Nontarget Analysis via LC-QTOF-MS to Assess the Release of Organic Substances from Polyurethane Coating

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1 Nontarget Analysis via LC-QTOF-MS to Assess the Release of Organic Substances from Polyurethane Coating Agnessa Luft, Kathrin Bröder, Uwe Kunkel,#, Manoj Schulz, Christian Dietrich, Roland Baier, Peter Heininger and Thomas A. Ternes, * Federal Institute of Hydrology (BfG), Koblenz, Germany # present address: Bavarian Environment Agency, Augsburg, Germany Federal Waterways Engineering and Research Institute, Karlsruhe, Germany * Corresponding author phone: ; fax: ; ternes@bafg.de Content Chemicals and Standards... 3 Test Materials... 4 HRMS Analysis via LC-QTOF-MS... 5 Quantification of the Identified Substances verified by Authentic Reference Standards... 6 Data Analysis of Qualitative Screening... 8 Bacterial Screening Toxicity Test... 9 Leaching Experiments DOC, TNb and LC-QTOF-MS Results...10 Identification...19 References...29 Pages: 29 Tables: 7 Figures: 12 Tables Table S1. Seven reference standards used in the study Table S2. Organic substances in the used 1C-PU coating according to manufacturer declaration Table S3. MS Parameters for measurements in positive and negative ESI mode Table S4. Overview of the quantified substances with their mass, limit of quantification (LOQ) and results of the preliminary environmental analysis of three surface waters. Rhine and Mosel samples are composite samples of 3 months (M). Teltowkanal samples are grab samples Table S5. Parameter for peak extraction and alignment (MarkerView ) Table S6. Parameter for gradient separation used a mobile phase consisting of methanol, ethyl acetate and n- hexane Table S7. Substances detected in leachates summarized with their retention time, mass, intensity, fragments, and group. The table consists of masses belonging to groups A E, including p-toluenesulfonic acid (1 30), masses without specific group assignment (31 41), masses with insufficient MS 2 spectra (42 47), and mass without MS 2 spectra (48). The masses are sorted by intensity (pos.) within the group. Chemical structures verified by reference standard are highlighted in gray Figures Figure S1. Leaching experiment of 1C-PU coating with varying hardening and leaching duration. Sum of peak intensities (I) with and (II) without adducts and isotopes and number of peaks (III) with and (IV) without adducts S1

2 and isotopes. MQ water was used as leaching water. Analysis was performed in the positive ionization mode using LC-QTOF-MS. The statistical errors of the measurements are given as 95% confidence intervals (n = 3). 10 Figure S2. TNb in samples of leachate from the leaching experiment with varying polymer hardening (t= 0, 24 h and 14 d) and leaching duration. MQ water was used as leaching water Figure S3. Peak intensities of masses belonging to groups A E in leachate samples from the leaching experiment with varying polymer hardening (t = 0, 24 h and 14 d). MQ water was used as leaching water. Analyses were performed in the positive ionization mode using LC-QTOF-MS (only No. 11 was measured in negative ionization mode). The statistical errors of the measurements are given as standard deviation (n = 3). Masses obtained by MarkerView. t R is given in min Figure S4. (I) Sum of peak intensities, (II) DOC, (III) sum of peak numbers and (IV) TNb in leachate samples from the leaching experiment with varying leaching water, MQ and river water (RW). The polymer hardening duration was 24 h. For (I) and (III), analyses were performed in the positive ionization mode using LC-QTOF- MS Figure S5. Peak intensities of masses belonging to groups A E in leachate samples from the leaching experiment with varying leaching water, MQ and river water. The polymer hardening duration was 24 h. Analyses were performed in the positive ionization mode using LC-QTOF-MS. Masses obtained by MarkerView. t R is given in min Figure S6. (I) Sum of peak intensities [%] after repetitive water renewals compared to the peak intensities prior to any water renewals. The same comparison using (II) DOC [%], (III) number of peaks [%] and (IV) TNb [%]. Polymer hardening duration was 24 h. Leaching duration before water renewals was 14 d. Leaching duration after each water renewal was 24 h. MQ and river water (RW) were used as leaching water. For (I) and (III), analyses were performed in the positive ionization mode using LC-QTOF-MS Figure S7. Final peaks (after subtraction steps) were detected in leachates from leaching experiment I. Peak intensities resulted from hardening duration t = 0 (direct water addition after final material preparation) and leaching duration t = 14 d. Analyses were performed in the positive and negative ionization mode using LC- QTOF-MS. The arrow indicates the one peak that was only visible in negative ionization mode Figure S8. Examples of one of the numerous possible structural composition of two of the declared prepolymers Figure S9. Chemical structures of the first three substances suggested by MetFrag for measured MS 2 spectrum in positive ionization mode Figure S10. MS 2 spectra of identified substances of (I) N-(tosyl)carbamate, (II) p-toluenesulfonamide, (III) p- toluenesulfonic acid, (IV) 4,4 -MDI, (V) TDI, and (VI) [C 2 H 4 O] n derivatives. Intensities are on a different scale from PeakView due to data conversion to mzml (open format) Figure S11. (I) Relative amounts (sum of peak intensities) of the five characterized groups and of the peaks without any structure proposal in total. (II VI) In addition, the relative amount of substances within the groups A E. The sum of peak intensities was formed by summarized the peak intensities over all leaching parameters (leaching duration and hardening). Substances that were identified by reference standard are highlighted in gray. t R is given in min Figure S12. Luminescent bacteria inhibition test with Aliivibrio fischeri of leachates from leaching experiments (I) with varying polymer hardening duration, (II) with varying water types and (III) with water renewal. Hardening duration was set to 24 h in (II). (IV) Gradient separation step with leachates from leaching experiments with varying hardening duration and standards. Only MQ was used as leaching water in (I, IV). Dark spots indicate toxicity to Aliivibrio fischeri. The darker the spot, the higher the degree of toxicity. Of all samples, an amount of 10 µl was sprayed on the TLC plate using an automatic TLC sampler. After an exposure time of 10 min the detection was performed with the TLC Visualizer S2

