Target Analysis, Suspected-Target, and Non-Target Screening for

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 17, 2016 | http://pubs.acs.org Publication Date (Web): December 12, 2016 | doi: 10.1021/bk-2016-1242.ch002

Chapter 2

Target Analysis, Suspected-Target, and Non-Target Screening for Evaluation and Comparison of Full-Scale Ozonation at Three Wastewater Treatment Plants Jochen Tuerk,*,1,2 Andrea Boergers,1 Juri Leonhardt,1 Christoph Portner,1,3 Linda Gehrmann,1 and Thorsten Teutenberg1 1Institut

für Energie- und Umwelttechnik e. V. (IUTA, Institute of Energy and Environmental Technology), Bliersheimer Straße 58-60, 47229 Duisburg, Germany 2Centre for Water and Environmental Research (ZWU), University of Duisburg-Essen, Universitätsstraße 2, 45117 Essen, Germany 3Current address: Tauw GmbH, Richard-Loechel-Str. 9, 47441 Moers, Germany *E-mail: [email protected]. Phone: +49 2065 418 179.

The entry of micropollutants into the water cycle is of growing concern. This is also reflected by the EU Water Framework Directive and new guidelines for wastewater treatment plant effluents. Ozonation seems to be a good answer to this problem. The wastewater treatment plants Bad Sassendorf, Duisburg-Vierlinden and Schwerte were extended by a full-scale ozonation. Removal efficiency and process optimization were assessed by target analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). Suspected-target and non-target screening using high resolution mass spectrometry (HRMS) were applied for chemical evaluation of transformation products and comparison of the three wastewater treatment plants. Data evaluation was done using an in-house created reference database as well as commercially and freely available databases. The different workflows are exemplarily discussed and a new concept using microscale two-dimensional liquid chromatography coupled to high resolution mass spectrometry is presented. © 2016 American Chemical Society

Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Introduction Due to extensive use of relevant amounts of micropollutants (e. g. pharmaceuticals, iodinated contrast media, pesticides, personal care products) and their transformation products can be detected in effluents of wastewater treatment plants (WWTP) and surface waters up to µg/L concentrations. The impact of environmentally relevant micropollutants on human health and aquatic organisms has been insufficiently studied. However, within the implementation of the EU Water Framework Directive (WFD), which forces a good chemical and biological status for all surface waters (2000/60/EC) (1), WWTP effluents as point sources become increasingly important. In 2013 a “watch-list” was introduced (2013/39/EU) and updated in 2015 (EU 2015/495) (2, 3). Besides diclofenac (analgesic) and the endocrine disrupting compounds 17ß-estradiol and 17α-ethinylestradiol (natural and synthetic hormone) some macrolide antibiotics, neonicotionoids and estrone were added to the watch-list. Contamination of the aquatic environment with toxic substances is considered as a major factor concerning the unsatisfactory ecological status of many rivers (4). Common wastewater treatment plants are not able to remove these substances, adequately. The question whether it is necessary to completely eliminate micropollutants from this point source is still under discussion. In several research studies it could be demonstrated that ozonation and the application of activated carbon are promising technologies for the elimination of the micropollutants. Against this background and in the context of the North-Rhine-Westphalian research project “Elimination of pharmaceutical residues in municipal wastewater treatment plants” ozonation as an advanced treatment for the removal of micropollutants was implemented on a large scale and tested in practical operation (5). The use of ozone as an oxidizing agent for the elimination of micropollutants at wastewater treatment plant effluents is a relatively new approach to reduce the emission of such substances for improving the water quality (6). However, using economically efficient operation conditions the oxidation generally does not result in a complete mineralization of organic substances but rather leads to partially oxidized transformation products (7–10). In the context of the joint research project “Study of metabolite formation during the use of ozone in municipal wastewater treatment plants“ the question was addressed, whether oxidation by-products which exhibit ecotoxicological or human toxicological effects are formed during ozonation (11). An important part of this project was the combination of suspected-target and non-target screening with an effect-based approach to assess the combined toxicity of transformation products formed by the ozonation in real wastewater on three large-scale sewage plants differing in their catchment areas. The ozonation was hereby incorporated in the conventional process of municipal wastewater treatment. Ozonation leads to two different oxidation mechanisms. The first one is the direct, specific and slow reaction of the compounds with ozone (O3). The second is the fast and unspecific reaction of the micropollutants with hydroxyl radicals (12). These radicals are formed by the reaction of ozone with the wastewater matrix. Both reaction pathways result in the formation of partially oxidized products. In the following these transformation products (TP) are named as oxidation by-products indicating the origin of the 30 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


