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: 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. ......................................................................................... 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.

CAS Substance Molecular weight Molecular Formula

- Ethyl N-(3-amino-2-methylphenyl)carbamate 194.23 C10H14N2O2

7450-62-6 Ethyl N-[5-(ethoxycarbonylamino)-2-methyl-

phenyl]carbamate 266.29 C13H18N2O4

5577-13-9 Ethyl N-(tosyl)carbamate 243.28 C10H13NO4S

14437-03-7 Methyl N-(tosyl)carbamate 229.25 C9H11NO4S

70-55-3 p-Toluenesulfonamide 171.22 C7H9NO2S

6192-52-5 p-Toluenesulfonic acid 172.20 C7H8SO3

10097-16-2 Diethyl 4,4'-methylenebis(N-phenylcarbamate) 342.40 C19H22N2O4

S4

Test Materials

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.

No./

Groupa

Substance Ionization

Mode

Massb

[M+H]+ or

[M–H]–

LOQ

[g L-1

]

Rhine, Koblenz 2016

[g L-1

]

Mosel, Koblenz 2016

[g L-1

]

Teltowkanal, Berlin

[g L-1

]

Surface water M1-3

M4-6

M7-9

M10-12

M1-3

M4-6

M7-9

M10-12

2016/09/09

2016/09/10

2016/09/11

2016/09/12

1

A Ethyl N-(tosyl)carbamate pos. 244.0638 0.06

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

2

A Methyl N-(tosyl)carbamate pos. 230.0482 0.06

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

10

B p-Toluenesulfonamide pos. 172.0427 10

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

11 p-Toluenesulfonic acid neg. 171.0121 0.1 < LOQ < LOQ < LOQ < LOQ 11 0.6

< LOQ < LOQ < LOQ < LOQ 4.8 < LOQ

12

C

Diethyl 4,4'-methylenebis(N-

phenylcarbamate) pos. 343.1652 0.03

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

16

D

Ethyl N-[5-(ethoxycarbonylamino)-

2-methyl-phenyl]carbamate pos. 267.1339 0.03

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ

19

D

Ethyl N-(3-amino-2-

methylphenyl)carbamate pos. 195.1128 0.1

< 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) –/+ 0.01

S8

Data Analysis of Qualitative Screening

MS data acquisition was controlled with Analyst TF 1.5.1 software (SCIEX). Data were

analyzed using MarkerView™ 1.2.1, MultiQuant™ 2.1 and PeakView™ 1.2.0.3 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 3.0.3 and Tinn-R Editor 2.4.1.7 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+NH4]

+, [M+K]

+ (adducts) and

13C,

15N,

34S,

37Cl,

81Br,

41K (isotopes). Here, the option was set to allow doubly charged ions.

S9

Table S5. Parameter for peak extraction and alignment (MarkerView™).

Peak extraction and alignment MarkerView™

Data to process

Period 1

Experiment 1

Min. Retention Time 1 min

Max. Retention Time 30 min

Subtraction Offset 10 scans

Subtraction Mult. Factor 1.3

Noise Threshold 100

Min. Spectral Peak Width 80 ppm

Min. RT Peak Width 4 scans

Alignment Retention Time Tolerance 0.3 min

Mass Tolerance 50 ppm

Filtering

Remove peaks < 3 a/ -

b

Use Global Exclusion List False

Max. Number of Peaks 9000

Normalization Perform RT correction False

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]

1 100 0 0 15 3

2 80 20 0 18 3

3 70 30 0 21 2

4 60 40 0 24 2

5 50 50 0 27 2

6 30 70 0 30 2

7 10 90 0 33 2

8 0 100 0 36 2

9 0 60 40 39 2

10 0 20 80 42 2

11 0 0 100 45 2

S10

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).

S11

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

S12

7 B

8 B

9 B

10 B

11

12 C

13 C

14 C

15 D

16 D

17 D

18 D

S13

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. tR is given in min.

S14

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.

S15

Group A

Group B

Group C

S16

Group D

S17

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. tR 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).

S18

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.

S19

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 MS2 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.

No./

Groupa

Proposed chemical

structures/

Identification

confidence levelb (L)

tR

[min]

Mass

measd.

(pos/neg)

[M+H]+/

[M-H]-

Intensity

in

samplec

Fragments

Five most intense fragments from MS2 spectra (pos/neg)

