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Identification of halogenated
disinfection byproducts of nonylphenol
in chlorinated wastewater effluent
using novel high resolution GC/Q-TOF
Christiane Hoppe-Jones, Shawn Beitel, Sofia
Nieto, Nathan Eno, Shane Snyder
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Overview
• Background
• Experimental design
• Linear 4-nonylphenol – model compound
• Technical mixture
• Bioassay analysis
– P53
– ER
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Disinfection By-Product Formation
“The formation and control of emerging disinfection by-products of health concern” – Krasner, S.W.
2009, Phil. Trans. R. Soc. A 367: 4077-4095.
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Toxicity of brominated and
iodinated DBPs
“Toxic Impact of Bromide and Iodide on Drinking Water Disinfected with Chlorine or Chloramines” –
Yang, Y. et al. 2014, ES&T 48:12362-12369.
Cytotoxicity/Genotoxicity:
I-DBPs > Br-DBPs > Cl-DBPs
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Why take a closer look and
Nonylphenol?
• Used in antioxidants, lubricating oils, detergents, emulsifiers
• Used in the production of nonylphenolethoxylates (detergents)
• Technical mixture of many isomers
• Known to have estrogenic activity
• On the EPA Drinking Water CCL4 Draft List
1999 Environ. Sci. Tech. 33(16) 2814-2829
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Why take a closer look and
Nonylphenol?
“Occurrence and Maternal Transfer of Chlorinated
Bisphenol A and Nonylphenol in Pregnant Women
and Their Matching Embryos” –
Chen, M. et al. 2016, ES&T 50(2):970-977.
“Ubiquitous Occurrence of Chlorinated Byproducts of
Bisphenol A and Nonylphenol in Bleached Food
Contacting Papers and Their Implications for Human
Exposure” –
Zhou, Y. et al. 2015, ES&T 49(12):7218-7226.
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Objectives
• Confirmation of halogenated by-products reported in the
literature
• Formation of brominated and iodinated by-products
• Identify preferred pathway for three different oxidants:
– Chlorine
– Monochloramine
– Chlorine dioxide
• Test Toxicity and Estrogenicity
“Accurate-mass identification of chlorinated and
brominated products of 4-nonylphenol, nonylphenol
dimers, and other endocrine disrupters” –
Thurman, M. 2006, J. Mass Spectrom. 41:1287-1297.
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Nonylphenol Oxidation
0.04M Phosphate buffer,
1ppm Nonylphenol
6.3uM Bromide, 6.3uM Iodide
4ppm Chlorine
10min
30min
1h
2h
5h
4ppm Chlorine Dioxide
10min
30min
1h
2h
5h
4ppm Monochloramine
10min
30min
1h
2h
5h
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Metrohm 850 IC+ Agilent 7900 ICP-MS
Agilent 7900 ICP-MS
Agilent 6540 LC-Q-TOF
Sample Preparation and Analysis
Samples
LLE w/ Hexane
GC-QTOF
GC-ICP-MS
SPE
LC-QQQLC-QTOF GC-QTOF BioassayLC-ICP-MS
Direct injection
IC-ICP-MS
Agilent 7200 GC-Q-TOF Agilent 7900 ICP-MS
Volatiles and
semi-volatiles
unknownsPolar
unknowns
Halogenated
volatiles and
semi-volatiles
Halogenated
organics
Oxahalides
(BrO3-; IO3
-) and
Ions (Cl-, Br-, I-)
Agilent 6490 LC-MS/MS
Targeted analysis
of identified DBPs
Mutagenicity
Genotoxicity
p53 induction
Cytotoxicity
Oxidative stress
Estrogenic effects
Glucocorticoid effects …
Bioassays
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Sample Preparation and Analysis
Agilent 7200 GC-Q-TOF
• Quenching with Na Thiosulfate
• SPE extraction (HLB)
• Elution (MTBE, MeOH)
• Analysis on GC-qTOF
– DB-5MS UI (30m x 0.25mm x 0.25um)
– Scan m/z 50-1000
• Aliquot transferred to DMSO
• Analysis in bioasssays
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GC Analysis
Agilent 7200 GC-Q-TOF
Column DB-5MS, 30 m, 0.25 mm, 0.25 µm
Injection volume 1 µL
Injection mode 10:1
Split/Splitless inlet temperature 280 °C
Oven temperature program50 °C for 3 min
10 °C/min to 300 °C, 7 min hold
Carrier gas Helium at 1.5 mL/min, constant flow
Transfer line temperature 300 °C
Ionization modeStandard EI at 70 eV;
