S1 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: +49 261-1306 5560; fax: +49 261-1306-5363; e-mail: [email protected]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. ......................................................................................... 3 Table S2. Organic substances in the used 1C-PU coating according to manufacturer declaration. ....................... 4 Table S3. MS Parameters for measurements in positive and negative ESI mode. ................................................. 5 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. .......................................................................................... 7 Table S5. Parameter for peak extraction and alignment (MarkerView™). ............................................................ 9 Table S6. Parameter for gradient separation used a mobile phase consisting of methanol, ethyl acetate and n- hexane. .................................................................................................................................................................... 9 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. ........................................................................................................... 19 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
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S1
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
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. ......................................................................................... 3 Table S2. Organic substances in the used 1C-PU coating according to manufacturer declaration. ....................... 4 Table S3. MS Parameters for measurements in positive and negative ESI mode. ................................................. 5 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. .......................................................................................... 7 Table S5. Parameter for peak extraction and alignment (MarkerView™). ............................................................ 9 Table S6. Parameter for gradient separation used a mobile phase consisting of methanol, ethyl acetate and n-
hexane. .................................................................................................................................................................... 9 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 MS2 spectra (42–47), and mass without
MS2 spectra (48). The masses are sorted by intensity (pos.) within the group. Chemical structures verified by
reference standard are highlighted in gray. ........................................................................................................... 19
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
S2
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. ............................................................. 11 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. tR is given in min. ............................................................................................ 13 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. ........................................................................................................................................................................ 14 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. tR is given in min. ............................................................................................................................ 17 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. ................................................. 18 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........................ 19 Figure S8. Examples of one of the numerous possible structural composition of two of the declared
prepolymers. .......................................................................................................................................................... 22 Figure S9. Chemical structures of the first three substances suggested by MetFrag for measured MS
2 spectrum
in positive ionization mode. .................................................................................................................................. 23 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) [C2H4O]n derivatives. Intensities are on a different scale
from PeakView due to data conversion to mzML (open format). ......................................................................... 26 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.
tR is given in min. .................................................................................................................................................. 27 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. ................................................................ 29
S3
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.
Table S2. Organic substances in the used 1C-PU coating according to manufacturer declaration.
S5
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.
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
Table S3. MS Parameters for measurements in positive and negative ESI mode.
Parameter positive negative
m/z range 100 - 2000 100 - 2000
m/z range for IDA 30 - 1200 30 - 1200
# of IDA experiments / spectra 8 8
Curtain gas / psi 40 40
Ion source gas 1 / psi 35 35
Ion source gas 2 / psi 45 45
IonSpray voltage floating / V 5500 -4500
Temperature / °C 550 550
Collision energy for IDA / eV 40 -40
Declustering potential / V 60 -100
S6
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 (0–1000 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).
S7
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.
48 15.79 101.0614 1.7E+04 a Peaks were assigned to groups as part of the identification process. b According to Schymanski et. al1
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 NH4+ adduct mass / parent ion [M+H]+ mass
CAS-No. 53317-61-6
CAS-No. 127821-00-5
Figure S8. Examples of one of the numerous possible structural composition of two of the declared
prepolymers.
S23
1st
2nd
3rd
Figure S9. Chemical structures of the first three substances suggested by MetFrag for measured MS2
spectrum in positive ionization mode.
S
O
O
NH
O
OH
S
O
O
NH
O
OH
S
O
O
NH
O
O
S24
(I)
(II)
S25
(III)
(IV)
(V)
S26
(VI)
Figure S10. MS2 spectra of identified substances of (I) N-(tosyl)carbamate, (II) p-toluenesulfonamide, (III) p-
toluenesulfonic acid, (IV) 4,4′-MDI, (V) TDI, and (VI) [C2H4O]n derivatives. Intensities are on a different
scale from PeakView due to data conversion to mzML (open format).
S27
(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. tR is given in min.
S28
(I)
(II)
(III)
(IV)
S29
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