Occurrence of Stable Foam in the Upper Rhine River Caused by Plant

Jul 3, 2002 - For 30 yr, a persistent foam cover has been observed during the summer months in the Rhine River beneath the. Rhine Fall, a waterfall ne...
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Environ. Sci. Technol. 2002, 36, 3250-3256

Occurrence of Stable Foam in the Upper Rhine River Caused by Plant-Derived Surfactants CHRISTIAN WEGNER AND MATTHIAS HAMBURGER* Institute of Pharmaceutical Biology, University of Jena, Semmelweisstrasse 10, 07743 Jena, Germany

For 30 yr, a persistent foam cover has been observed during the summer months in the Rhine River beneath the Rhine Fall, a waterfall near Schaffhausen, Switzerland. This phenomenon has been a matter of public concern ever since its first appearance, but all previous attempts to clarify the origin of this foam had remained inconclusive. With the aid of electrospray LC-MS, triterpene saponins and mono- and digalactosyldiacylglycerolipids (MGDAG and DGDAG), two classes of tensioactive metabolites occurring in the aquatic plant Ranunculus fluitans Lamk. (Ranunculaceae), were detected in river water and foam samples. Saponin concentrations in water and foam samples were monitored at regular intervals during the years 1998 and 2000. Other compound classes with surfactant properties such as proteins, humic acids, and synthetic detergents were also analyzed. Foam occurrence paralleled with saponin concentration and with the amounts of detached Ranunculus biomass accumulating at the dam of the hydroelectric power plant of Schaffhausen located just above the Rhine Fall but not with the concentration of synthetic detergents. The ecotoxicological potential of Ranunculus constituents, water, and foam samples was checked with a representative range of aquatic indicator organisms. No acute toxicity was observed at concentrations that were at least 50-fold higher than those found in the environmental samples.

Introduction The presence of a stable foam beneath the Rhine Fall, a waterfall in the Rhine River near Schaffhausen, Switzerland, has been a recurring phenomenon from June to September ever since its first appearance in 1970-1971. The intensity varies strongly from day to day. At its worst, the foam may cover up to half of the river’s surface and only gradually disappear several kilometers downstream. Accumulation of massive foam lumps is observed in backwater areas of the river (Figures 1 and 2). There has been considerable public concern (1) and fears of potentially harmful effects of this foam. Also, the Rhine Fall being a major tourist attraction, the occurrence of a foam cover during the summer season has been rather disturbing. From the early 1970s onward, the authorities have been looking into the causes for this phenomenon. Initial discussions focused on industrial pollution: synthetic detergents and effluents from a nearby wastewater treatment plant. Subsequently, these potential sources were ruled out by monitoring of possible sources rather than by analytical * Corresponding author phone: ++49-(0)3641-949840; fax: ++49(0)3641-949842; e-mail: [email protected]. 3250

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evidence from water and foam samples. In 1976, a causal link between the aquatic plant Ranunculus fluitans Lamk. and the occurrence of the foam was postulated (2). This hypothesis, however, was not supported by experimental data. Subsequently, further attempts were undertaken to clarify the nature and origin of surfactants responsible for the formation of the foam. They remained inconclusive because of the lack of adequate analytical methodologies available at that time (3, 4). There have been numerous reports on the occurrence of foam in rivers, streams, and marine estuaries. Foam formation was linked to industrial effluents from paper and leather industries (5), mass development of phytoplankton in heavily polluted rivers (6), or algal blooms in estuarine areas (7, 8). However, foam also occurs in nonpolluted ecosystems (9, 10). Given the various underlying causes, the composition of these foams varied considerably. Foams due to industrial effluents contained tannins, polyamides, amino acids, and lipids (5), whereas in cases of algal blooms high contents of particulate organic matter and massive release of dissolved organic carbon were found (6, 11). In pristine ecosystems, foams contained lipids and stabilizing mucilages from kelp (9), phenolics, carbohydrates and proteins (12, 13), and humic substances originating from higher plants, plankton, and microorganims (10). Given the unresolved issue of foam formation in the Rhine River, we recently reconsidered the hypothesis of a biogenic origin of this foam phenomenon and its possible link to R. fluitans along two converging lines of thought (14): The first appearance of foam in 1970-1971 coincided with a period of dramatic changes in the aquatic flora in the Rhine River between Schaffhausen and the Lake of Constance situated upstream. R. fluitans, which up to the late 1960s had been a regularly but sparsely occurring species in the Rhine River, became the most abundant aquatic macrophyte in the flora that was previously dominated by Potamogeton species and Zannichellia palustris (3). Within a short period, R. fluitans settled primarily in the previously nonoccupied, faster flowing stretches of the river where it formed large banks of dense growth. It is now generally accepted that this almost explosive propagation was mainly due to significantly increased phosphate concentrations in the river (see Figure 1 in Supporting Information) (15). It is likely that the simultaneously occurring reduction of suspended matter, leading to an increased light exposure of the aquatic flora, provided further favorable growth conditions. The reduced turbidity apparently was a consequence of the installation of several wastewater treatments plants upstream in the late 1960s and in the following years, leading to a decrease in suspended organic matter (personal communication). Chemotaxonomic considerations provided a second line of support for a possible link between the foam phenomenon and R. fluitans. Species of the Ranunculaceae characteristically accumulate triterpene saponins, a group of tensioactive metabolites (16), although there is little data on saponins in the genus Ranunculus itself (17). The aims for our investigations were thus to adress the longstanding public concerns about the foam phenomenon by clarifying the underlying causes. The possible link to R. fluitans and the role of additional factors contributing to foam formation were to be analyzed. Given the fears about potential harmful properties of the foam, the acute toxicity was to be probed with the aid of recognized organismic assays used in aquatic toxicology. 10.1021/es025532p CCC: $22.00