3 Chemicals and Standards Stock solutions of reference standards (Table S1) with concentration of 10 mg L -1 were prepared in methanol. Further dilution steps were done to prepare final reference standard solutions to a concentration of 1 µg L -1, 10 µg L -1 and 1000 µg L -1 for calibration. To compare the reference standards with analytes from leachate samples, ultrapure water (MQ) was also used as solvent to avoid any negative effects on the chromatography. The stock solutions were stored in the freezer at 20 C. The diluted standard solutions were stored in the dark at 4 C. Table S1. Seven reference standards used in the study. CAS Substance Molecular weight Molecular Formula - Ethyl N-(3-amino-2-methylphenyl)carbamate C 10 H 14 N 2 O Ethyl N-[5-(ethoxycarbonylamino)-2-methylphenyl]carbamate C 13 H 18 N 2 O Ethyl N-(tosyl)carbamate C 10 H 13 NO 4 S Methyl N-(tosyl)carbamate C 9 H 11 NO 4 S p-toluenesulfonamide C 7 H 9 NO 2 S p-toluenesulfonic acid C 7 H 8 SO Diethyl 4,4'-methylenebis(N-phenylcarbamate) C 19 H 22 N 2 O 4 S3

4 Test Materials Table S2. Organic substances in the used 1C-PU coating according to manufacturer declaration. S4

5 HRMS Analysis via LC-QTOF-MS MQ water with 0.1% formic acid was used as mobile phase A and acetonitrile with 0.1% formic acid as mobile phase B. The gradient of mobile phase A was as follows: start with 98%, after 3 min decrease to 2% within 15 min, kept isocratic for 6 min, returned to the initial conditions (98%) within 0.5 min which was held for the last 5.5 min. The total run time was 30 min. The flow rate was adjusted to 200 µl min -1 and the column oven temperature to 25 C. The injection volume of the sample was 10 µl. A Luna 3 µm C18 column (150 x 2 mm, 3 µm; Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation. MS was performed in full scan TOF-MS and MS/MS mode (high resolution) with information dependent acquisition (IDA) experiments (product ion). The resolution of measurements was 35,000 at m/z = 400 and the mass accuracy below 5 ppm. Further important MS parameters for measurements in positive and negative ESI mode are presented in Table S3. Table S3. MS Parameters for measurements in positive and negative ESI mode. Parameter positive negative m/z range m/z range for IDA # of IDA experiments / spectra 8 8 Curtain gas / psi Ion source gas 1 / psi Ion source gas 2 / psi IonSpray voltage floating / V Temperature / C Collision energy for IDA / ev Declustering potential / V An automated external calibration system (Calibrant Delivery System, CDS) was used for mass calibration of the mass spectrometer to maintain the mass accuracy during the batch S5

