Source of Toxic and Carcinogenic Vocs, Phenols and Heavy Metals?

Waterpipe Smoke:

Source of toxic and carcinogenic VOCs, phenols and heavy metals?

Jens Schubert*, Frederic D. Müller, Roman Schmidt, Andreas Luch and Thomas G. Schulz

German Federal Institute for Risk Assessment (BfR), Department of Chemicals and Product Safety

Max-Dohrn-Straße 8-10, 10589 Berlin, Germany

*Corresponding author

Jens Schubert

German Federal Institute for Risk Assessment (BfR)

Department of Chemicals and Product Safety

Max-Dohrn-Straße 8-10, 10589 Berlin, Germany

Tel.: +49 30 18412 3325

Fax.: +49 30 18412 4928

Table of Contents for Supplementary Data

Table S-1. CAS registry numbers and suppliers of the chemicals used.

Table S-2. Analyte-specific parameters for 12 VOCs and their corresponding internal standards.

Table S-3. GC-MS operating parameters for analysis of VOC groupI.

Table S-4. GC-MS operating parameters for analysis of VOC groupII.

Table S-5. Coefficients of determination, precision, limits of detection (LODs) and limits of quantification (LOQs) of 12 VOCs in clean solvent standards.

Table S-6. Analyte-specific parameters for eight phenolic compounds.

Table S-7. RP-HPLC-FLD operating parameters for analysis of phenolic compounds.

Table S-8. Composition and timing of the gradient applied for the analysis of phenolic compounds.

Table S-9. Coefficient of determination, precision, limits of detection (LODs) and limits of quantification (LOQs) of eight phenolic compounds in clean solvent standards.

Table S-10. Recovery of eight phenolic compounds in fortified solvent spikes.

Table S-11. Analyte-specific parameters for 17 elements.

Table S-12. Microwave digestion conditions for unburned tobacco.

Table S-13. Microwave digestion conditions for unburned charcoal.

Table S-14 Microwave digestion conditions for burned tobacco and charcoal.

Table S-15 ICP-MS operating parameters for analysis of elements.

Table S-16 Coefficient of determination, precision, limits of detection (LODs) and limits of quantification (LOQs) of 17 elements in clean aqueous standards.

Figure S-1. Experimental set-up for determination of VOCs: wind cover (1a), charcoal (1b), tobacco (1c), head (1d), ash tray (1e), steam (2), bowl (3), 92mm filter pad holder equipped with 92mm glass fiber filter pad (4), hose (5), impingers filled with methanol (6), cryogenic bad (dry ice / isopropyl alcohol, at or below -72°C) (7), pump (8, Borgwaldt Shisha Smoker).

S-2

Table S-1. CAS registry numbers and suppliers of the chemicals used.

Chemical / CAS number / Supplier
Methanol / 67-56-1 / A
Acetonitrile / 75-05-8 / A
Isopropyl alcohol / 67-63-0 / B
Acetic acid / 64-19-7 / A
Nitric acid / 7697-37-2 / B
Hydrogen peroxide / 7722-84-1 / C
1,3-Butadiene / 106-99-0 / A
Isoprene / 78-79-5 / A
Acrylonitrile / 107-13-1 / A
Benzene / 71-43-2 / A
Toluene / 108-88-3 / A
Ethylbenzene / 100-41-4 / A
p-Xylene / 106-42-3 / A
m-Xylene / 108-38-3 / A
Pyridine / 110-86-1 / A
o-Xylene / 95-47-6 / A
Styrene / 100-42-5 / A
Quinoline / 91-22-5 / A
Benzene-d6 (IS) / 1076-43-3 / A
Toluene-d8 (IS) / 2037-26-5 / A
p-Xylene-d10 (IS) / 41051-88-1 / A
Pyridine-d5 (IS) / 7291-22-7 / A
Styrene-d8 (IS) / 19361-62-7 / A
Quinoline-d7 (IS) / – / A
Hydroquinone / 123-31-9 / A
Resorcinol / 108-46-3 / A
Catechol / 120-80-9 / A
Phenol / 108-95-2 / A
Guaiacol / 90-05-1 / A
m-Cresol / 108-39-4 / A
p-Cresol / 106-44-5 / A
o-Cresol / 95-48-7 / A
Multi-element standarda / – / D
Scandium standard for ICP (IS)b / – / A
Rhodium ICP standard (IS)c / – / C
Bismuth ICP standard (IS)c / – / C

a Containing: 500µg/ml (Al, Zn); 200µg/ml (Cu); 180µg/ml (Mn); 120µg/ml (Ba); 31.25µg/ml (Sn); 25µg/ml (Cr); 14µg/ml (Ni); 12µg/ml (Mo); 4.8µg/ml (Li); 2µg/ml (Co); 1µg/ml (Be, Pb, V); 0.5µg/ml (Cd); 0.2µg/ml (As); 0.01µg/ml (Tl); 5.0% HNO3 (v/v); 0.4% HF (v/v).

b Containing: 1000µg/ml (Sc); 5.0% HNO3 (w/w).

c Containing: 1000µg/ml (Rh, Bi); 2-3% HNO3.