 2002 American Chemical Society Published on Web 07/03/2002

FIGURE 1. Massive foam lumps on the rivers surface approximately 2 km downstream of the Rhine Fall.

FIGURE 2. Sampling of foam beneath the Rhine Fall.

Materials and Methods Chemicals. HPLC-grade solvents were from Roth, Karlsruhe, Germany. Samples of the synthetic detergents were purchased from Hu¨ls, Marl, Germany (SDS, LAS, and AEO/APEO), and Merck, Darmstadt, Germany (PEG).

Reference Compounds. Oleanolic acid (1) and hederagenin (2) were of HPLC reference grade (Roth, Karlsruhe, Germany). Glycosides 3-8, 11, and 12 are naturally occurring compounds of R. fluitans and were previously isolated from the plant (14). Monogalactosyl glycerol (9) and digalactosyl glycerol (10) were prepared ad hoc from MGDAG or DGDAG by mild basic hydrolysis (25% NH3, reflux for 1 h (see below)). Purified fractions of saponins, MGDAG, and DGDAG used for acute toxicity assays were obtained from R. fluitans as follows: Freeze-dried plant material (400 g) was milled, defatted with CH2Cl2 (1.5 L) for 48 h, and extracted with MeOH (1.5 L, 3 × 24 h) at room temperature to afford 60 g of MeOH extract. The extract was dissolved in H2O and extracted with BuOH (saturated with H2O, 3 × 500 mL). The BuOH layers were combined and evaporated to afford a crude saponin extract (12 g). A portion (8 g) was submitted to open column chromatography on silica gel 60 (40-63 µM, Macherey-Nagel, Du ¨ ren, Germany) eluted with CH2Cl2/MeOH (98:2) and CH2Cl2/MeO/H2O (80/20/2 and 60/40/10). Fractions were analyzed by TLC on silica gel 60 F254-coated Al sheets (Merck, Darmstadt, Germany) using CH2Cl2/MeO/H2O (70/30/3) as mobile phase. Godins reagent (18) was used for visualization of compounds. Saponin-containing fractions were combined and purified by gel chromatography on Sephadex LH 20 (Pharmacia Biotech, Uppsala, Sweden) to afford a saponin fraction (494 mg) and MGDAG (214 mg) and DGDAG (421 mg) fractions. Sampling. Water and foam were collected at monthly intervals from January to March and from July to December 1998 and from April to December 2000. Between July 31 and August 4, 2000, a daily monitoring was carried out. Samples (5-10 L of water, 3 L of settled foam) were taken in the middle of the river, approximately 300 m below the Rhine Fall. Plant material was collected at the same location throughout 1998. All samples were frozen immediately after sampling and freeze-dried to yield dry residues of 150-180 mg/L for water samples and 300-800 mg/L for foam samples. Dry mass of the plant samples was 7.5%. Samples of potable water were VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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taken at the pump stations of Schaffhausen and Neuhausen and processed like the river water samples. Sample Preparation for the Qualitative Analysis (Saponins, MGDAG, DGDAG, and Synthetic Detergents). Freeze-dried water and foam samples, corresponding to 2.5 L of the original volume, were extracted with MeOH (5 mL). After evaporation of solvent, the dry residue was reconstituted in 250 µL of MeOH. Acid Hydrolysis of Saponins. The freeze-dried water samples (corresponding to 1 L of the original volume) were refluxed in 2 N HCl (5 mL) for 1 