6 measurements. This was performed by using an APCI calibration solution (positive and negative polarity solutions). The TOF was calibrated every 2.5 h during the batch measurement in an air-conditioned room at 24 C ± 1 C. When measuring the samples of the first leaching experiment, the samples were injected in triplicate, for subsequent samples no repeat injections were used. Quantification of the Identified Substances verified by Authentic Reference Standards An external calibration with 17 calibration points ( µg L -1 ) was used. The limit of quantification (LOQ) was defined as the lowest point in the calibration curve with a signal to noise ratio (S/N) of at least 10. Leachate samples were used for analysis, undiluted as well as diluted (1:10, 1:100). S6

7 Table S4. Overview of the quantified substances with their mass, limit of quantification (LOQ) and results of the preliminary environmental analysis of three surface waters. Rhine and Mosel samples are composite samples of 3 months (M). Teltowkanal samples are grab samples. No./ Substance Ionization Group a Mode Mass b [M+H] + or [M H] LOQ [ g L -1 ] Rhine, Koblenz 2016 [ g L -1 ] Surface water M1-3 M7-9 Mosel, Koblenz 2016 [ g L -1 ] M1-3 M7-9 Teltowkanal, Berlin [ g L -1 ] 2016/09/ /09/11 M4-6 M10-12 M4-6 M /09/ /09/12 1 A 2 A 10 B Ethyl N-(tosyl)carbamate pos Methyl N-(tosyl)carbamate pos p-toluenesulfonamide pos < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 11 p-toluenesulfonic acid neg < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 4.8 < LOQ 12 C Diethyl 4,4'-methylenebis(Nphenylcarbamate) pos < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 16 D Ethyl N-[5-(ethoxycarbonylamino)- 2-methyl-phenyl]carbamate pos < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 19 D Ethyl N-(3-amino-2- methylphenyl)carbamate pos < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ a SI Table S7, b mass range for extraction of ion chromatograms (EICs) / S7

8 Data Analysis of Qualitative Screening MS data acquisition was controlled with Analyst TF software (SCIEX). Data were analyzed using MarkerView 1.2.1, MultiQuant 2.1 and PeakView software (SCIEX). With the MarkerView software the data of all samples measured in one batch were analyzed and presented in lists as so called features consisting of extracted peaks with their respective mass and retention time. Parameters for peak extraction (see Table S5) were set to obtain these data lists. MultiQuant software was used to perform automatic peak integration and, if necessary, to integrate manually. Thus, the peak data was completed by peak intensity in addition to mass and retention time obtained from MarkerView data. Peaks which were also detected with similar intensity in control samples (the ratio of peak intensity of sample and control sample less than 10) were deleted. This was done to eliminate peaks originating from the test system. The programs R and Tinn-R Editor in combination with the software R package nontarget 1.5 (Eawag, Dübendorf, Switzerland) were used to detect, filter and determine adduct and isotope relations in the MS data set. The following adducts and isotopes were considered in the positive mode: [M+H] +, [M+Na] +, [M+NH 4 ] +, [M+K] + (adducts) and 13 C, 15 N, 34 S, 37 Cl, 81 Br, 41 K (isotopes). Here, the option was set to allow doubly charged ions. S8

9 Table S5. Parameter for peak extraction and alignment (MarkerView ). Peak extraction and alignment Data to process Alignment Filtering Period 1 Experiment 1 Min. Retention Time Max. Retention Time Subtraction Offset MarkerView 1 min Subtraction Mult. Factor min Noise Threshold 100 Min. Spectral Peak Width Min. RT Peak Width Retention Time Tolerance Mass Tolerance Remove peaks < Use Global Exclusion List 10 scans 80 ppm 4 scans 0.3 min 50 ppm 3 a / - b False Max. Number of Peaks 9000 Perform RT correction False Normalization Perform normalization False a remove peaks for measurements with sample injection in triplicate b no remove peaks for measurements with single sample injection Bacterial Screening Toxicity Test Table S6. Parameter for gradient separation used a mobile phase consisting of methanol, ethyl acetate and n-hexane. Step Methanol [Vol %] Ethyl acetate [Vol %] n-hexane [Vol %] Migration distance [mm] Drying time [min] S9

10 Leaching Experiments DOC, TNb and LC-QTOF-MS Results LC-QTOF-MS - Subtraction of Adducts and Isotopes. After a blank subtraction step, adducts and isotope ions were additionally subtracted. By doing so, the sum of peak intensities and peak numbers was reduced as shown in the Figure S1. However, the shape of the curves remained the same. (I) (II) (III) (IV) Figure S1. Leaching experiment of 1C-PU coating with varying hardening and leaching duration. Sum of peak intensities (I) with and (II) without adducts and isotopes and number of peaks (III) with and (IV) without adducts and isotopes. MQ water was used as leaching water. Analysis was performed in the positive ionization mode using LC-QTOF-MS. The statistical errors of the measurements are given as 95% confidence intervals (n = 3). S10