formed compounds.. In contrast to this notation transformation products from biological processes are named metabolites. Metabolites are formed in the human body during the metabolism of active pharmaceutical ingredients or as product of the biological waste water treatment. Besides classical target analyses of 156 micropollutants using GC-MS and LC-MS/MS a suspected-target screening and a non-target screening approach were performed. For process optimization and routine monitoring of the different WWTP effluents 12 representative substances were selected. Seven of them were analyzed using LC-MS/MS and five using GC-MS. Most of the substances were typically detectable in WWTP effluents in high concentration. For the evaluation of the ozonation six substances analyzed using LC-MS were selected. These substances were the corrosion inhibitor 1H-benzotriazole, the anti-epileptic carbamazepine, the analgesic diclofenac, the beta-blocker metoprolol, the antibiotic sulfamethoxazole and the contrast media diatrizoic acid. Melperone, a psychotropic drug, which is also analyzed by LC-MS/MS was not taken into consideration because of the very low concentrations in WWTP effluents. Substances analyzed using GC-MS were the two musk fragrances galaxolide and tonalide, the flame retardant tris(2-chloroethyl) phosphate (TCEP), the complexing agent ethylenediaminetetraacetic acid (EDTA) and the plasticizer bisphenol A. For suspected-target screening a high resolution mass spectrometer (HRMS) was used for measurement of the exact mass. Taking the isotopic pattern into consideration, the most probable sum formula could be calculated. For identification of suspects a comparison of the molecular formula and if available the retention time as well as the fragmentation pattern was done using different databases. Non-target screening was used for characterization and comparison of the different wastewater samples of the three investigated WWTPs equipped with an ozonation as tertiary treatment step for micropollutant removal. Statistical differences and cluster analysis were performed by means of principle component analysis (PCA). As required for further characterization of the detected features in silico methods were used for comparison of calculated and measured fragmentation pattern.

Full Scale Ozonation Wastewater ozonation for micropollutant removal is usually located downstream of the final clarification. On the basis of the current level of knowledge a biological post-treatment is demanded for the treatment of the formed TP (13). For an efficient usage of ozone, low organic carbon content and an efficient and reliable nitrification are required, because residual nitrite and the organic matrix can both react with ozone. Dosage of ozone is calculated by the specific concentration zspez, which describes the relation of consumed ozone (ingas concentration minus offgas concentration) to the dissolved organic carbon (DOC). Abegglen and Siegrist showed that for the removal of 80% of the micropollutants a specific ozone dosage of zspez = 0.7 – 0.9 mgO3/mgDOC is sufficient (14). Ozone can be added into the aqueous phase by diffusers or by a 31


ventury-injection system. Using the injection system, ozone is introduced to the water phase by a reduction of the fluid pressure, caused by a constricted section of the fluid pipeline. The three different WWTP with the different polishing steps are described subsequently.


