Biodegradation of the artificial sweetener acesulfame in biological wastewater treatment and sandfilters

A considerable removal of the artificial sweetener acesulfame (ACE) was observed during activated sludge processes at 13 wastewater treatment plants (WWTPs) as well as in a full-scale sand filter of a water works. A long-term sampling campaign over a period of almost two years revealed that ACE removal in WWTPs can be highly variable over time. Nitrifying/denitrifying sequencing batch reactors (SBR) as well as aerobic batch experiments with activated sludge and filter sand from a water works confirmed that both activated sludge as well as filter sand can efficiently remove ACE and that the removal can be attributed to biologically mediated degradation processes. The lab results strongly indicated that varying ACE removal in WWTPs is not associated with nitrification processes. Neither an enhancement of the nitrification rate nor the availability of ammonium or the inhibition of ammonium monooxygenase by N-allylthiourea (ATU) affected the degradation. Moreover, ACE was found to be also degradable by activated sludge under denitrifying conditions, while being persistent in the absence of both dissolved oxygen and nitrate. Using ion chromatography coupled with high resolution mass spectrometry, sulfamic acid (SA) was identified as the predominant transformation product (TP). Quantitative analysis of ACE and SA revealed a closed mass balance during the entire test period and confirmed that ACE was quantitatively transformed to SA. Measurements of dissolved organic carbon (DOC) revealed an almost complete removal of the carbon originating from ACE, thereby further confirming that SA is the only relevant final TP in the assumed degradation pathway of ACE. A first analysis of SA in three municipal WWTP revealed similar concentrations in influents and effluents with maximum concentrations of up to 2.3 mg/L. The high concentrations of SA in wastewater are in accordance with the extensive use of SA in acid cleaners, while the degradation of ACE in WWTPs adds only a very small portion of the total load of SA discharged into surface waters. No removal of SA was observed by the biological treatment applied at these WWTPs. Moreover, SA was also stable in the aerobic batch experiments conducted with the filter sand from a water works. Hence, SA might be a more appropriate wastewater tracer than ACE due to its chemical and microbiological persistence, the negligible sorbing affinity (high negative charge density) and its elevated concentrations in WWTP effluents.


Information about the WWTPs
Tab. S1 S3 Quantitative analysis of ACE by LC-MS/MS S1.1 S4 Quantitative analysis of ACE and the TPs SA and ANSA by IC-MS/MS S1.2 S4-S5 Monitored mass transitions and compound specific MS parameters used for quantification of acesulfame Tab. S2 S5 Calibration and determination of the limit of quantification S1.3 S5 TP identification by high-resolution mass spectrometry S1.4 S5-S6 Determination of basic operational parameters S1.5 S6

Results and discussion
Basic operational parameters measured in laboratory batch experiments for the approaches A and B Tab. S3 S7 Basic operational parameters measured in laboratory batch experiments for the approach C Tab. S4 S8 Basic operational parameters measured in bench scale reactors in batch mode Tab. S5 S9 Basic operational parameters measured in influent and effluent of the bench scale reactors in sequencing batch mode, Tab. S6 S10 Concentration [µg/L] of ACE in surface water and in the respective collection well after slow sand filtration and short underground passage.   List of retention times (RTs) and precursors and product ions from MS and MS 2 spectra obtained from ACE and ACE TPs using IC-LTQ-Orbitrap-MS with electrospray ionization in negative ionization mode.

S3
Tab. S1: Overview about the biological treatment steps, selected operational parameters as well as the average ACE concentrations (± standard deviation) detected in the influents and effluents of 13 municipal WWTPs which were sampled over a period of two weeks (24 h composite samples, n=6). The operational parameters were provided by the WWTPs. Values of the chemical oxygen demand (COD) and NH4-N represent either the annual average or (in parentheses) the average of the sampling period. Influent samples were taken from raw wastewater before biological treatment and effluent samples were taken from the final effluent before discharge into the receiving water (i.e. after secondary clarification or sand filtration).