h, and the mixture were extracted with CHCl3 (3 × 5 mL). The organic layer was evaporated to dryness and redissolved in 250 µL of MeOH. Quantitative Analysis of Saponins as Triterpenoids 1 and 2. LC-MS conditions are given below. External standard method; range of calibration curves 100-10 000 ng/mL; R2 > 0.9986, for a quadratic curve function. All LC-MS analyses were carried out in triplicate (standards and analytes); RSD ) 1% were determined for all samples. Limit of quantification: 1 ng on-column, corresponding to 50 ng/mL of 1 and 2, respectivley (signal-to-noise ratio ) 4:1). Recovery rates: 96% for 1, 85% for 2. For the determination of recovery rates, 100 µL of stock solutions (1.0 mg/mL) of 1 and 2, respectively, were refluxed with 2 N HCl (5 mL) for 1 h. After extraction with CHCl3 (3 × 5 mL), the organic layer was evaporated to dryness, redissolved in MeOH (250 µL), and analyzed by LCMS. The total saponin content in the environmental samples was calculated as saponin 5, on the basis of concentrations determined for 1 and 2 and the known molecular masses of aglycons and saponin 5. Basic Hydrolysis of MGDAG and DGDAG. The freezedried sample, corresponding to 1 L of the original volume of water or foam sample, was refluxed in 25% NH3 (10 mL) for 1 h to liberate mono- and digalactosylglycerol (MGG and DGG, 9 and 10), respectively. After centrifugation, the supernatant was collected and the residue was washed with H2O (3 × 5 mL). Solutions were combined and evaporated to dryness, and the residue was reconstituted in H2O (250 µL) prior to analysis. Limit of quantification: 3 ng on-column for 9 and 4 ng for 10, respectively (signal-to-noise ratio ) 4:1). Reference compounds (MGDAG, DGDAG) and environmental samples were treated in an identical way. Liquid Chromatography-Mass Spectroscopy (LC-MS). The system consisted of a series 1100 HPLC (Agilent, Waldbronn, Germany) including an autosampler, highpressure mixing pump, column oven, and DAD detector connected to a Perkin-Elmer API 165 single quadrupole mass spectrometer with a PE Sciex Turbo ion spray interface operated with a split ratio of 1:4. For all analyses, the columns were thermostated at 22 °C, and the source temperature was set at 350 °C. The following columns were used: LiChrospher 100 RP 18e (5 µm, 4.6 × 125 mm) (I); LiChrospher 100 RP 8e (5 µm, 4.6 × 125 mm) (II); LiChrospher 100 Diol (5 µm, 4.6 × 250 mm) (III). All columns were from Merck, Darmstadt, Germany. Analysis of saponins and triterpenoids: 1 and 2 (triterpenoids): column (II), MeCN/NH4OAc 0.01 N, 70/30, 1 mL/ min; 3-8 (saponins): column (I), MeCN/NH4OAc 0.01N, 20: 80 f 40:60 over 15 min, 1 mL/min. Positive/negative ion mode, ion spray voltage 5 kV, focusing potential 230 V, declustering potential 30 V, TIC m/z 250-1300 (0.8 s/scan), SIM (0.1 s/scan). Analysis of glyceroglycolipids 9 and 10: column (III), n-PrOH/NaOAc 0.01 N, 90/10, 0.8 mL/min, positive ion mode, ion spray voltage 5 kV, focusing potential 230 V, declustering potential 30 V, SIM (0.1 s/scan). Analysis of 11 and 12: column (II) MeOH/NH4OAc 0.01 N 96/4 over 15 min. MS conditions as for saponins. Poly(ethylene glycol)s (PEG) and alkylethoxylates (AEO): column (I), MeCN/NH4OAc 0.01 N, 1 mL/min, 0:100 f 40:60 3252

