 Quantification of PSMs in river water
The target PSMs were detected in a concentration range of 1–3400 ng/L (Table 1). The concentrations of identified metabolites in individual samples is given in SI (Table S4). The highest concentrations were detected for lycorine and narciclasine with maximum concentrations of 2 and 3 µg/L during the times of high abundance of Galanthus sp. (snowdrop) and Leucojum vernum (spring snowflake). The concentrations of these phytotoxins strongly exceed the TTC of 0.1 µg/L for non-genotoxic and non-endocrine disrupting compounds and would be of concern, if these water resources would be used for drinking water production. Both compounds are highly bioactive and toxic causing among others nausea and emesis in human and animals (Kornienko and Evidente 2008; Kretzing et al. 2011). Acetylcholinesterase inhibition effects of lycorine and narciclasine were reported (Hostettmann et al. 2006; Cahlikova et al. 2013). So, apart from its toxicity, lycorine also has more positive effects, as many SPMs, such as antibacterial, anti-viral, anti-malarial, anti-allergy effects, inhibits protein and DNA synthesis and has cardiovascular protection and antitumor effects (Jahn et al. 2012).
Table 1
The concentration range (min – max, ng/L) of identified plant metabolites in river water.
Plant secondary metabolite | Chemical Formula | CAS No | m/z (M + H+) | Retention time (min) | MDL (ng/L) | Concentration range (min – max, ng/L) | Frequency of detection |
Leipzig | Bode | Leipzig | Bode |
Coumarin | C9H6O2 | 91-64-5 | 147.0441 | 7.3 | 11 | 12 | 43 | 1 | 1 |
Esculetin | C9H6O4 | 305-01-1 | 179.0336 | 4.2 | 50 | 116–1658 | 104–157 | 2 | 3 |
Fraxidin | C11H10O5 | 525-21-3 | 223.0600 | 7.8 | 4 | 56–1145 | 19–155 | 9 | 8 |
Scopoletin | C10H8O4 | 92-61-5 | 193.0496 | 7.1 | 2 | 9 – 47 | 7–49 | 9 | 9 |
Bergapten | C12H8O4 | 484-20-8 | 217.0495 | 10.1 | 4 | 510 | 541 | 1 | 1 |
Psoralen | C11H6O3 | 66-97-7 | 187.0388 | 9.1 | 3 | ND | 141–224 | 0 | 2 |
Lycorine | C16H17NO4 | 476-28-8 | 288.1225 | 1.0 | 3 | 1015–2331 | 11 | 2 | 1 |
Narciclasine | C14H13NO7 | 29477-83-6 | 308.0765 | 5.7 | 150 | 507–3353 | ND | 2 | - |
Nicotine | C10H14N2 | 54-11-5 | 163.1228 | 0.9 | 1.6 | 2–6 | 4 – 35 | 2 | 2 |
Piperine | C17H19NO3 | 94-62-2 | 286.1434 | 11.9 | 0.9 | 1–338 | 4 – 294 | 4 | 2 |
Formononetin | C16H12O4 | 485-72-3 | 269.0804 | 10.8 | 3 | 8–35 | 123 | 5 | 1 |
Coniferyl aldehyde | C10H10O3 | 458-36-6 | 179.0701 | 7.6 | 8 | 13–46 | ND | 2 | - |
ND – Not Detected |
The second group of PSMs exceeding the TTC of 0.1 µg/L is the coumarin derivatives with fraxidin and esculetin concentrations of 19 to 1145 ng/L and 116 to 1658 ng/L, respectively, while coumarin and scopoletin remained below 50 ng/L (Table 1). In general, samples from the ELP catchment showed higher concentrations of coumarins than those from the Bode catchment. All four compounds have been isolated from Fraxinus excelsior (Qazi et al. 2018; Venugopala et al. 2013; Klimo and Hager 2001; Whang et al. 2005), a characteristic tree along the rivers in both catchments. Coumarins comprise a very large class of substances, found in several higher plants and constitute fused benzene and pyrone rings (Nakamura et al. 2013; Shinbo et al. 2006; Whang et al. 2005). Simple coumarins have been found to be biologically active with anti-stress, anti-fatigue, anti-gastric ulcer, anti-depressive, immuno-enhancing and anti-inflammatory effects (Whang et al. 2005; Witaicenis et al. 2010). Scopoletin, isolated from Scopolia carniolica (Solanaceae), was shown to inhibit acetylcholinesterase (Hostettmann et al. 2006).