Short Description of the Three Full-Scale Advanced Treatment Procedures The mechanical-biological treatment plant Bad Sassendorf (BS) has a design capacity of approximately 12,000 population equivalents. The catchment area of the treatment plant consists of six hospitals and health clinics. The mean age of the inhabitants and especially of the resort guests is high. At the WWTP Bad Sassendorf a full scale ozonation with a reaction basin of 65 m3 was built to treat the WWTP effluent (15). Ceramic diffusers are used for the application of ozone to the reaction tank with residence times of about 13 minutes. The maximum dry weather flow (Qmax) is 300 m3/h. The biological post-treatment is done by a polishing pond with a volume of 8,400 m³. A general sketch is given in Figure 1, a. The WWTP Duisburg-Vierlinden (DU) is also equipped with a full scale ozonation after the final clarification (Figure 1, b). It has a design capacity of 30,000 population equivalents and the wastewater is almost municipal. The characteristic of this system was a two-line structure with two different ozone entry systems at each reaction basin (V = 100 m3, hydraulic retention time (HRT) = 30 min, Qmax = 400 m3/h, water depth = 5 m). The two different systems were pursued separately and a comparison of the entry systems was possible. The first system was a ventury-injection with a fluidized bed for biological post-treatment. Ozone gas with a maximum gas flow of 13.4 m³/h was introduced into a partial water flow of 17 m³/h by an injector. A static mixer merged the partial water flow with the influent of the ozonation (maximum of 100 m³/h) The water was pumped through the reaction basin. After the reaction basin the biological post treatment was located in the same basin separated by a wall with a perforated plate at the bottom. Polypropylene growth bodies with a specific surface of 750 m²/m³ and a density of 1 kg/m² were added for biofilm formation. The second line used ceramic diffusers. The ozone was injected through 14 diffusers with a diameter of 17.8 cm (16). Permanent operation of the full-scale ozonation at WWTPs DuisburgVierlinden and Bad Sassendorf was done by flow proportional ozone dosages. The third WWTP with an advanced treatment step is the WWTP Schwerte (S) with a total design capacity of 50,000 population equivalents. The wastewater contains municipal and industrial wastewater. The characteristic of the WWTP Schwerte are two completely separated lines for biological wastewater treatment. To compare an advanced treatment with a conventional wastewater treatment the tertiary treatment procedures were equipped at the WWTP Schwerte only at one line. The research plant consists of an ozonation followed by a powdered activated carbon (PAC) unit. The two systems can be operated either separately or in a combination of both. A special characteristic is the possibility to do the advanced treatment after the final clarification, or the treatment by “dynamic recirculation”. Operation in the recirculation mode is done by treating the effluent 32 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of the WWTP with ozone and/or PAC and recirculate the advanced treated wastewater into the biological treatment step of the WWTP (see Figure 1, c). For the oxidative treatment six reactors with a total volume of 192 m3 were equipped with ceramic diffusers at the first and third reactor. The total dry weather capacity is 900 m3/h with a hydraulic retention time of 12 minutes (17). The adsorption step is done in three reaction tanks, which were not used during this study. The hydraulic capacity of the final clarification is the limitation for the recalculated partial flow. Therefore, the recirculation rate changes dynamically with the inflow of the WWTP.

Figure 1. Schematic overview of the three WWTPs containing catchment area, biological treatment and final clarification equipped with an advanced treatment step for micropollutant removal. a) Bad Sassendorf, b) Duisburg-Vierlinden, c) Schwerte.

Materials and Methods Target Analysis Micropollutant analysis was performed after solid phase extraction (SPE) by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). Sample clean-up and enrichment was done by solid phase extraction (SPE) using comparable polymeric sorbents (200 mg Strata XL, Phenomenex, Aschaffenburg, Germany and 150 mg Oasis HLB, Waters, Eschborn, Germany). A 150 mm x 2.1 mm Synergi 4u Polar-RP 80A column (Phenomenex, Aschaffenburg, Germany) was used for the chromatographic separation. A flow rate of 400 µL/min was adjusted. The mobile phase consists of 0.1% formic acid in water (%A) and methanol (%B). The column oven was set to 30 °C. The detection was done using an API 3000 tandem mass spectrometer (Sciex, Darmstadt, Germany). The 33



analysis of iodinated contrast media was done after enrichment on a 6 mL, 200 mg Isolute ENV+ SPE cartridge (Biotage, Uppsala, Sweden) with LC-MS/MS. The chromatographic separation was done on a Synergi 4µ Hydro HPLC column (Phenomenex, Aschaffenburg, Germany) followed by the detection on the API 3000. Besides the LC-MS/MS analysis further micropollutants and the two oxidation by-products bromate and N-nitrosodimethylamine (NDMA) were quantified by ion chromatography and gas chromatography mass spectrometry (data not shown).

