Occurrence and removal of lidocaine, tramadol, venlafaxine, and their metabolites in German wastewater treatment plants
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- Rúa-Gómez, P.C. & Püttmann, W. Environ Sci Pollut Res (2012) 19: 689. doi:10.1007/s11356-011-0614-1
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Some of the pharmaceuticals that are not extensively investigated in the aquatic environment are the anesthetic lidocaine (LDC), the analgesic tramadol (TRA), and the antidepressant venlafaxine (VEN). LDC metabolizes to 2,6-xylidine (2,6-DMA) and monoethylglycinexylidine (MEGX), TRA to O-desmethyltramadol (ODT), and VEN to O-desmethylvenlafaxine (ODV). Within this study, the distribution and behavior of these compounds in German wastewater treatment plants (WWTPs) were investigated.
Samples of influents and effluents from WWTPs in Hesse, Germany were collected between January and September 2010. Analytes were extracted from wastewater samples by solid-phase extraction and from solid samples by sonication. Extracts were measured using gas chromatography/mass spectrometry.
LDC, TRA, VEN, ODT, and ODV were detected in all analyzed influent and effluent samples. 2,6-DMA could not be identified. MEGX was not detected. TRA and ODV were present in untreated wastewater at the highest concentrations (max, 1,129 (TRA) and 3,302 ng L−1 (ODV)), while the concentrations of LDC and VEN were all significantly lower (mean, 135 (LDC) and 116 ng L−1 (VEN)). All of the analytes were only partially removed in the WWTPs. The mean ratios between the concentrations of the metabolites and their respective parent compounds in influents were 4.7 (ODV/VEN) and 0.7 (ODT/TRA). These values remain approximately constant comparing influents and effluents.
LDC, TRA, VEN, ODT, and ODV are only partially removed from sewage water by WWTPs and thus are continuously discharged in respective recipient rivers. A further transformation of TRA and VEN into the known metabolites during treatment in the WWTPs is not observed.
KeywordsWastewaterLidocaineTramadolVenlafaxineMetabolitesWastewater treatment plantRemoval rates
A wide range of pharmaceutical compounds is used for the treatment of various diseases in both humans and domestic animals. These compounds and/or their metabolites are excreted through urine and feces into domestic wastewater and make their way to wastewater treatment plants (WWTPs). Some of them are not totally removed in the WWTPs and remain in the effluents entering surface waters (González Alonso et al. 2010; Zhou et al. 2010) and even groundwater (Einsiedl et al. 2010). The potential for a drug to reach the aquatic environment depends on three factors: the sales amount, the pharmacokinetic behavior (half life, urinary and fecal excretion, metabolism, etc.), and the rate of degradation of the compounds in the sewage system and in WWTPs.
In the meantime, a high amount of drugs and related metabolites have been detected in the aquatic environment and the list is continuously increasing (Choi et al. 2008; Gros et al. 2007; Kasprzyk-Hordern et al. 2008). Some non-extensively investigated drugs are the amino compounds lidocaine, tramadol, and venlafaxine. Lidocaine (2-(diethylamino)-N-(2,6-dimethylphenyl)ethanamide (LDC)), originally marketed by ASTRA AB as Xylocaine®, is widely used as a local anesthetic and antiarrhythmic agent by cardiac disorders. Both humans and animals metabolize LDC to various metabolites, most of them quite unstable at room temperature. One of its major active metabolites is 2-(ethylamino)-N-(2,6-dimethylphenyl)ethanamide (monoethylglycinexylidide (MEGX)), which has demonstrated stability at room temperature (Tam et al. 1990). MEGX metabolizes further into 2,6-dimethylaniline (2,6-xylidine (2,6-DMA)), suspected to be a potential carcinogen (Bangoluri et al. 2005). Tramadol ((1RS, 2RS)-2-(dimethylaminomethyl)-1-(3-methoxyphenyl)cyclohexanol (TRA)), marketed by Grünenthal GmbH as Tramal®, is a centrally acting analgesic. The metabolic fate of tramadol is unusually complex, O-desmethyltramadol (ODT) being its major active metabolite (Chytil et al. 2009). Venlafaxine (1-[2-(dimethylamino)-1-(4-methoxyphenyl)ethyl]cyclohexanol (VEN)), marketed by Pfizer as Effexor®, represents a new form of antidepressants which have the ability to affect the uptake of the neurotransmitters serotonin and norepinephrine. Its major active metabolite is O-desmethylvenlafaxine (ODV), a demethyl form of the parent compound.