A: Sigma Aldrich (Munich, Germany).

B: VWR International GmbH (Darmstadt, Germany).

C: Merck KGaA (Darmstadt, Germany).

D: Inorganic Ventures (Madrid, Spain).

S-10

Table S-2. Analyte-specific parameters for 12 VOCs and their corresponding internal standards.

Analyte / Purity
(%) / CAS number / IARC groupa / Internal standard / Retention time (min) / Ion mass
(m/z) / Confirmation ion
(m/z)
Volatile organic compounds group I (VOC groupI)
1,3-Butadiene / 99.5 / 106-99-0 / 1b / benzene-d6 / 3.641 / 54 / 39
Isoprene / 99.0 / 78-79-5 / 2Bc / benzene-d6 / 5.218 / 67 / 53
Acrylonitrile / 99.0 / 107-13-1 / 2Bc / benzene-d6 / 6.235 / 53 / 52
Benzene / 99.9 / 71-43-2 / 1b / benzene-d6 / 11.782 / 78 / 77
Toluene / 99.9 / 108-88-3 / 3c / toluene-d8 / 18.627 / 91 / 92
Benzene-d6 (IS) / 99.6 / 1076-43-3 / – / – / 11.636 / 84 / 52
Toluene-d8 (IS) / 99.6 / 2037-26-5 / – / – / 18.335 / 98 / 100
Volatile organic compounds group II (VOC groupII)
Ethylbenzene / 99.9 / 100-41-4 / 2Bd / p-xylene-d10 / 7.705 / 91 / 106
p-Xylene / 99.9 / 106-42-3 / 3c / p-xylene-d10 / 7.943 / 91 / 106
m-Xylene / 99.9 / 108-38-3 / 3c / p-xylene-d10 / 8.138 / 91 / 106
Pyridine / 99.8 / 110-86-1 / 3d / pyridine-d5 / 9.294 / 79 / 52
o-Xylene / 99.9 / 95-47-6 / 3c / p-xylene-d10 / 9.489 / 91 / 106
Styrene / 99.9 / 100-42-5 / 2Be / styrene-d8 / 11.802 / 104 / 78
Quinoline / 98.0 / 91-22-5 / – / quinoline-d7 / 19.165 / 129 / 102
p-Xylene-d10 (IS) / 99.0 / 41051-88-1 / – / – / 7.798 / 98 / 116
Pyridine-d5 (IS) / 99.5 / 7291-22-7 / – / – / 9.370 / 84 / 56
Styrene-d8 (IS) / 98.0 / 19361-62-7 / – / – / 11.815 / 112 / 84
Quinoline-d7 (IS) / 97.0 / – / – / – / 19.177 / 136 / 108

a IARC classification groups: 1=carcinogenic to humans; 2A=probably carcinogenic to humans; 2B=possibly carcinogenic to humans; 3=not classifiable as to its carcinogenicity to humans.

b IARC, 2012. A review of human carcinogens: chemical agents and related occupations. The International Agency for Research on Cancer, Lyon.

c IARC, 1999. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. The International Agency for Research on Cancer, Lyon.

d IARC, 2000. Some industrial chemicals. The International Agency for Research on Cancer, Lyon.

e IARC, 2002. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. The International Agency for Research on Cancer, Lyon.

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Table S-3. GC-MS operating parameters for analysis of VOC groupI.

Parameter / Description
GC column / DB-5ms (60mx0.32mmi.d.x1.00µm film)
Injector / Cold Injection System (CIS)
Injection mode / Split
Split ratio / 5:1
Injection volume / 1.0µl
Initial injector temperature / 45°C
Injector ramp rate / 12°C/s
Injector final ramp / 295°C
Final time / 6.0min
Carrier gas / Helium
Flow mode / Constant flow
Flow / 2.0ml/min
Initial oven temperature / 35°C
Initial time / 6.0min
Oven ramp rate / 3°C/min
Oven final first temperature / 100°C
Final time first ramp / 0.0min
Oven ramp rate / 30°C/min
Oven final temperature / 255°C
Final time / 2.0min
Total run time / 34.83min
GC outlet / MSD
MSD acquisition mode / SIM
MSD transfer line / 295°C
MSD ion source temperature / 230°C
MSD quadrupole temperature / 150°C
MSD solvent delay / 3.0min

Table S-4. GC-MS operating parameters for analysis of VOC groupII.