Suspected-Target and Non-Target Screening For the suspected-target and non-target screening approach the samples were analyzed as native sample after filtration using a 0.45 µm cellulose filter and after enrichment by a factor of 1,000 using solid phase extraction (6 mL, 150 mg Oasis HLB, Waters, Eschborn, Germany). The native and enriched samples were measured together with blanks and a reference substance mixture. For chromatographic separation an ultra-high pressure system (Thermo Scientific Aria Transcend) was used. A sample volume of 60 µL was injected onto a 100 x 2.1 mm analytic column (Thermo Scientific Hypersil Gold aQ). A 7 minute solvent gradient (eluent A: water + 0.1% formic acid, B: methanol + 0.1% formic acid) from 5-99% B was applied resulting in a total cycle time of 15 minutes for chromatographic separation at a flow rate of 400 µL/min. The column oven was set to 25 °C. For mass spectrometric detection an Exactive Plus and a Q-Exactive (Thermo-Fisher Scientific, Bremen, Germany) HRMS with positive and negative electrospray ionization were used. Full scan and all ion fragmentation (AIF) were performed in permanently alternated mode with the Orbitrap and data independent acquisition (DIA) with the quadrupole Orbitrap system. A resolution setting of 70.000 (FWHM at m/z 200) was used. A mass range of m/z 100 to 1,500 was applied to detect a high number of possible contaminants. The mass axis of the system was calibrated with the standard calibration mix once prior to each measurement. The data were analyzed in a widely automated workflow using TraceFinder 3.1, SIEVE 2.1 and Compound Discoverer 1.0 software (Thermo-Fisher Scientific, Bremen, Germany). For identification of detected sum formulas a database search using Chemspider and a self-created transformation products database (IGF database) were applied (15). For further identification and plausibility check mzCloud, Norman Massbank and Stoff-Ident were used. DAIOS, Drugbank, HMDB and Metlin were only tested for evaluation of the workflows. In addition a data evaluation by annotation of possible and detected transformation products (metabolites from the biological wastewater treatment as well as oxidation by-products from the ozonation) with vendor software and the public databases Metfusion was tested for further characterization of detected features and plausibility checking. 34



2D-LC-QTOF Measurements The conventional 1D-LC separations were performed on an Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany). The separation was carried out on a Luna C18(2) column (150 mm x 2.0 mm, 3 µm particles, Phenomenex, Aschaffenburg, Germany). The injection volume was 20 µL. The flow rate was 200 µL/min and the oven temperature was set to 30 °C. The mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile (eluent B). The 2D-LC separations were performed on an Eksigent NanoLC-Ultra 2D pump system (Sciex, Dublin, CA). A 50 mm x 0.1 mm, 5 µm Hypercarb column (Thermo-Fisher Scientific, Dreieich, Germany) was used for the first dimension (D1) separation with a flow rate of 200 nL/min and an oven temperature of 60 °C for the first and second dimension (D2). The mobile phase consisted of 0.1% formic acid in water (mobile phase A) and methanol (B). The injection volume on the D1 column was 1.57 µL. A solvent gradient was applied according to the following program: 8 min hold at 1% B, in 45 min 1-99% B, 35 min hold at 99% B, in 5 min 99-1% B. For the second dimension separation a superficially porous 2.6 µm SunShell C18 particle (ChromaNik Technologies, Osaka, Japan), packed by Grace Davison (Worms, Germany) into a 50 mm x 0.3 mm hardware, was used. The flow rate was 40 µL/min. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). A solvent gradient was applied according to the following program: 3-97% B in 0.5 min, 0.1 min hold at 97% B, in 0.1 min 97-3% B, re-equilibration at 3% B for 0.3 min. The complete gradient cycle time took 1 min and was usually repeated without flow-stop until the end of the D1 program. For the mass spectrometric detection a TripleTOF 5600 hybrid HRMS system (Sciex, Darmstadt, Germany) with a DuoSpray ion source and a TurboIonSpray probe for ESI experiments was used. For the 1D-LC experiments with flow rates of 200 µL/min, the standard probe was used. This standard electrospray emitter tip with an inner diameter (ID) of 130 µm was replaced by an emitter with an ID of 50 µm for all 2D-LC experiments. MS data acquisition was controlled by Sciex Analyst TF 1.5.1 and the data were analyzed using Sciex PeakView 1.2.0.3 and MultiQuant 2.1.1742.0. The data acquired by the 2D-LC approach had to be manually evaluated due to the lack of commercially available 2D software packages. A suspected-target screening approach with information dependent acquisition was performed to obtain additional structural information. All measurements were performed in positive electrospray ionization (ESI +) mode. More detailed method information can be found in Leonhardt et al. (2015) (18).