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over 20 min, positive ion mode, ion spray voltage 5.5 kV, focusing potential 280 V, declustering potential 35 V, TIC m/z 150-1200 (0.8 s/scan). Sodium dodecyl sulfate (SLS) and linear alkylbenzylsulfonates (SDS/LAS): column (I), MeCN/NH4OAc 0.01 N, 1 mL/min, 30:70 f 100:0 over 25 min, negative ion mode, ion spray voltage 4.5 kV, focusing potential 220 V, declustering potential -20 V, TIC m/z 150-1200 (0.8 s/scan). Analysis of Humic Substances. Automated size exclusion chromatography with UV and organic carbon detection (IR) was used for the analysis of the humic substances (19, 20). Separation was carried out on a Toyopearl HW-50S resin column (250 × 20 mm). Phosphate buffer (0.029 mol/L, pH 6.5) was used as eluent at a flow rate of 1 mL/min. Total dissolved organic carbon (DOC) was determined by injection of 0.45-µm filtered samples bypassing the chromatographic column. Polysaccharides and humic substances were fractionated, characterized by UV detection (254 nm), and quantified by IR detection after UV oxidation in an cylindrical UV thin film reactor (wavelength 185 nm; Gra¨nzel, Germany). Full experimental details are given in ref 21. Protein Analysis. Protein content was determined according to the method of Bradford (22). Freeze-dried samples were dissolved in water at 10 mg/mL. The assay was carried out in 96-well microtiter plates. Absorption was measured at 595 nm on a UV 3550 microplate reader (Bio-Rad, Munich, Germany). Bovine albumin (albumin fraction V; Merck, Darmstadt, Germany) was used as a reference, and water was used as a blank. Detection limit for bovine albumin was 10 µg/mL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to ref 23. Reagents used were from a commercially available SDSPAGE set (Biometra, Go¨ttingen, Germany). The gel consisted of a stacking gel (3% N,N′-bismethyleneacrylamide) and a separating gel (12.5% N,N′-bismethyleneacrylamide). To 20 µL of sample, 20 µL of sample buffer was added and heated for 2 min in a boiling water bath. After the sample was cooled to room temperature, 4 µL of dithioerythritiol was added, and 7 µL of the mixture was applied on every gel slot. Electrophoresis was carried out on a power supply model 1000/500 (Bio-Rad) with 10 min at 10 mA and then 20 mA with variable voltage. Samples were running from cathode to anode. Staining of gels with alkaline silver nitrate was performed according to ref 24 with a commercial silver stain kit (Bio-Rad). Coomassie staining of gels was performed using a commercial Coomassie Brillant Blue R-250 Staining Solutions Kit (Bio-Rad). Bioassays. Kits for the ecotoxicological tests were obtained from Creasel (Deinze, Belgium). Acute toxicity tests on Daphnia magna, Artemia salina (brine shrimp, second instar larvae), Brachyonus calyciflorus (rotifer), and Selenastrum capricornutum (unicellular algae) were performed according to the standard operational procedures of the commercially available test kits Daphtoxkit, Artoxkit, Rotoxkit, and Algaltoxkit. Briefly, eggs of Daphnia, Artemia, and Brachyonus were hatched in the appropriate nutrient solution and kept until they reached the required development stage. The appropriate number of organisms were pipetted to the test solutions and kept in the dark at 20 or 25 °C, as required. Evaluation was carried out under a stereomicroscope. The number of live organisms was counted, and survival was calculated in comparison to blank controls. In the Selenastrum assay, algae were suspended in an algal medium and diluted to 106 cells/mL. A Casy 1 TT cell counter (Scha¨rfe Systems, Reutlingen, Germany) with a capillary i.d. of 150 µm was used for the determination of the cell number. The algae (final concentration in the assay 104 cells/mL) were incubated for 72 h at 25 °C and 8000 lux. Algal cell counts were carried out after 24, 48, and 72 h.

FIGURE 3. Saponins (3-8) and glycoglycerolipids (11 and 12) in R. fluitans, their hydrolysis products (1, 2, 9, and 10), and their molecular ions observed in ESI (positive or negative ion mode). Larvicidal and molluscicidal assays on Aedes aegypti (instar larvae) and Biomphalaria glabrata (mollusc) were carried out according to refs 25 and 26. Briefly, eggs of Aedes were incubated in tap water at 27 °C for 24 h. Larvae were then placed into the test solutions, and motility was assessed after 30 min and 24 h under a stereomicrosope. The Biomphalaria assay was carried out according to a protocol published by the World Health Organization. Molluscs of defined size (diameter of shell 9 mm) were placed into test solutions and kept at room temperature for 24 h. The survival was assessed by observation of the heartbeat under a dissecting microscope. Test solutions of saponin, MGDAG and DGDAG fractions, and MeOH and BuOH extract were prepared in H2O or artificial seawater (Artemia salina) at concentrations of 0.01, 0.10, 1.00, 10.00, and 100.00 ppm. Foam and water samples were reconstituted in H2O to original concentration.

Results and Discussion A thorough phytochemical investigation of R. fluitans, in particular the search for compounds with surfactant properties, was a precondition for probing the link between this plant and the foam phenomenon. We therefore characterized the plant’s metabolite profile, whereby two major classes of amphiphilic compounds were identified, namely, triterpene saponins (3-8) and mono- (MGDAG, 11) and digalactosyldiacylglycerols (DGDAG, 12) (Figure 3) (14). The saponins were structurally closely related bidesmosides (27) (saponins bearing two sugar chains at different positions) derived from oleanolic acid (1) and hederagenin (2).