1

A L1 15.61

244.0659 8.3E+06 198.0238 155.0179 119.0629 91.0567 65.0427

242.0517 9.5E+06 196.0090 170.0293 155.0180 105.9615 42.0027

2

A L1 14.80

230.0500 6.7E+05 198.0243 155.0180 119.0630 91.0566 65.0423

228.0364 1.4E+06 196.0095 170.0320 155.0189 105.9615 42.0024

3

A L3 16.33

256.0668 5.6E+05 173.0292 155.0181 119.0633 91.0568 65.0422

4

A L3 14.03

274.0772 2.3E+05 198.0252 155.0182 119.0615 91.0571 65.0414

272.0631 1.4E+06 196.0093 170.0297 106.0674 73.0314 42.0022

S20

5

A

Side-chain structure

could not be proposed

due to missing

characteristic MS2

fragments. L4

17.16

471.0927 1.4E+05 256.0666 198.0258 155.0180 102.0578 91.0561

6

A L3 17.27

272.0976 8.8E+04 198.0249 155.0173 119.0630 91.0567 65.0408

270.0838 4.1E+05 196.0089 170.0293 155.0176 105.9611 42.0018

7

B

L3

17.35

280.1395/

298.1506d 1.8E+06 155.0178 124.1141 109.1034 91.0569 65.0421

8

B 16.58

280.1387/

298.1503d 1.5E+06 155.0180 124.1141 109.1034 91.0568 65.0424

9

B 16.05

280.1393/

298.1507d 2.4E+05 155.0183 124.1123 109.1036 91.0565 65.0438

10

B L1 13.16

172.0437 1.0E+05 155.0177 119.0646 106.0657 91.0567 65.0424

170.0296 7.3E+05 106.0673 78.9752 61.9730

11 L1

15.85

171.0135e 3.3E+06 107.0516 79.9588

12

C L1 17.74

343.1686 2.4E+06 297.1269 251.0842 150.0567 132.0460 104.0516

13

C L2 14.99

225.1038f 1.8E+05 180.0829 132.0466 106.0674 104.0519 77.0413

14

C L2

14.57 402.2070 6.6E+04 384.1950 178.0884 150.0574 132.0464 106.0881

400.1917 5.1E+03 354.1508 295.1101 269.1300 249.0693 223.0895

15

D L3 13.39

429.1794f 4.5E+05 323.1181 219.0784 193.0993 175.0521 149.0726

427.1649 9.3E+04 321.1038 279.1014 173.0365 147.0572 105.0564

16

D L1 16.04

267.1365 1.4E+05 193.0637 175.0522 147.0571 132.0473 106.0674

17

D L3

12.83

429.1796f 9.1E+04 323.1185 219.0792 193.0996 175.0523 149.0730

427.1670 2.6E+03 321.1023 279.1011 173.0370 147.0573 105.0567

18

D L2 15.13

267.1371 7.2E+04 193.0626 175.0523 147.0574 132.0455 104.0523

19

D L1 9.63

195.1144 6.0E+04 149.0730 134.0497 121.0779 106.0672 77.0409

20

D L2

10.70

195.1146 3.4E+04 149.0726 121.0775 106.0675 94.0675 77.0412

S21

21

D

L3

13.31 475.2230 1.5E+04 265.1203 221.0941 193.0987 175.0520 149.0725

473.1712 3.7E+03 321.1030 279.1007 173.0365 147.0574 105.0566

22

D 14.01

475.2244 1.2E+04 265.1206 221.0948 193.0992 175.0504 149.0726

23

D

L3

9.66 403.2029 6.5E+03 255.1361 193.0996 171.1408 149.0756 106.0673

24

D 10.25

403.2022 2.3E+03 255.137 193.1000 171.1398 149.0728 106.0687

25

E

L4 12.35

336.1688/

319.1416g 1.9E+05 187.0609 143.0350 99.0091 89.0629 45.0375

26

E

L4 11.99

275.1150 1.7E+05 143.0354 133.0862 99.0085 89.0610 45.0394

27

E

L4 12.59

380.1947/

363.1682g 1.7E+05 231.0867 187.0610 143.0357 99.0094 89.0618

28

E

L4 11.36

231.0881 1.6E+05 143.0369 133.0873 99.0099 89.0617 45.0379

29

E

L4 12.98

336.1688/

319.1424g 9.3E+04 275.1129 231.0886 187.0620 143.0354 99.0101

30

E

L4 13.06

380.1960/

363.1693g 5.6E+04 275.1137 231.0888 187.0621 143.0355 99.0094

31 10.44 180.0825 2.1E+05 152.0638 128.0512 101.0410 77.0412

32 13.13 276.5357 1.5E+05 209.9912 155.0194 119.9446 91.0564 65.0422

33 14.67 133.0668 1.3E+05 115.0564 105.0724 79.0572 77.0415 51.0273

34 11.83 129.0925 1.1E+05 111.0816 91.0569 77.0423 57.0372 43.0233

35 12.42 133.0872 1.1E+05 73.0679 61.0308 45.0394 41.0446 39.0295

36 16.72 263.1092 9.1E+04 226.0801 215.0878 202.0800 133.0309 115.0577

37 13.13 255.0403 7.6E+04 214.0129 168.0382 149.0188 133.0249 121.0249

38 13.02 386.3051 5.9E+04 231.1361 187.1467 170.1192 100.1148 88.0421

39 17.09 247.1127 4.8E+04 228.0970 204.0957 191.0868 178.0793 91.0568

40 10.85 184.1141 4.3E+04 168.0823 156.0826 143.0754 128.0638 115.0563

41 19.49 263.1091 3.8E+04 245.0984 226.0793 215.0887 202.0790 91.0595

42 12.39 101.0612 3.3E+05 43.0237 39.0414

43 9.28 195.1248 2.8E+04 89.0610 45.0379

44 13.45 101.0613 1.3E+04 72.9421 43.0238 41.0524 39.0285

45 14.11 323.2011 7.9E+03 149.0726

46 15.76 271.1463 7.3E+03 108.9642 106.0670 91.0582 79.0584

S22

47 13.14 362.0555 7.1E+04 227.9987 209.9919 191.0170 154.0340 91.0569

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

confidence Environ. Sci. Technol. 2014, 48 (4) 2097– 2098, DOI: 10.1021/es5002105.