Target Analysis - Results and Data Assessments Assessment Criteria The discussed environmental quality standards (EQS) for surface waters in context of the EU Water Framework Directive (2, 3) were used for the evaluation. The concept for the evaluation is shown in Table 1. 35 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Table 1. Assessment Criteria for the Concentrations of the Micropollutants in Surface and Wastewater c

< ½ EQS

½ - 1 EQS

1 - 2 EQS

2 - 4 EQS

> 4 EQS

Assessment

Excellent

good

moderate

unsatisfactory

poor

The water quality is often expressed by colors: excellent = blue, good = green, moderate = yellow, unsatisfactory = orange and poor = red. Because of nonexistent values for WWTP effluents, in the federal state of North-Rhine Westphalia EQS for surface waters are often used for the evaluation of WWTP effluents on a precautionary basis. The reason for this approach is a huge amount of drinking water production from surface waters. Beside this hard criterion also the consideration of wastewater amount respectively the dilution of the WWTP effluent in the receiving water for the calculation of quality criteria is still under discussion. EQS taken into consideration were derived from the oekotoxzentrum, Switzerland (19). For most of the micropollutants no legal EQS were available. Predicted no effect concentrations (PNEC) could be taken into consideration, too. For several micropollutants a precautionary value (PV) of 0.1 µg/L is suggested in NRW, Germany. The EQS, PNEC and PV values for assessment of monitoring results are given in Table 2.

Table 2. EQS, PNEC, and PV Values for Surface Waters Substance

Unit

EQS

PNEC

PV

Diatrizoic acid

µg/L

n.a.

11,000

0.1

1H-Benzotriazole

µg/L

30

30

0.1

Carbamazepine

µg/L

0.5

2.5

0.1

Diclofenac

µg/L

0.05

0.05

0.1

Metoprolol

µg/L

64

3.2

0.1

Sulfamethoxazole

µg/L

0.6

0.59

0.1

The EQS was set in relation to the concentration of the micropollutants of the water. The assessment reflects the chemical state of the water body or the WWTP effluent, respectively. In Switzerland a different approach for the assessment of the cleaning capacity of the tertiary treatment was realized. The cleaning capacity was reviewed by 12 chosen substances. They are divided into two groups. The first group were “excellent eliminable” and the second group “good eliminable” substances. The cleaning of the wastewater was sufficient if six of the 12 substances were eliminated by 80% in average for each sampling campaign. Four 36 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of the substances had to be first group substances and two of the substances had to be second group substances (20). A similar approach for the evaluation of tertiary treated WWTP effluent with the requirement of 80% elimination will be probably established also in NRW. In opposite to the Swiss approach, the micropollutants were not divided into two groups. At the moment following six compounds were chosen: 1H-benzotriazole, carbamazepine, diclofenac, metoprolol, clarithromycin and sulfamethoxazole. The needed elimination rate of 80% has to be reached in annual average. Results of the Target Analysis The mean effluent concentrations of the WWTP without an additional treatment step are given in Table 3 together with the number of analyzed samples and the discussed EQS. The EQS were exceeded for the anti-epileptic carbamazepine, the analgesic diclofenac and for the antibiotic sulfamethoxazole. The assessment was done using the average value. The standard deviation was not used for the assessment.

Table 3. Concentrations (µg/L) of the Selected Micropollutants in the Effluents of the Different WWTP and Assessment of the Concentrations in Relation to the EQS WWTP