11 I) Leaching Experiment of varying Polymer Hardening and Leaching Duration. Figure S2. TNb in samples of leachate from the leaching experiment with varying polymer hardening (t= 0, 24 h and 14 d) and leaching duration. MQ water was used as leaching water. 1 A 2 A 3 A 4 A 5 A 6 A S11

12 7 B 8 B 9 B 10 B C 13 C 14 C 15 D 16 D 17 D 18 D S12

13 19 D 20 D 21 D 22 D 23 D 24 D 25 E 26 E 27 E 28 E 29 E 30 E Figure S3. Peak intensities of masses belonging to groups A E in leachate samples from the leaching experiment with varying polymer hardening (t = 0, 24 h and 14 d). MQ water was used as leaching water. Analyses were performed in the positive ionization mode using LC-QTOF-MS (only No. 11 was measured in negative ionization mode). The statistical errors of the measurements are given as standard deviation (n = 3). Masses obtained by MarkerView. t R is given in min. S13

14 II) Leaching Experiment with varying Leaching Water. The measured DOC showed no difference between MQ water and river water. A maximum DOC level of 1.1 g C L -1 was measured in contact with MQ water and river water at a leaching duration of 14 d (Figure S4- II). The measured TNb was different in MQ water and river water at a leaching duration of 14 d, whereby a maximum TNb level of 18.2 mg L -1 in river water and 13.9 mg L -1 in MQ water was measured (Figure S4-IV). At 6 h of leaching duration the sum of peak intensities was comparable in MQ water and river water, while between 24 h and 14 d the sum of peak intensities increased in river water with the leaching duration more than in MQ water (Figure S4-I). The number of peaks remained relatively constant at all leaching durations and was similar in MQ water and river water (Figure S4-III). (I) (II) (III) (IV) Figure S4. (I) Sum of peak intensities, (II) DOC, (III) sum of peak numbers and (IV) TNb in leachate samples from the leaching experiment with varying leaching water, MQ and river water (RW). The polymer hardening duration was 24 h. For (I) and (III), analyses were performed in the positive ionization mode using LC-QTOF-MS. S14

15 Group A Group B Group C S15

16 Group D S16

17 Group E Figure S5. Peak intensities of masses belonging to groups A E in leachate samples from the leaching experiment with varying leaching water, MQ and river water. The polymer hardening duration was 24 h. Analyses were performed in the positive ionization mode using LC-QTOF-MS. Masses obtained by MarkerView. t R is given in min. III) Leaching Experiment with Water Renewal. In MQ water, after the first water renewal, the leached DOC was only 6% of what was released during the first 14 d. This was similar in river water. After the third water renewal, the released DOC was less than 2% of the DOC released after 14 d. As with the initial 14 d leaching period (Figure S4-II), after each water renewal, the DOC in MQ water was relatively comparable with the DOC in river water (Figure S6-II). However, with respect to TNb a difference was observed between the two water types (Figure S6-IV) as was observed after the initial 14 d leaching period (Figure S4- IV). After the first water renewal, the TNb was in MQ water 15% of the value before any water renewal and in river water was 5%. A similar difference was also observed after the subsequent two water renewals (Figure S6). S17

18 The LC-QTOF-MS results showed, as with the DOC results, that further compounds were leached out of the test material (Figure S6-I and S6-III). In MQ water, after the first water renewal, the sum of peak intensities (Figure S6-I) was still 53% of what was released during the first 14 d. Even after the third water renewal, the sum of peak intensities was 37% of the sum prior to any water renewals. Although the trends in DOC and sum of peak intensities corresponded well to one another, the two values measure different properties and therefore are not expected to be proportional. This can be seen when comparing Figure S6-I and S6-II. (I) (II) (III) (IV) Figure S6. (I) Sum of peak intensities [%] after repetitive water renewals compared to the peak intensities prior to any water renewals. The same comparison using (II) DOC [%], (III) number of peaks [%] and (IV) TNb [%]. Polymer hardening duration was 24 h. Leaching duration before water renewals was 14 d. Leaching duration after each water renewal was 24 h. MQ and river water (RW) were used as leaching water. For (I) and (III), analyses were performed in the positive ionization mode using LC-QTOF-MS. S18