In recent studies LDC, TRA, and VEN have been detected in effluents from WWTPs and in some rivers and lakes in both Europe and North America. However, there is only a limited data available about the reduction of these compounds and their metabolites in the WWTPs. A report from Eawag (German acronym for the Swiss Federal Institute of Aquatic Science and Technology) indicates the presence of LDC, TRA, and VEN at various locations in Lake Constance, Switzerland, at concentrations of 1, 9, and 2 ng L−1, respectively (Eawag 2009). Kasprzyk-Hordern et al. (2008) detected TRA at concentrations up to 7,000 ng L−1 in rivers of South-Wales, UK, impacted by treated wastewater discharges. Lajaunesse et al. (2008) reported the occurrence of VEN and its demethylated metabolite in municipal wastewater and in recipient waters in Montreal, Canada. More recently, VEN has been identified in various surface waters samples in Colorado, USA, at concentrations ranging between 102 and 690 ng L−1 (Schultz et al. 2010). Moreover, VEN and ODV were shown to be present in influents and effluents from WWTPs in southern Ontario, Canada, with effluent concentrations up to 800 (VEN) and 1,600 ng L−1 (ODV). The removal rates for both compounds in the WWTP were about 40% (Metcalfe et al. 2010).
The aim of the present study was to investigate the distribution and behavior of LDC, TRA, VEN, and the dominating metabolites in three WWTPs in Hesse, Germany. Daily samples from influents and effluents from the WWTPs were collected and analyzed for LDC, 2,6-DMA, MEGX, TRA, ODT, VEN, and ODV using solid-phase extraction (SPE) followed by gas chromatography–mass spectrometry (GC-MS). Moreover, solid samples obtained from the micro-screen rotating drum filter system (MRF) of one WWTP were extracted by sonication, in order to analyze the possible absorption of the analytes in particulate matter.
LDC, 2,6-DMA, TRA hydrochloride, VEN hydrochloride, and the internal standard squalane were purchased from Sigma-Aldrich (Steinheim, Germany). MEGX was kindly supplied by AstraZeneca (Wedel, Germany). ODT, ODV, and the internal standard d6-TRA were provided by Toronto Research Chemical Inc. (Ontario, Canada). Individual stock solutions (1 μg μL−1) of target compounds and of the internal standard d6-TRA were prepared in methanol. The stock solution of the internal standard squalane (1 μg μL−1) was prepared in hexane. Working standards of the analytes were prepared from these stock solutions by dilution with methanol. All reported concentrations of target compounds and internal standards are concentrations of the hydrochloride-free analytes.
Acetone was obtained from LS Labor-Service (Griesheim, Germany). All other organic solvents were analytical grade (Carl Roth, Karlsruhe, Germany) and were distilled before use. Ultrapure water was produced from de-ionized water using an Astacus ultrapure water purification system (MembraPure, Bodenheim, Germany).
2.2 Study sites and sample collection
Characteristics and operating conditions of the WWTPs sampled for influent and effluent
20 Jan 2010–02 Mar 2010 and 16 Sept 2010–23 Sept 2010
20 Jan 2010–26 Feb 2010
23 Feb 2010–09 Apr 2010
Population served (PE)
Average flow (m3 day−1)
68% R and 32% I
62% R and 38% I
94% R and 6% I
1 pharmaceutical lab
12 specialist clinics
1 nursing home
5 nursing homes
6 nursing homes
The wastewater samples were collected using refrigerated autosamplers. Defined volumes of influent and effluent samples were collected every 60 min to obtain a composite sample over a 24-h period. In WWTP2, the volume collected every 60 min was proportional to the flow rates at a collection time on the influent and on the effluent, respectively. In WWTP1 and WWTP3, the sample volume was the same every 60 min. The water samples were collected in 1-L brown glass bottles and stored cooled at 4°C in the dark until processing in laboratory within 7 days after sampling. The bottles were cleaned before use with a laboratory glassware washer G 7835 CD (Miele, Gütersloh, Germany), then flushed with distilled methanol and dried by heating to 110°C for a minimum of 1 h.