Substance

Bad Sassendorf

DuisburgVierlinden

Schwerte

n = 46

n = 19

n = 13

0.62 ± 0.71

2.3 ± 1.4

13 ± 5.2

EQS

Diatrizoic acid 1H-Benzotriazole

30

3.1 ± 1.7

3.2 ± 1.9

5.4 ± 0.85

Carbamazepine

0.5

0.71 ± 0.27

1.8 ± 0.61

0.88 ± 0.50

Diclofenac

0.05

3.3 ± 1.5

2.1 ± 0.70

2.5 ± 0.55

Metoprolol

64

2.4 ± 1.4

1.5 ± 1.1

2.7 ± 1.1

Sulfamethoxazole

0.6

0.79 ± 0.82

0.95 ± 0.41

1.1 ± 0.3

Because of the different treatment at the WWTP Schwerte, only the results of the WWTP Bad Sassendorf and Duisburg-Vierlinden were described in table 4. By equipping the WWTP with an additional treatment process the effluent concentrations were reduced. Ozonation was done with different specific ozone dosages. The results were split into zspez up to 0.5 mgO3/mgDOC, 0.5 - 0.7 mgO3/mgDOC; 0.7 - 0.9 mgO3/mgDOC and above 0.9 mgO3/mgDOC. The effluent concentrations after ozonation of selected micropollutants were given in Table 4. 37 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 4. Mean Concentrations (µg/L) of the Selected Micropollutants after Ozonation at the WWTP Bad Sassendorf and Duisburg-Vierlinden with Different Specific Ozone Dosage zspez

< 0.5

0.5 - 0.7

0.7 - 0.9

> 0.9

16

15

9

23

Substance

c (µg/L)

c (µg/L)

c (µg/L)

c (µg/L)

Diatrizoic acid

2.1 ± 1.4

2.0 ± 1.3

0.55 ± 0.53

0.67 ± 0.86

1H-Benzotriazole

1.7 ± 0.76

0.68 ± 0.52

1.0 ± 1.2

0.75 ± 0.38

Carbamazepine

0.42 ± 0.44

0.056 ± 0.085

0.013 ± 0.015

0.014 ± 0.011

Diclofenac

0.44 ± 0.47

0.070 ± 0.067

0.12 ± 0.094

0.065 ± 0.071

Metoprolol

0.85 ± 0.30

0.29 ± 0.24

0.32 ± 0.49

0.17 ± 0.17

Sulfamethoxazole

0.20 ± 0.19

0.07 ± 0.041

0.022 ± 0.022

0.021 ± 0.015


n

The EQS for carbamazepine and sulfamethoxazole were no longer exceeded. The quality of the water can be improved up to “excellent” for carbamazepine and sulfamethoxazole. Without the tertiary treatment step the assessment was “moderate”. For diclofenac the EQS of 0.05 µg/L cannot be reached even by using high specific ozone dosages of more than 0.9 mgO3/mgDOC. The initial status is “poor” without an additional wastewater treatment. The additionally treated effluent quality is still “poor” for zspez < 0.5,”moderate” for zspez = 0.5 - 0.7 and > 0.9 and “unsatisfactory” for zspez = 0.7 - 0.9. After ozonation with zspez ≥ 0.5 mgO3/mgDOC only some values were above the EQS of 0.1 µg/L. The elimination rates for diclofenac were above 80% for zspez ≥ 0.3 mgO3/mgDOC. The elimination rates using different zspez for the selected micropollutants are given in Figure 2. It is shown that higher ozone dosages lead to a higher elimination of the substances 1H-benzotriazole and metoprolol. Carbamazepine, diclofenac and sulfamethoxazole can be eliminated even using lower specific ozone dosages. The elimination of the iodinated contrast media diatrizoic acid is not efficient, even when high ozone dosages are applied. The discussed elimination of 80% in average could be observed in single experiments using specific ozone dosages of approximately 0.5 mgO3/mgDOC. Because of the recirculation mode the results of the WWTP Schwerte were not directly comparable to the effluent concentrations of Bad Sassendorf and Duisburg-Vierlinden. A comparison to the non-treated effluent of the WWTP Schwerte showed reduced concentrations of the selected micropollutants. The concentrations were reduced by ozonation for 23% for diatrizoic acid, 60% for carbamazepine, 56% for diclofenac, 52% for metoprolol, 48% for sulfamethoxazole (n = 14 for the five compounds) and 35% for 1H-benzotriazole (n = 12). 38



Figure 2. Elimination of the selected micropollutants using different specific ozone dosages including minimum and maximum value, 25th and 75th quartile, median and mean value.