Water and foam samples were collected below the Rhine Fall at regular intervals over the years 1998 and 2000 (see Materials and Methods). We then developed methods for the qualitative and quantitative analyses of these surfactants in water and foam samples. Considering the low concentrations to be anticipated, the structural complexity of saponins and glyceroglycolipids, the inherent difficulties for detection of these substances (27), and possible matrix interferences, LC-MS analysis was the most appropriate approach (28, 29). Electrospray ionization (ESI) of triterpene saponins in the negative ion mode produces reasonably intense [M - H]ions (28). ESI LC-MS analysis of a R. fluitans extract and the corresponding mass spectrum for saponin 5 are shown in Figure 4a. Apart from the [M - H]- signal, diagnostic fragment ions at m/z 633 and 471 resulted from the loss of the esterlinked trisaccharide moiety at C-28 and the subsequent elimination of the glucosyl residue at C-3. The chromatogram of a foam sample revealed a very similar saponin pattern whereby the identity with Ranunculus saponins 3-8 (14) was further substantiated with the aid of authentic reference compounds and by the characteristic ESI-MS fragmentation pattern (Figure 4b). In the corresponding water sample, the saponin concentration was too low for analysis in the fullscan mode. However, monitoring for the anticipated molecular ions of 3-8 revealed a saponin pattern comparable to that in plant and foam samples (Figure 5). Thus, R. fluitans saponins were present in foam and water samples taken beneath the Rhine Fall. Glycoglycerolipids could be detected in foam samples only (see Figure 2 in Supporting Information) despite the fact that their content in the plant is higher than that of saponins. The reasons are presently not known, but we assume that they may undergo a rapid biological breakdown. Monodesmosidic saponins (saponins without ester glycosidic moiety R3; see Figure 3), which are the most likely degradation products of bidesmosides 3-8, were not detected (27). Next, a method for quantitative analysis of the saponin content in water samples was developed. All Ranunculus saponins are structurally closely related. Hence, no major differences with respect to surfactant properties were to be expected between the individual compounds. Considering the fact that foam formation is a sum effect of all surfactants present, a dosage of individual saponins appeared not to produce substantially more information than the determination of their common moieties. For these reasons, the aglycons oleanolic acid (1) and hederagenin (2) liberated by acid hydrolysis of saponins in the samples were quantified, and the total saponin concentration was calculated as major compound 5. The saponin concentrations in water samples from the year 1998 (Figure 6a) showed a distinct seasonal variation. The peak during the summer months coincided with the occurrence of foam in the river. The water flow was fairly constant during the summer months of 1998 but slightly below the long-term average (see Figure 3 in Supporting Information). The saponin concentrations paralleled the quantities of detached R. fluitans biomass retained by the dam of the hydroelectric power plant at Schaffhausen, located 1.5 km upstream of the Rhine Fall (Figure 6a) (30). The data of the year 2000 revealed a rather different situation. An exceptional high-water situation in 1999 had destroyed most of the plant root systems because of shifting gravel in the riverbed. R. fluitans populations and, in consequence, the amounts of biomass were thus heavily reduced in the following year (Figure 6b). Saponin concentrations were also much lower than in 1998. A slight increase in the saponin concentrations during the summer was still detectable, but there was no distinct peak as in 1998 (Figure 6a). Foam formation was also significantly reduced in comparison to VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. ESI LC-MS of saponins in negative ion mode. (a) Saponin pattern of the crude plant extract (TIC, m/z 250-1400) (left) and fragmentation pattern of saponin 5 (right). (b) Saponin pattern in a reconstructed ion chromatogram ([M - H]- ions of saponins 3-8, recorded in full-scan mode as in panel a in a foam sample of July 27, 1998 (left), and fragmentation pattern of saponin 5 (right).

FIGURE 5. Saponin pattern in a water sample of July 27, 1998 (selected ion monitoring of [M - H]- ions for saponins 3-8). previous years. In addition, the water flow in August was well above average (see Figure 3 in Supporting Information) and resulted in an additional dilution effect. A parallel between saponin concentration and foam occurrence alone could not be considered as proof for a causal link between the two phenomena. To further clarify the role of the R. fluitans saponins, additional surfactants that could possibly contribute to foam formation had to be taken into account. These could either be of natural origin, such as humic substances and proteins, or of anthropogenic origin, such as synthetic detergents. We therefore screened various water and foam samples for humic substances (see Materials and Methods and Table 1 in Supporting Information) (19, 20). Protein analysis was carried out with the aid of a colorimetric assay (22) and by gel electrophoresis (see Materials and Methods). Protein was not detectable in any of the samples analyzed. The concentrations of humic substances were low. Dissolved organic carbon (DOC) was approximately 10% of that reported from some surface waters in Germany (21). The proportion of humic substances of the DOC was only 30-50%. Typically, proportions of 70% or higher have been found (21). It was thus unlikely that protein and humic substances would play a role in foam formation. Synthetic detergents and their breakdown products occur in household and industrial effluents and hence in surface water. To assess their possible role in the foam phenomenon, water and foam samples were analyzed for the presence of all major classes of synthetic detergents such as linear alkylbenzylsulfonates (LAS), alkylphenylethoxylates (APEO), alkylethoxylates (AEO), sodium dodecyl sulfate (SDS), and poly(ethylene glycol)s (PEG) as characteristic breakdown products of AEO and APEO (31, 32). These compounds are amenable to LC-MS analysis (32-35) and were thus 3254