In order to study the variation of the concentrations of the analytes in the influent during a 24-h period, composite samples were collected on 18 Mar 2010 every 2 h from one sampling point on the sewerage system of WWTP2 using a refrigerated autosampler. This sample point receives residential wastewater from a population of about 4,000 inhabitants.
2.3 Sample preparation
SPE was carried out using PPL Bond Elute extraction cartridges (1 mL, Varian, Darmstadt, Germany). Extraction volumes were 250 and 500 mL for sewage influent and effluent, respectively. Insoluble components and suspended particulate matter were removed by filtering the samples through a 1-μm borosilicate glass fiber filter (Type A/E, Pall, Dreieich, Germany). The filters were pre-washed with dichloromethane and then heated in an oven for 2 h at 400°C. Before sample extraction, the solid-phase adsorbent was conditioned with 1 mL methanol, 1 mL methanol/acetone (1/1, v/v), and 1 mL distilled water. The samples were introduced to the cartridges by means of PTFE tubes at low pressure (approximately 750 mbar). After sample loading, the SPE cartridges were dried in a nitrogen stream and then eluted three times with 333 μL methanol/acetone (1/1, v/v). The extracts were dried and then dissolved in 400-μL methanol. Subsequently, 2-μg squalane and 2-μg d6-TRA were added to each sample as internal quantification standards.
The samples from the MRF (90% liquid and 10% solid) were first centrifuged at 2,500 rpm for 30 min. The liquid fractions were prepared for analysis using SPE. The solid fraction was freeze-dried using a lyophilization apparatus Christ Alpha I-5 (Martin Christ, Osterode, Germany) for 24 h. Due to the small amount of solid fraction per sample, all fractions were combined and analyzed as one. The solid fraction was extracted three times with 20 mL methanol/dichloromethane (1/1, v/v) using sonication. The sample was immersed in an ultrasound cleaning bath SONOREX SUPER RK 510 H (Bandelin, Berlin, Germany) at a frequency of 35 kHz for 15 min. After the extractions, the sample was centrifuged at 2,500 rpm for 5 min, and then the liquid extracts were combined and evaporated to dryness under vacuum at room temperature. The residue was dissolved in 400-μL methanol; 2-μg squalane and 2-μg d6-TRA were added to each sample as internal quantification standards.
2.4 GC-MS analysis
The samples were analyzed by a Trace GC Ultra gas chromatograph coupled to an ion trap mass spectrometer ITQ 900 (Thermo Scientific, Dreieich, Germany). The gas chromatograph was equipped with a TG-5MS capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness, Thermo Scientific, Dreieich, Germany). Helium (≥99.999%) was used as the carrier gas at a constant flow of 1.1 mL min−1. The initial column oven temperature (80°C) was increased at 4°C min−1 to a final temperature of 300°C, which was maintained for 30 min. The sample solution (1 μL) was injected using a Tripluss auto sampler in the splitless mode at an injector temperature of 240°C. The mass spectrometer was operated in electron impact mode with 70 eV ionization energy, and full scan spectra were collected in the range from m/z 50 to 650.
Main electron impact ionization fragment masses used for identification of the substances and their metabolites
Fragment masses (m/z)
58, 86, 120, 234
77, 91, 106, 120, 121
58, 77, 106,120, 163
58, 77, 135, 263
58, 77, 114, 121, 249
58, 91, 119, 134, 179
58, 77, 91, 120
2.5 Determination of recoveries
To determine recovery rates over the instrument (GC-MS), 1-L surface water samples (n = 6) were extracted by SPE and the extracts were spiked with the analytes and the internal standards. Extraction procedure by SPE enrichment and measurements by GC-MS were performed as described in Sections 2.3 and 2.4. The recoveries were determined in comparison to a non-enriched standard solution providing values of 86 ± 6% (LDC), 54 ± 5% (2,6-DMA), 64 ± 10% (MEGX), 86 ± 8% (TRA), 95 ± 7% (VEN), 85 ± 10% (ODT), and 95 ± 3% (ODV). The spiked extracts were placed in a fume hood for 24 h at room temperature and then assessed again. Except for 2,6-DMA, no noteworthy differences in the peak areas were found. For 2,6-DMA, the observed peak area in the spiked extracts remeasured was 98 ± 1% lower than the peak area observed in the spiked extracts measured initially. Thus, the metabolite of lidocaine 2,6-DMA appears to be unstable at room temperature and cannot be identified in water samples using this method (because the compound is degraded before analysis).