S1.1 Quantitative analysis of ACE by LC-MS/MS
Quantitative analysis of ACE in samples from WWTPs and the waterworks was conducted by LC-MS/MS. After addition of the surrogate standard (4 µg/L ACE-d 4 ), 80 µL of the samples were injected into an Agilent 1260 Series liquid chromatography system (Agilent Technologies, Waldbronn, Germany) coupled to a SCIEX QTrap 5500 mass spectrometer (SCIEX, Darmstadt, Germany). Chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column (2.1 x 150 mm, 3.5 µm, Agilent Technologies, Waldbronn, Germany). Ultrapure water was used as mobile phase A and methanol as mobile phase B, both acidified with 0.1% formic acid.
The gradient elution applied was as follows: 100% A for 1 min, decrease to 80% A within 1 min, further decrease to 0% A within 14.5 min, hold isocratic at 0% A for 5.5 min, increase to initial conditions (100% A) within 0.1 min and hold isocratic for 6 min. The total run time was 28 min and the flow rate and the column temperature were set to 0.3 mL/min and to 30°C, respectively. The mass spectrometer was operated with negative ion electrospray ionization (ESI) using multiple reaction monitoring (MRM). The ion source conditions were as follows: collision gas, medium; curtain gas 40 psi; ion source gas 1, 40 psi; ion source gas 2, 45 psi; source temperature 500°C; ion spray voltage, -4.5 kV; entrance potential, -10 V. The monitored mass transitions and compound specific MS parameters are described in Tab. S2.

S1.2 Quantitative analysis of ACE and the TPs SA and ANSA by IC-MS/MS
Quantitative analysis of ACE and the TPs SA and ANSA in samples from batch experiments was realized with an ion chromatography system (Metrohm 881 Compact IC pro with chemical suppressor, Metrohm GmbH, Filderstadt, Germany), coupled to an API 4000 triple quadrupole mass spectrometer (SCIEX, Darmstadt, Germany). ACE-d 4 was used as surrogate standard.

Chromatographic separation was achieved on a Metrosep A Supp 5 anion exchange column
(150 x 4 mm) connected to a Metrosep A Supp 4/5 guard column (Metrohm GmbH, Filderstadt, Germany). Ultrapure water and a carbonate buffer (5 mM Na 2 CO 3 and 3.1 mM NaHCO 3 ) both amended with 10% methanol were used as mobile phase A and B, respectively. The gradient elution applied was as follows 36% A for 8 min, decrease to 0% A within 2 min, hold isocratic at 0% A for 30 min, increase to initial conditions (36% A) within 1 min and hold isocratic at 36% A for 4 min. The total run time was 45 min and the flow rate and the column temperature were set to 0.7 mL/min and to 50°C, respectively. The injection volume was 10 µL. The mass spectrometer was operated with negative electrospray ionization (ESI) using multiple reaction monitoring (MRM). The ion source conditions were as follows: collision gas, medium; curtain S5 gas 30 psi; ion source gas 1, 50 psi; ion source gas 2, 50 psi; source temperature 600°C; ion spray voltage, -4.2 kV; entrance potential, -10 V. The monitored mass transitions and compound specific MS parameters are described in Tab. S2.

S1.3 Calibration and determination of the limit of quantification (LOQ)
For quantification of ACE, SA and ANSA an internal standard calibration with linear fitting and a weighing factor of 1/x was used. The limit of quantification was derived from the signalto-noise (S/N) ratio in the native samples. At the LOQ, the S/N ratio of the mass transitions used for quantification (MRM 1) and confirmation (MRM 2, not available for SA) had to be at least 10 and 3, respectively.