Suspected-Target Screening LC-HRMS data of WWTP influent, after final clarification, ozonation and biological post-treatment were measured in triplicate. Following criteria for a positive hit for the suspected-target screening were used for selecting the suspects from the infinite frame list: mass accuracy of 5 ppm, detection of the suspects at all replicates with a signal-to noise ratio above 3 (S/N > 3) and consideration of the isotopic distribution in a widely automated workflow using the vendor software packages. Table 5 summarizes the number of detected suspects from the in-house created IGF database (15). Compared to the high number of detected frames, about 12% in the ESI positive and about 4% in the ESI negative mode were confirmed. The detected suspects in all WWTPs were transformation products from the target substances carbamazepine, ciprofloxacin, diclofenac, metoprolol and bisphenol A. Further verification by the fragmentation pattern or comparison of the retention time was not performed, because no toxicological relevant effects to the investigated end points vitality, estrogenicity, mutagenicity, or chronic toxicity were observed in the samples after ozonation by using an effect-based approach. The water quality after ozonation was unchanged or improved (11). 39



Table 5. Number of Detected Suspects from the IGF Database after Treatment with a High Specific Ozone Dosage of zspez = 0.9 mgO3/mgDOC n (ESI+)

n (ESI-)

WWTP Bad Sassendorf

31

12

WWTP Duisburg-Vierlinden (line 1, injector)

34

13

WWTP Duisburg-Vierlinden (line 2, diffuser)

31

14

WWTP Schwerte

18

13

Besides the transformation products also several active pharmaceutical ingredients (API) were analyzed by the use of the vendor software (Tracefinder). Most of the APIs were also on the target list and therefore known. Depending on matrix effects and different ionization efficiency limit of detection of the suspected-target screening ranged between 0.1 and 2 µg/L. Venlafaxine, which was not on the target list, was detected by a suspected-target screening. The measured mass and the calculated sum formula were compared with Chemspider database. For m/z 278.21149 ([M+H]+ → C17H27NO2) the compound name venlafaxine was proposed as hit number one by more than 10,000 entries. This was also confirmed by the Stoff-Ident database, which revealed additional information about 4 metabolites. For improvement of the level of confidence these results were added to a quest in mzCloud for comparison of the fragmentation pattern. Further sample characterization was done by the vendor software packages Sieve and Metworks for identification of the metabolites. Under consideration of phase 1 metabolites from venlafaxine the metabolite N,N-didesmethylvenlafaxine (C15H23NO2, exact mass 249.172882 Da, detected [M+H]+ m/z 250.18022 → M = 249.17294 Da, Δppm = 0.25) was identified at the WWTP Duisburg-Vierlinden. The same metabolite was also found in the river Rhine by Ruff et al. (21). A source could not be located although we propose that WWTP effluents are a point source for the venlafaxine metabolites. We also tested this in silico transformation workflow for carbamazepine. The vendor software Compound Discoverer 1.0 annotates the phase 1 and 2 metabolites. In the influent of the WWTP Duisburg-Vierlinden i.e. the glucuronide metabolite was detected. After full scale ozonation TP250 (C15H10N2O2) and TP254 (C15H14N2O2) could be detected. Further verification of the two postulated transformation products from Ruff et al. (21) and our own measurements were done by comparison with an ozonated laboratory sample of the reference substance venlafaxine.

Non-Target Screening For screening of unknown compounds the elimination of background signals and insignificant peaks is a major issue. In true non-target screening, where no initial information about the analytes are available, automated peak detection and 40 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


spectra deconvolution algorithms are mandatory to reveal the typically detected several thousands of frames or features in a complex sample. The number of frames depends on the filter settings for peak finding and deconvolution. First step of visualization would be the comparison of the total ion current (TIC). In comparison to GC-MS screening analysis, the TIC of an LC-MS screening normally shows no significant peaks. Therefore, consideration of TICs is not useful and often m/z vs. retention time plots are shown to demonstrate the high number of frames. Instead of such non-specific visualization of a single sample a characterization of each treatment procedure could give a better overview. After blank subtraction the evaluation of the investigated samples from the WWTP Duisburg-Vierlinden was done by scatter-plot visualization (Figure 3). Using this volcano plot focusing on the peak list of formed compounds during the ozonation was possible.