In order to determine recovery rates over the total method, 1-L surface water samples (n = 6), 1-L treated wastewater samples (n = 6), and 0.2-L untreated wastewater samples (n = 6) were spiked before extraction with the analytes. SPE enrichment and detection by GC-MS were performed as described above. Mean recoveries in surface water were: 70 ± 6% (LDC), 61 ± 3% (MEGX), 73 ± 6% (TRA), 95 ± 8% (VEN), 60 ± 9% (ODT), and 96 ± 3% (ODV); in treated wastewater (n = 6): 65 ± 8% (LDC), 52 ± 8% (MEGX), 62 ± 5% (TRA), 81 ± 3% (VEN), 56 ± 4% (ODT), and 85 ± 3% (ODV); and in untreated wastewater: 56 ± 4% (LDC), 49 ± 9% (MEGX), 61 ± 6% (TRA), 79 ± 6% (VEN), 51 ± 9% (ODT) and 71 ± 11% (ODV).
Recoveries of LDC, TRA, and ODT calculated over the instrument are higher than the recoveries calculated over the total method, indicating that the extraction procedure could be enhanced using another SPE material. For MEGX, the recoveries calculated over the instrument showed no appreciable difference to the recoveries calculated over the total method. Thus, matrix-induced signal suppression appears to be responsible for the loss of MEGX by GC-MS.
Recovery of d6-TRA was calculated over the total method in surface water samples (n = 6) resulting in a value of 55 ± 4%. Because recoveries of the analytes exceeded significantly the recovery of d6-TRA, the use of d6-TRA as surrogate standard would lead to an overestimation. For this reason, this compound was used as internal standard and was added after SPE.
In order to calculate recovery rates of the analytes from solid fractions, 0.5-g freeze-dried activated sludge samples (n = 6) were spiked with the analytes and then stirred to spread over the spiking solution. The spiked samples were placed in a fume hood for 24 h at room temperature. Extraction procedures were performed as described in Section 2.3 for solid fractions. The recoveries were determined in relation to a non-enriched standard solution. Mean recoveries of the analytes in activated sludge were 85 ± 13% (LDC), 60 ± 5% (MEGX), 73 ± 9% (TRA), 83 ± 14% (VEN), 61 ± 7% (ODT), and 76 ± 9% (ODV), at a spiking level of 200 ng g−1.
2.6 Limit of quantification and calibration
Calibration curves for each substance in water samples were prepared by spiking 1-L of ultrapure water (n = 10) with various amounts of stock solutions of target compounds to yield final concentrations between 1 and 2,000 ng L−1. The samples were extracted and analyzed in the same way as field samples. Blank samples were analyzed and the target analytes were not detected. The limit of detection (LOD) of each analyte was calculated in accordance with the German standard method DIN 32645 German Institute for Standardization (1994), with a confidence interval of 99% using the standard deviation of the linear regression curve. The limit of quantification (LOQ) was estimated as three times the LOD and provided 15 (LDC), 48 (MEGX), 26 (TRA), 18 (VEN), 40 (ODT), and 28 ng L−1 (ODV).
For the activated sludge samples, a six-point calibration was used ranging from 1 to 200 ng g−1. LOQs were calculated as three times the LODs in accordance to DIN 32645 (1994) providing values of 26 (LDC), 63 (MEGX), 35 (TRA), 25 (VEN), 61 (ODT), and 39 ng g−1 (ODV).
3 Results and discussion
3.1 Analytes in wastewater
The measured concentrations of TRA, VEN, and ODV in wastewater from the investigated WWTPs are consistent with other studies conducted in Europe and North America. Lajaunesse et al. (2008) reported the presence of VEN and ODV in untreated wastewater in Canada at similar concentrations. Metcalfe et al. (2010) and Schultz et al. (2010) reported much higher concentrations of VEN and ODV in treated wastewater of WWTPs in North America (>1,000 and >2,500 ng L−1, respectively). The lower concentrations of the compounds in samples from WWTP1, WWTP2 and WWTP3 may be explained by higher consumption of pharmaceuticals in North America, different sewage collection systems or variations in rainfall rates. According to the weather station of the German Meteorological Service at the Frankfurt Airport (DWD German Meteorological Service 2011), irregular mild raining events occurred during the first sample collection (between 20 Jan 2010 and 09 Apr 2010). All of the investigated WWTPs use a sewage collection system through which wastewater and rain water are mixed before treatment. Thus, concentrations of the pharmaceuticals and their metabolites could have been slightly diluted by rainfalls. Hummel et al. (2006) found TRA in both influents and effluents of German WWTPs at concentrations of 1,500 and 610 ng L−1, respectively. However, the data obtained in the present study from WWTP1, WWTP2, and WWTP3 indicate that the demethylated metabolite ODT has also an environmental relevance in wastewater.