19 Identification Substances detected in positive and negative ionization mode are plotted in Figure S7 and listed in Table S7. Some substances could be measured in both positive and negative ionization mode while one additional substance was detected exclusively in negative ionization mode. Figure S7. Final peaks (after subtraction steps) were detected in leachates from leaching experiment I. Peak intensities resulted from hardening duration t = 0 (direct water addition after final material preparation) and leaching duration t = 14 d. Analyses were performed in the positive and negative ionization mode using LC-QTOF- MS. The arrow indicates the one peak that was only visible in negative ionization mode. Table S7. Substances detected in leachates summarized with their retention time, mass, intensity, fragments, and group. The table consists of masses belonging to groups A E, including p-toluenesulfonic acid (1 30), masses without specific group assignment (31 41), masses with insufficient MS 2 spectra (42 47), and mass without MS 2 spectra (48). The masses are sorted by intensity (pos.) within the group. Chemical structures verified by reference standard are highlighted in gray. Proposed chemical Mass No./ t structures/ R Intensity measd. Fragments in Group a Identification (pos/neg) [min] confidence level b (L) [M+H] + sample / c Five most intense fragments from MS 2 spectra (pos/neg) [M-H] - 1 A L E E A L E E A L E A L E E S19

20 5 A Side-chain structure could not be proposed due to missing characteristic MS 2 fragments. L E A L E E B / d 1.8E B / d 1.5E B L / d 2.4E B L E E L e 3.3E C L E C L f 1.8E C L E E D L f 4.5E E D L E D L f 9.1E E D L E D L E D L E S20

21 21 D E E D L E D E D L E E L / g 1.9E E L E E L / g 1.7E E L E E L / g 9.3E E L / g 5.6E E E E E E E E E E E E E E E E E S21

22 E E+04 a Peaks were assigned to groups as part of the identification process. b According to Schymanski et. al 1 c Sample used for intensity: hardening duration t = 0 (direct water addition) and leaching duration t = 14 d d In-source fragment mass / parent ion ([M+H] + ) mass e This mass was detected only in the negative ionization mode. f In-source fragment mass, parent ion mass unknown g NH 4 + adduct mass / parent ion [M+H] + mass CAS-No CAS-No Figure S8. Examples of one of the numerous possible structural composition of two of the declared prepolymers. S22

23 1 st O NH S O O OH 2 nd O NH S O O OH 3 rd O NH O S O O Figure S9. Chemical structures of the first three substances suggested by MetFrag for measured MS 2 spectrum in positive ionization mode. S23

24 (I) (II) S24

25 (III) (IV) (V) S25

26 (VI) Figure S10. MS 2 spectra of identified substances of (I) N-(tosyl)carbamate, (II) p-toluenesulfonamide, (III) p- toluenesulfonic acid, (IV) 4,4 -MDI, (V) TDI, and (VI) [C 2 H 4 O] n derivatives. Intensities are on a different scale from PeakView due to data conversion to mzml (open format). S26

27 (I) (II) (III) (IV) (V) (VI) Figure S11. (I) Relative amounts (sum of peak intensities) of the five characterized groups and of the peaks without any structure proposal in total. (II VI) In addition, the relative amount of substances within the groups A E. The sum of peak intensities was formed by summarized the peak intensities over all leaching parameters (leaching duration and hardening). Substances that were identified by reference standard are highlighted in gray. t R is given in min. S27

28 (I) (II) (III) (IV) S28

29 Figure S12. Luminescent bacteria inhibition test with Aliivibrio fischeri of leachates from leaching experiments (I) with varying polymer hardening duration, (II) with varying water types and (III) with water renewal. Hardening duration was set to 24 h in (II). (IV) Gradient separation step with leachates from leaching experiments with varying hardening duration and standards. Only MQ was used as leaching water in (I, IV). Dark spots indicate toxicity to Aliivibrio fischeri. The darker the spot, the higher the degree of toxicity. Of all samples, an amount of 10 µl was sprayed on the TLC plate using an automatic TLC sampler. After an exposure time of 10 min the detection was performed with the TLC Visualizer. References (1) Schymanski, E. L.; Jeon, J.; Gulde, R.; Fenner, K.; Ruff, M.; Singer, H. P.; Hollender, J. Identifying small molecules via high resolution mass spectrometry: communicating confidence Environ. Sci. Technol. 2014, 48 (4) , DOI: /es S29