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FIGURE 6. Saponin concentrations in water samples (columns) and R. fluitans biomass (line) retained by the dam of the hydroelectric power plant, Schaffhausen: (a) 1998 and (b) 2000. Water samples were collected at the dates indicated; Ranunculus biomass indicates cumulative amounts (tons of fresh weight) for the corresponding month. monitored by ESI LC-MS. In the context of this investigation, relative concentrations only were determined via identical workup for all samples and comparison of the absolute MS signal intensities (see Figure 7 and Figures 4 and 5 in Supporting Information). LAS, SDS, and PEG were found in all water and foam samples, whereas APEO and AEO were below the detection limit. Concentrations of LAS, SDS, and PEG in water samples showed considerable day-to-day variations but, unlike the saponin concentrations, did not follow a seasonal pattern correlating with foam occurrence. Additional analyses (data not shown) revealed the presence of synthetic surfactants throughout the year. Samples collected during the winter of the year 2000 contained PEG, SDS, and LAS at concentrations that were comparable to those of the summer months. Daily monitoring of saponins

FIGURE 7. ESI LC-MS detection of PEGs in environmental samples: (a) total ion chromatogram (TIC) of PEG standard, (b) TIC of the foam sample August 2, 2000, (c) TIC of the water sample August 2, 2000. (d) TIC of water sample June 5, 2000. No foam formation was observed on June 5, 2000. Workup procedure and analytical settings were strictly identical for all samples. and detergents in June and August 2000 revealed that concentrations of PEG, SDS, and LAS were in some cases severalfold higher on days without foam occurrence than on days with foam formation (Figure 7 and Figures 4 and 5 in Supporting Information). Saponins, however, were only detected on days of foam occurrence. The analytical data collected provide strong evidence that the R. fluitans saponins have to be considered as the causative agents for foam formation because saponin concentration in water samples was only one of several possible factors that clearly paralleled foam occurrence. However, other factors may contribute to the stabilization of the saponin foam generated under the massive energetic impact of the Rhine Fall. Such factors could be colloidal or ultra-fine particulate matter protecting foam lamellae from confluence or solutes such as other surfactants accumulating at the air/ water interface (36). Indeed, visual examination of settled foam samples revealed a high proportion of finely suspended particles, and the concentrations of PEG, SDS, and LAS were approximately 2 orders of magnitude higher than in water samples collected simultaneously (see Figure 7b,c and Figure 5c,d in Supporting Information). There were, however, substantial differences in surfactant concentrations and composition between the foam samples and in enrichment factors as compared to the corresponding water samples. Enrichment factors of about 2 orders of magnitude were also found for the saponins. The possible negative effects of the saponins on the quality of potable water and a potential ecotoxicological impact of the foam have been a longstanding public concern. Potable water supply in the region is mainly based on underground water taken in vicinity of the riverbed. Analysis of water samples from the pump stations in Schaffhausen and Neuhausen revealed that saponins were indeed detectable (37). However, in comparison to river water sampled on the same day, the concentrations were at least 5-fold lower. The possible toxicity of the foam toward aquatic organisms has also been a matter of discussion. Plant-derived surfactants of the saponin type may show substantial acute toxicity toward aquatic organisms and have therefore been used in Africa and Latin America as fish poisons and molluscicides (26, 27). We assayed R. fluitans extracts, purified compounds, and water and foam samples for acute toxicity against a range of recognized indicator organisms used in aquatic toxicology (38). Toxic effects in none of these organisms were observed at concentrations that were at least 50-fold higher than those