Figure 3. Volcano plot of the samples “effluent after biological treatment” vs. “after ozonation” from WWTP Duisburg-Vierlinden at a specific ozone dosage of zspez = 0.5 mgO3/mgDOC.

Here, the intense investigation of the compounds formed during ozonation would normally start. As described at the suspected-target screening, we did not investigate these new peaks in detail, because no toxicological relevant effects were observed in the samples after ozonation by in vitro and in vivo assays (11). A second approach was the comparison of the different sample types from the three investigated WWTPs. After retention time alignment and mass deviation framing statistical evaluation procedures like PCA enable to identify differences and correlations between a set of samples. Figure 4 shows a PCA from all sample types (also treatment with powdered activated carbon at the WWTP Schwerte). It could be clearly seen from Figure 4 that all samples from the WWTP Schwerte are different and as expected no statistical correlation could be observed. 41



Figure 4. PCA from wastewater samples of the investigated WWTP.

Unexpectedly, the samples after ozonation at the WWTPs DuisburgVierlinden (diffuser and injector) and Bad Sassendorf showed similar results at the multivariate statistical data evaluation. Instead of the WWTP Schwerte, where different treatment procedures were applied, the samples after ozonation of WWTP effluents are broadly similar. This example showed that besides the comparison of equal samples from different sampling campaigns PCA is also a powerful tool for identification of similarities. After this enclosed research project we will continue HRMS data evaluation for identification of the characteristic compounds after ozonation. Also retrospective data analysis would be possible for new emerging contaminants and their behavior at full-scale ozonation using these data sets. Besides the described screening approach in silico methods for annotation of typical human or microbial metabolites and typical oxidation by-products after ozonation can help to evaluate the transformation reactions of micropollutants in every step – from application to WWTP. In this context Singer et al. recently published a workflow for rapid screening and combined modelling (22), which should be applied in the future.

2D-LC-QTOF At the previous sections one-dimensional liquid chromatography (1D-LC) coupled to high resolution mass spectrometry could be demonstrated as powerful tool for different screening approaches of complex wastewater samples that might contain hundreds to thousands of different components. However, the analysis of such complex environmental samples with one-dimensional liquid chromatography has limitations in terms of selectivity and peak capacity. In that regard, two-dimensional liquid chromatography (2D-LC) might also be a powerful tool for the comprehensive analysis of wastewater samples (23). In 42 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 5 TIC chromatograms and 2D-plots of a standard mixture and an influent sample from the WWTP Schwerte shows the chromatographic resolution and advantage of 2D-LC-QTOF for suspected-target and non-target screening (18, 23).

Figure 5. Total ion current chromatograms of a standard mixture and a native real wastewater sample. (a) Standard mixture using 1D-HPLC-MS; (b) wastewater sample using 1D-HPLC-MS (solid lines = detected target compounds without MS/MS spectra; dashed lines = detected target compounds with MS/MS spectra); (c) standard mixture using 2D-nLC x µLC-MS; (d) wastewater sample using 2D-nLC x mLC-MS (stars = detected target compounds without MS/MS spectra; rings = detected target compounds with MS/MS spectra). Reproduced with permission from Reference (23). Copyright (2013) American Chemical Society.

A comparison between one-dimensional and microscale comprehensive two-dimensional liquid chromatography coupled to high resolution mass spectrometry was performed. First of all, a reference standard mix containing 99 compounds was analyzed to obtain the retention time and additional MS/MS information. In the second step, a native influent sample of the WWTP Schwerte was analyzed on the basis of a suspected-target screening approach. Data evaluation for characterization and final identification of suspects in the wastewater samples was based on following criteria: 43 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

• • •

Accuracy of the exact mass: ± 5 ppm Retention time deviation: ± 2.5% Comparison of MS/MS spectra with the reference standard


The number of positive results in Table 6 showed that using the 2D-LC approach the identification was significant better than the 1D-LC measurements (18).

Table 6. Comparison of Identified Suspects by 1D-HPLC-MS and 2DnLC x mLC-MS. Detailed List of Detected Suspects Is Given in the Supporting Information Table S-2 of the Original Article (23). 1D-LC

2D-LC

Mass accuracy of the ref. standard (

Target Analysis, Suspected-Target, and Non-Target Screening for

Recommend Documents