S1.4 TP identification by high-resolution mass spectrometry
High-resolution mass spectra and MS 2 fragmentation experiments for the detection, identification and characterization of ACE TPs were obtained by Orbitrap-MS (LTQ Orbitrap Elite, Thermo Scientific, Bremen, Germany) operated in negative and positive ionization mode.
The Orbitrap system was coupled to an ion chromatography system (Metrohm 881 Compact IC pro with chemical suppressor, Metrohm GmbH, Filderstadt, Germany). Chromatographic conditions were the same as described above (S.1.2) for quantification of the TPs SA and ANSA.
Data dependent acquisition was used to give rise to spectra as follows: A full scan (50-300 m/z) was performed followed by MS 2 scans for the two most intense ions with intensities of > 1000 and > 500, respectively. CID (collision-induced dissociation) and HCD (higher energy collisional dissociation) with normalized collision energies of 35% and 120%, respectively, S6 were used for fragmentation. In addition, dynamic exclusion was applied (exclusion of masses for which two MS 2 experiments have been performed; exclusion duration, 15 s; repeat duration, 30 s). External calibration was performed before the analysis of each batch to assure accurate mass determinations with a resolution of 60,000.
To avoid the missing of TPs not amenable to chromatographic separation by IC, the Orbitrap system was also interfaced with a Thermo Scientific Accela U-HPLC system (Accela pump and autosampler) equipped with a reversed-phase (RP) column (Hydro-RP, 150 x 4.6 mm, 4 µm) from Phenomenex (Aschaffenburg, Germany). Ultrapure water and acetonitrile (both amended with 0.1% formic acid) were used as mobile phase A and B, respectively. The gradient elution applied was as follows 100% A for 5 min, decrease to 60% A within 10 min, further decrease to 5% A within 3 min, hold isocratic at 5% A for 5 min, increase to initial conditions (100% A) within 0.1 min and hold isocratic at 100% A for 6.9 min. The total run time was 30 min and the flow rate and the column temperature were set to 0.5 mL/min and to 50°C, respectively. The injection volume was 10 µL. In batch-experiments with sand, analyses of dissolved organic carbon (DOC) was performed according to German standard DIN EN 1484 (DIN, 1997) by combustion catalytic oxidation using a TOC analyser (TOC-L series, Shimadzu, Kyoto, Japan).   --211  --214  -8  -188  --192  --197  -24  -176  --172  --176  -48  -150  --147  --153  -96 -

Setup replicate time 1 2 3 [h] NH 4 -N NO 3 -N DOC NH 4 -N NO 3 -N DOC NH 4 -N NO 3 -N DOC TSS
Tab. S4: Basic operational parameters measured in laboratory batch experiments for the approach C (all concentrations as mg/L).     indicated that the additional oxygen is located in α-position (C-5) of the keto group of ACE.
For TP178 (RT 14.7 min) an exact mass of 177.9814 was determined and indicated an hydroxylation either at the C-5 position or at the C-7 position. The former would lead to 6methyl-4,5-dioxo-1,2,3-oxathiazinan-3-ide 2,2-dioxide which differs from the assumed structure of TP180b only by a keto group instead of an alcohol group at the C-5 position.
However, since the MS 2 spectrum of TP 178 only exhibited one dominant fragment (m/z 98.0249, C 4 H 4 NO 2 , [M-SO 3 ] -) which was comparable to that of acesulfame but not to any of those of TP180b, it was assumed that TP 178 still bears the double bond between C-5 and C-6 and is more likely hydroxylated at the C-7 position. It was therefore tentatively identified as 6-(hydroxymethyl)-4-oxo-4H-1,2,3-oxathiazin-3-ide 2,2-dioxide.
The measured exact mass of TP192 (m/z 191.9613, C 4 H 2 NO 6 S) suggested the introduction of two additional oxygens and the cleavage of 2 hydrogens. TP192 was also detected doubly charged (m/z 95.4769) which indicated the presence of an acidic moiety. However, the MS 2 spectrum gave no indication of a carboxyl group (no cleavage of CO 2 or CH 2 O 2 ). Instead the only plausible structure was that of 6-formyl-4,5-dioxo-1,2,3-oxathiazinan-3-ide 2,2-dioxide exhibiting a 1,3-dicarbonyl moiety. The deprotonation of TP 192 leading to the double charged species in the ESI source can be explained by the formation of enolate forms and their stabilization by delocalization of the charge over three atoms. Except for SA, TP 192 was the only TP for which no further degradation was observed.
However, the proposed chemical structures of the minor TPs TP180b, TP178 and TP192 could not be further confirmed, since reference standards were not available and the quantities were too low for structural elucidation by NMR.