measured in the environmental samples (see Materials and Methods and Table 2 in Supporting Information). As already mentioned, the data collected lead to the conclusion that the foam phenomenon is linked to the occurrence of R. fluitans. The species however is widely distributed in many European rivers. R. fluitans is a dominating aquatic plant in lower stretches of Karst River and Riss-Moraine streams of southwestern Germany and in the faster flowing stretches of the Danube (39). It also massively occurs in major rivers of Switzerland (40), central Germany (personal observation), northeastern France (41), and England and Wales (42). R. fluitans populations in these rivers underwent major changes over the last four decades. Receding growth has been linked to channelization (40), whereas expansion in some rivers resulted from a decreased sediment load by particulate pollutants (43). These rivers, however, differ considerably from the situation of the Rhine, and there has indeed been no report on foam formation in connection with the presence of R. fluitans. The foam phenomenon encountered at the Rhine Fall appears to be the result of a set of particular circumstances, namely, a massive growth of the surfactant-producing R. fluitans combined with an efficient extraction of these water-soluble substances and the energetic impact required for efficient foam formation. The parallel between the R. fluitans biomass retained at the power plant and the saponin concentrations in water strongly suggests that release of saponins occurs primarily from detached shoots rather than from the intact plants. Reduction of Ranunculus growth in the Rhine should, in principle, lead to disappearance of foam. Mechanical removal by a specially designed cutting machine was carried out for many years to improve navigation but did not diminish Ranunculus populations (44) (see also Figure 7 in Supporting Information). Phosphate concentration is apparently a major limiting factor for growth of R. fluitans. Stricter environmental policies have significantly lowered the phosphate load in the Rhine (see Figure 1 in Supporting Information), and R. fluitans populations have indeed somewhat receded over the past few years (37). The very low turbidity of the Rhine, however, provides favorable light conditions for R. fluitans and may have counteracted to a certain extent the effects of reduced phosphate concentrations. The next few years will reveal whether the current trend continues, which should lead to a reduction or disappearance of the highly disturbing but fortunately innocuous foam phenomenon. VOL. 36, NO. 15, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Acknowledgments Financial support was provided by the Amt fu ¨ r Lebensmittelkontrolle und Umweltschutz des Kantons Schaffhausen; the Amt fu ¨ r Abfall, Wasser, Energie und Luft des Kantons Zu¨rich; and the Federal Office for the Environment (BUWAL), Bern, Switzerland. H. Hardmeier, S. di Gregorio, and staff of the Amt fu ¨ r Lebensmittelkontrolle und Umweltschutz des Kantons Schaffhausen are gratefully acknowledged for organizational support and sampling. We thank H. Hardmeier also for numerous discussions and unpublished information on the Rhine foam phenomenon. Thanks are due to W. Ma¨ndli (Ma¨ndli-Boote, Neuhausen) for providing boats for sample collection, to B. von Felten (EKW Schaffhausen) and K. Wa¨chter (Limnex AG, Zu ¨ rich) for provision of data on Ranunculus populations and plant biomass, and to Prof. C. Steinberg and collaborators (Institut fu ¨ r Gewa¨ssero¨kologie und Binnenfischerei, Berlin) for analysis of humic substances. The authors are grateful to Prof. K. Hostettmann (University of Lausanne) and co-workers for carrying out the larvicidal and molluscicidal bioassays.

Supporting Information Available ESI LC-MS chromatograms of glyceroglycolipids in R. fluitans and foam, monthly and cumulative plant biomass at hydroelectric power plant of Schaffhausen, monthly average flow of Rhine River, SDS and LAS analyses, yearly average of phosphate concentrations in Rhine River, analyses of humic substances, and results of toxicological testing. This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Anonymous. Neue Zu ¨ rcher Zeitung 1997, 13/14 (Sept.), 57. (2) Eichenberger, E. Wasser Energ. Luft 1976, 10, 234-239. (3) Huber, M. Diploma Thesis, University of Zurich, Zurich, Switzerland, 1976. (4) di Gregorio, S. Diploma Thesis, Kantonales Laboratorium fu ¨ r Lebensmittelkontrolle und Umweltschutz, Schaffhausen, Switzerland, 1996. (5) Madrange, L.; Chaboury, P.; Ferrandon, O.; Mazet, M.; Rodeaud, J. Rev. Sci. Eau 1993, 6, 315-333. (6) Buhse, G. Courier Forschungsinst. Senckenberg 1980, 43, 1-226. (7) Eberlein, K.; Leal, M. T.; Hammer, K. D.; Hickel, W. Mar. Biol. 1985, 89, 311-316. (8) Wyatt, T. Nat. Resour. 1998, 34, 40-51. (9) Velimirov, B. Mar. Ecol. 1982, 3, 97-108. (10) Mills, M. S.; Thurman, E. M.; Ertel, J.; Thorn, K. A.; Gaffney, J. S. In Humic and Fulvic Acids: Isolation, Structure, and Environmental Role; Marley, N. A., Clark, S. B., Eds.; ACS Symposium Series 651; American Chemical Society: Washington, DC, 1996; pp 151-192. (11) Baetje, M.; Michaelis, H. Mar. Biol. 1986, 93, 21-28. (12) Barlocher, F.; Gordon, J.; Ireland, R. J. J. Exp. Mar. Biol. Ecol. 1988, 115, 179-186. (13) Craig, D.; Ireland, R. J.; Barlocher, F. J. J. Exp. Mar. Biol. Ecol. 1989, 130, 71-80. (14) Wegner, C.; Hamburger, M.; Kunert, O.; Haslinger, E. Helv. Chim. Acta 2000, 83, 1454-1464.