Concentrations were generally higher at the WWTP1 influent while WWTP3 had significantly lower concentrations than the other WWTPs. These results may be explained by the potential sources of the respective pharmaceuticals in each WWTP (Table 2). The number of entities associated with the use of pharmaceuticals, e.g., hospitals and laboratories, is greater in the catchment area of the WWTP1 than in the catchment areas of WWTP2 and WWTP3. Thus, the more entities related with the use of pharmaceuticals in the catchment area of a WWTP, the higher the entry of the pharmaceuticals into the sewerage system.
The concentration data and the information about effluent flows have been used to calculate the loads of analytes which are daily discharged into the respective recipient waters. The loads ranged between 0.9 and 8.8 g day−1 for LDC, 1.8 and 18.3 g day−1 for TRA, 0.6 and 20.9 g day−1 for ODT, 0.5 and 6.1 g day−1 for VEN, and 2.2 and 38.7 g day−1 for ODV. These values confirm WWTPs as a major input source of the investigated analytes and their metabolites into surface waters.
Mean concentrations (ng L−1) of the target analytes in the samples of wastewater recirculated by the MRF
Liquid fraction (n = 3)
3.2 Concentration profiles from 24-h sampling
3.3 Parent compounds and their metabolites
Calculated ratios between the concentrations of the metabolites and the parent substances measured in WWTPs influents and effluents
WWTP1 (n = 74)
WWTP2 (n = 66)
WWTP3 (n = 82)
For each WWTP, the ODT/TRA ratios calculated in influents are similar to the ratios calculated in effluents. The ODV/VEN ratios are slightly higher in the effluents compared to the influents of all three WWTPs. In WWTP1 the ratio increased from 5.8 to 6.3, in WWTP2 from 5.3 to 5.9 and in WWTP3 from 2.9 to 4.2. Given that in all investigated WWTP the removal rates of the metabolites are lower than the removal rates of their respective parent compound, a small increase of the ratios calculated in the effluents in comparison with the ratios calculated in the influents can be expected. Only a very high increase in the ratios calculated in the effluents with respect to the ratios calculated in the influents would imply further degradation of the parent compound in the WWTP, which is not the case for the analyzed pharmaceutical compounds. Thus, ODT and ODV are metabolized only in the liver and are not formed through the cleaning processes carried out at the investigated WWTPs.
The results of the study show that LDC, TRA, VEN, and the metabolites ODT and ODV are present in both influents and effluents from three WWTPs in Hesse, Germany. The concentration of analytes in influents differed between the plants, which may be explained by the diverse pharmaceuticals sources in each WWTP. As expected for pharmaceutical compounds, remarkable variations in concentrations in the influent of a WWTP during a 24-h period are found. LDC, TRA, VEN, ODT, and ODV are only partially removed trough cleaning processes at the investigated WWTPs and thus are continuously discharged into the respective recipient waters. The recirculation of treated wastewater to the primary clarifier appears to have a positive effect on the removal process of some analytes. The results indicate that during the treatment of wastewater in the WWTPs both parent compounds and metabolites are degraded. An enrichment of metabolites in relation to the parent compounds TRA and VEN is not observed. 2,6-DMA appears to be unstable in wastewater and could not be identified. MEGX was not detected in any effluent or influent samples. It is probably excreted at very low concentrations and/or degraded rapidly in the sewerage system. Further work is needed to determine the distribution of the investigated compounds and their metabolites in surface water, groundwater, and drinking water.
The first author thanks Deutscher Akademischer Austauschdienst and particularly Katholischer Akademischer Ausländerdienst for financial support. The support of colleagues and staff of the Institute of Atmospheric and Environmental Sciences at the J.W. Goethe University Frankfurt am Main is gratefully acknowledged. Thanks are due to the staff from the three WWTPs for support with sample collection.