Parameter / Description
GC column / forte SolGel-WAX
(30mx0.25mmi.d.x0.25µm film)
Injector / Cold Injection System (CIS)
Injection mode / Split
Split ratio / 5:1
Injection volume / 1.0µl
Initial injector temperature / 35°C
Injector ramp rate / 12°C/s
Injector final ramp / 285°C
Final time / 6.0min
Carrier gas / Helium
Flow mode / Constant flow
Flow / 1.4ml/min
Initial oven temperature / 40°C
Initial time / 5.0min
Oven ramp rate / 5°C/min
Oven final first temperature / 85°C
Final time first ramp / 0.0min
Oven ramp rate / 25°C/min
Oven final temperature / 280°C
Final time / 3.0min
Total run time / 24.80min
GC outlet / MSD
MSD acquisition mode / SIM
MSD transfer line / 295°C
MSD ion source temperature / 230°C
MSD quadrupole temperature / 150°C
MSD solvent delay / 4.0min

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Table S-5. Coefficients of determination, precision, recovery of the internal standards, limits of detection (LODs) and limits of quantification (LOQs) of 12 VOCs in clean solvent standards.

Analyte / Determination
coefficient
(r2)a / Precision
(%)b / Recovery of the internal standards
(%) / LOD
waterpipe
(ng/ml) / LOQ
waterpipe
(ng/ml) / LOQ
waterpipe
(µg/session)
1,3-Butadiene / 0.993 / 7.7 / 88 / 58.5 / 134 / 2.67
Isoprene / 0.996 / 6.3 / 88 / 15.3 / 41.6 / 0.832
Acrylonitrile / 0.997 / 4.2 / 88 / 64.8 / 145 / 2.90
Benzene / 0.998 / 0.57 / 88 / 13.2 / 36.0 / 0.721
Toluene / 0.999 / 0.88 / 90 / 15.5 / 41.9 / 0.837
Ethylbenzene / 0.999 / 1.1 / 87 / 6.75 / 18.9 / 0.755
p-Xylene / 0.999 / 1.6 / 87 / 3.96 / 11.4 / 0.455
m-Xylene / 0.999 / 2.0 / 87 / 7.50 / 20.8 / 0.832
Pyridine / 0.999 / 2.1 / 96 / 11.0 / 29.4 / 1.18
o-Xylene / 0.999 / 0.59 / 87 / 4.42 / 12.7 / 0.506
Styrene / 0.999 / 0.65 / 89 / 4.69 / 13.4 / 0.535
Quinoline / 0.999 / 0.68 / 93 / 5.99 / 16.9 / 0.675

LOD, limit of detection; LOQ, limit of quantification.

a n=3.

b n=6.

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Table S-6. Analyte-specific parameters for eight phenolic compounds.

Analyte / Purity
(%) / CAS number / IARC groupa,b / Retention time (min) / Excitation wavelength (nm) / Emission wavelength (nm)
Hydroquinone / 99.0 / 123-31-9 / 3 / 3.635 / 304 / 338
Resorcinol / 99.0 / 108-46-3 / 3 / 6.194 / 274 / 298
Catechol / 99.0 / 120-80-9 / 2B / 7.045 / 274 / 298
Phenol / 99.5 / 108-95-2 / 3 / 10.095 / 274 / 298
Guaiacol / ≥ 98.0 / 90-05-1 / – / 11.172 / 274 / 298
m-Cresol / 99.0 / 108-39-4 / – / 16.080 / 274 / 298
p-Cresol / 99.0 / 106-44-5 / – / 16.266 / 274 / 298
o-Cresol / 99.0 / 95-48-7 / – / 17.630 / 274 / 298

a IARC classification groups: 1=carcinogenic to humans; 2A=probably carcinogenic to humans; 2B=possibly carcinogenic to humans; 3=not classifiable as to its carcinogenicity to humans.

b IARC, 1999. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. The International Agency for Research on Cancer, Lyon.

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Table S-7. RP-HPLC-FLD operating parameters for analysis of phenolic compounds.