3256

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 15, 2002

(15) Mulligan, H. F.; Baranowski F. Int. Ver. Theor. Angew. Limnol. Verh. 1968, 17, 802-810. (16) Hegnauer, R. Chemotaxonomie der Pflanzen; Vol. 9; Birkha¨user: Basel, 1990; p 328. (17) Texier, O.; Ahond, A.; Regerat, F.; Pourrat, H. Phytochemistry 1984, 12, 2903-2905. (18) Godin, P. Nature 1954, 174, 134. (19) Huber, S. A.; Frimmel, F. H. Vom Wasser 1996, 86, 277-290. (20) Huber, S. A.; Frimmel, F. H. Anal. Chem. 1991, 63, 2123-2130. (21) Sachse, A.; Babenzien, D.; Ginzel, G.; Gelbrecht, J.; Steinberg, C. E. W. Biogeochemistry 2001, 54, 279-296. (22) Bradford, M. Anal. Biochem. 1976, 72, 248-254. (23) Laemmli, U.K. Nature 1970, 227, 680-685. (24) Gottlieb, M.; Chavko, M. Anal. Biochem. 1987, 165, 33-37. (25) Cepleanu, F. Dissertation, University of Lausanne, Lausanne, Switzerland, 1993. (26) Mott, K. E., Ed. Plant Molluscicides; Wiley: Chichester, 1983. (27) Hostettmann, K.; Marston, A. Saponins; Cambridge University Press: Cambridge, 1995. (28) Perret, C.; Wolfender, J. L.; Hostettmann, K. Phytochem. Anal. 1999, 10, 272-278. (29) Zhou, S.; Hamburger, M. J. Chromatogr. A 1996, 755, 189-204. (30) The particularities of R. fluitans growth appear to play a decisive role in this context. The species forms extensive root systems in the riverbed. Flexible shoots up to 6 m long grow from these roots from spring to early summer, when populations reach their maximum. Then, seasonal growth begin to massively detach and are carried away by the strong current. A weak region of the stem near the base acts thereby as a “mechanical fuse” (45). Lumps of decaying plant material get entangled along the river shore, whereas the major part of Ranunculus biomass is carried downstream (Figures 6 and 7 in Supporting Information). (31) Marcomini, A.; Zanette, M. J. Chromatogr. A 1996, 733, 193206. (32) Richardson, S. D. Chem. Rev. 2001, 101, 211-254. (33) Creszenzi, C.; Di Corcia, A.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1997, 31, 2679-2685. (34) Di Corcia, A.; Casassa, F.; Crescenzi, C.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1999, 33, 4112-4118. (35) Di Corcia, A.; Capuani, L.; Casassa, F.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1999, 33, 4119-4125. (36) Jo¨nnsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; Wiley: Chichester, 1999. (37) Wegner, C. Dissertation, University of Jena, Jena, Germany, 2001. (38) Wells, P. G., Lee, K., Blaise, C., Eds. Microscale Testing in Aquatic Toxicology: Advances, Techniques and Practice; CRC: New York, 1998. (39) Schu ¨ tz, W. Acta Bot. Gallica 1995, 142, 571-584. (40) Lauber, K.; Wagner, G. Flora Helvetica; Haupt: Bern, 1996. (41) Grasmu ¨ ck, N.; Haury, J.; Le´glize, L.; Muller, S. Hydrobiologia 1995, 300/301, 115-122. (42) Spink, A. J.; Murphy, K. J.; Westlake, D. F. Arch. Hydrobiol. 1997, 139, 509-525. (43) Whitton, B. A.; Boulton, P. N. G.; Clegg, E. M.; Gemmell, G. J.; Graham, G. G.; Gustar, R.; Moorhouse, T. P. Sci. Total Environ. 1998, 210/211, 411-426. (44) Kranich, L. Wasser Energ. Luft 1976, 10, 239-241. (45) Usherwood, J. P.; Ennos, A. R.; Ball, D. J. J. Exp. Bot. 1997, 48, 1469-1475.

Received for review January 16, 2002. Revised manuscript received May 23, 2002. Accepted May 31, 2002. ES025532P