Two furanocoumarins, bergapten and psoralen, have been detected in only two samples, each, from both catchments but in all cases above the TTC with 510 and 541 ng/L for bergapten and 141 and 224 ng/L for psoralen. They are synthesized by several plants, especially by those of the Apiaceace family (Schlatter et al. 1991). They are generally known for their strong photosensitizing activity when applied topically or accidentally get in contact to the skin. The exposure to furanocoumarins combined with long UV radiation causes cytotoxic reactions (e.g. erythema) and genotoxic responses by binding to nucleobases in DNA (Schlatter et al. 1991; Walter et al. 1982).
The flavonoid, formononetin was detected in five samples from the ELP catchment at a concentration range of 8–35 ng/L and in one sample from Bode catchment with 123 ng/L again exceeding the TTC. The latter was taken from an agricultural area (Kolpin et al. 2010; Hoerger et al. 2009b). The PSM occurs in many leguminous plants such as clover (Trifilium), an abundant species in fertile meadows and pastures but also beans such as green beans, lima beans and soy (Afendi et al. 2011, Nakamura et al. 2013, Puri et al. 1998). Formononetin has been shown to display estrogenic properties and induce angiogenesis activities (Li et al. 2015).
In both catchments, also the PSMs nicotine and piperine have been found in concentrations of 2 to 35 ng/L and 1–338 ng/L again with two samples exceeding the TTC. The input of both metabolites to the river water is very likely due to human activities, while no plants containing these compounds in the catchments are known. Both PSMs are widely consumed by humans and related to tobacco smoking and food flavouring, respectively. Nicotine is highly addictive and acts as a receptor agonist at most nicotinic acetylcholine receptors (nAChRs) (Godin et al. 2019). Piperine is a major component of Piper species (e.g Piper nigrum, Piper longum, Piper officinarum and Piper retrofractum), which are globally marketed as flavoring agent and cooking spice with a long history of human health benefits and a wide consumption (Shoba et al. 1998; Schnabel et al. 2020). Piperine has been found to have numerous medicinal applications such as antioxidant, antiplatelet, anti-inflammatory, antihypertensive, hepatoprotective, antithyroid, antitumor, antiasthmatic activity and has also been used as fertility enhance (Derosa et al. 2016). Apart from its numerous benefits, it may also have adverse effects including hemorrhagic necrosis and edema in gastrointestinal tract, urinary bladder and adrenal glands observed in animal tests with rats (Piyachaturawat et al. 1983). Zwart et al detected piperine in waste water treatment plant effluent and classified it as one of the most potent nonsteroidal estrogens (Zwart et al. 2018).
3.4 Co-occurrence of PSMs
Similar to anthropogenic compounds, also PSMs occur in mixtures. In all of the samples, where we detected our target PSMs, we found at least two of them, at two sites (11% of all samples), we detected even six co-occurring PSMs (Fig. 4(a)). The compounds fraxidin and scopoletin were common to all samples, with only one exception in the Bode catchment. Based on TTC of 0.1 µg/L, mixture risks exceeded a RQ of 1 at 16 out of 18 sites, at 5 sites mixture RQ was above 5. At one site each, even RQs of 10 and 50 were exceeded (Fig. 4(b)). This may indicate that toxic risks by frequently occurring PSMs may not be negligible and should be included in risk assessment of chemical mixtures in water resources.