Parameter / Description
LC column / Develosil 3u RP-Aqueous C30 140A (150mmx3.0mm, 3µm particle size)
Injection volume / 10.0µl
Oven temperature / 40°C
Flow rate / 0.55ml/min
Solvent A / 1.0% Acetic acid in water
Solvent B / 0.1% Acetic acid in acetonitrile
1. Excitation wavelength / 304nm
1. Emission wavelength / 338nm
2. Excitation wavelength / 274nm
2. Emission wavelength / 298nm
1. Detection window (304/338nm) / 0–4.8min
2. Detection window (274/298nm) / 4.8–24.0min

Table S-8. Composition and timing of the gradient applied for the analysis of phenolic compounds.

Time (min) / Solvent A (%) / Solvent B (%)
0.00 / 97 / 3
3.00 / 97 / 3
5.00 / 81 / 19
15.00 / 81 / 19
17.00 / 15 / 85
19.00 / 15 / 85
21.00 / 97 / 3
24.00 / 97 / 3

S-10

Table S-9. Coefficient of determination, precision, limits of detection (LODs) and limits of quantification (LOQs) of eight phenolic compounds in clean solvent standards.

Analyte / Determination
coefficient
(r2)a / Precision
(%)b / LOD
waterpipe
(ng/ml) / LOQ
waterpipe
(ng/ml) / LOQ
waterpipe
(µg/session) / LOQ
water
(µg/session)
Hydroquinone / 0.999 / 4.3 / 5.54 / 14.8 / 1.11 / 11.1
Resorcinol / 0.999 / 4.2 / 3.02 / 8.49 / 0.637 / 6.37
Catechol / 0.999 / 3.1 / 11.6 / 27.9 / 2.09 / 21.0
Phenol / 0.999 / 3.6 / 1.29 / 3.77 / 0.283 / 2.83
Guaiacol / 0.999 / 3.1 / 1.97 / 5.67 / 0.426 / 4.26
m/p-Cresol / 0.999 / 2.8 / 4.79 / 13.7 / 1.02 / 10.2
o-Cresol / 0.999 / 4.1 / 3.39 / 9.46 / 0.710 / 7.1

LOD, limit of detection; LOQ, limit of quantification.

a n=3.

b n=6.

S-17

Table S-10. Recovery of eight phenolic compounds in fortified solvent spikes.

Analyte / Recovery (%)
25ng/ml
n=5; (RSD, %) / Recovery (%)
250ng/ml
n=5; (RSD, %) / Recovery (%)
1000ng/ml
n=5; (RSD, %)
Hydroquinone / 86.6 (5.7) / 96.9 (4.1) / 92.2 (0.6)
Resorcinol / 94.3 (4.3) / 94.2 (1.6) / 96.5 (0.2)
Catechol / – / 90.5 (4.1) / 93.9 (0.6)
Phenol / 104 (2.7) / 92.5 (0.9) / 91.8 (1.4)
Guaiacol / 92.7 (12) / 91.6 (0.9) / 89.9 (2.7)
m/p-Cresol / 90.0 (3.1) / 84.2 (1.0) / 85.1 (0.4)
o-Cresol / 96.9 (4.1) / 80.7 (2.3) / 80.2 (1.8)

RSD, relative standard deviation.

Table S-11. Analyte-specific parameters for 17 elements.

Analyte / Purity
(%) / IARC groupa / Internal standard / Ion mass
(m/z)
Lithium (Li) / – / – / Scandium / 7
Beryllium (Be) / – / 1b / Scandium / 9
Aluminum (Al) / – / – / Scandium / 27
Vanadium (V) / – / – / Scandium / 51
Chromium (Cr) / – / 3c / Scandium / 52
Manganese (Mn) / – / – / Scandium / 55
Cobalt (Co) / – / 2Bd / Scandium / 59
Nickel (Ni) / – / 1b / Scandium / 60
Copper (Cu) / – / – / Scandium / 65
Zinc (Zn) / – / – / Scandium / 66
Arsenic (As) / – / 1b / Rhodium / 75
Molybdenum (Mo) / – / – / Rhodium / 95
Cadmium (Cd) / – / 1b / Rhodium / 111
Tin (Sn) / – / – / Rhodium / 117
Barium (Ba) / – / – / Rhodium / 135
Thallium (Tl) / – / – / Bismuth / 205
Lead (Pb) / – / 2Be / Bismuth / 206, 207, 208
Scandium (Sc; IS) / TraceCERT® / – / – / 45
Rhodium (Rh; IS) / Certipur® / – / – / 103
Bismuth (Bi; IS) / Certipur® / – / – / 209

a IARC classification groups: 1=carcinogenic to humans; 2A=probably carcinogenic to humans; 2B=possibly carcinogenic to humans; 3=not classifiable as to its carcinogenicity to humans.