low energy EI at 15 eV and 12 eV
Source temperature, 70eV/15 eV
or less 240°C/200°C
Quadrupole temperature 150°C
Mass range 50 to 1200 m/z
Spectral acquisition rate 5 Hz
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Data Analysis
• Data processed using SureMass in Unknowns Analysis B.08.00
• Compound identification using NIST14 EI library, confirmation by retention index (RI)
• Molecular ions of unknown brominated compounds were identified with the help of low electron energy
• Molecular ions were confirmed by evaluating the complete cluster for m/z, relative isotope abundance and isotope ratios using Molecular Formula Generator (MFG)
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Chlorine/Chlorine Dioxide Residual
0 30 60 90 120 150 180 210 240 270 300
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
3.5
4.0
4.5
0 30 60 90 120 150 180 210 240 270 300
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
3.5
4.0
4.5
Cl- (
mg
/L)
Time (min)
Chlorine Residual
ClO
2 (
mg
/L)
Time (min)
Chlorine Dioxide Residual
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Bromide, Iodide and Iodate Results
0 30 60 90 120 150 180 210 240 270 300
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
0 30 60 90 120 150 180 210 240 270 3000 30 60 90 120 150 180 210 240 270 300 0 30 60 90 120 150 180 210 240 270 300
0.02
0.04
0.06
0.08
0.10
0.12
0.14
7
8
0 30 60 90 120 150 180 210 240 270 300
1
2
3
4
5
6
7
8
0 30 60 90 120 150 180 210 240 270 300
0.05
0.10
0.15
0.20
0.25
7
8
0 30 60 90 120 150 180 210 240 270 3000 30 60 90 120 150 180 210 240 270 300
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 30 60 90 120 150 180 210 240 270 300
Br- (
M
)
Time (min)Time (min)Time (min)
Chlorine
Time (min)
IO3
- (M
)
Chlorine Dioxide
I- (M
)
Time (min)
Monochloroamine
Time (min) Time (min)Time (min)Time (min)
Br- I- IO3-
Chlorine Chlorine Dioxide Monochloramine
“Iodate and Iodo-Trihalomethane Formation during
Chlorination of Iodide-Containing Waters: Role of
Bromide” – Criquet, J. et al. 2012, Environmental
Science & Technology 46, 7350-7357.
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Chlo
rine
Chlo
rine
dioxi
de
Monoch
lora
min
e
Chlo
rine
Chlo
rine
dioxi
de
Monoch
lora
min
e0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Rela
tive C
on
cen
trati
on
of
DB
Ps
brominated DBPs iodinated DBPs
GC-ICP-MS
Br-DBPs I-DBPs
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Oxidation with chlorine
Nonylphenol
Br-Cl-I-benzene
DBP formation after oxidation with chlorine:
- 10min
- 30min
- 1h
- 2h
- 5h
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Oxidation with Chlorine
Nonylphenol
Monohalogenated DBPs:
Monoisotopic mass:
220.1827
Error: -2.27ppm
NP+Cl2
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Oxidation with Chlorine
Monohalogenated DBPs:
Monoisotopic mass:
254.1432
Error: +1.57ppm
NP+Cl2
Cl-Nonylphenol
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Oxidation with Chlorine
Monohalogenated DBPs:
Monoisotopic mass:
298.0927
Error: -2.01ppm
NP+Cl2+Br-
Br-Nonylphenol
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Oxidation with Chlorine
Monohalogenated DBPs:
Monoisotopic mass:
346.0788
Error: -3.18ppm
NP+Cl2+I-
Br-NonylphenolI-Nonylphenol
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Oxidation with Chlorine
Monohalogenated DBPs:NP+Cl2+Br-+I-
I-Nonylphenol
Br-Nonylphenol
Cl-Nonylphenol
Nonylphenol
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl2-NonylphenolNonylphenol
NP+Cl2
Monoisotopic mass:
288.1042
Error: -1.74ppm
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl,Br-Nonylphenol
Monoisotopic mass:
332.0537
Error: 0.60ppm
NP+Cl2+Br-
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl,Br-Nonylphenol
Monoisotopic mass:
332.0537
Error: 0.60ppm
NP+Cl2+Br-
Br2-Nonylphenol
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl,I-Nonylphenol
Monoisotopic mass:
380.0398
Error: -2.63ppm
NP+Cl2+I-
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl,I-Nonylphenol
Monoisotopic mass:
471.9755
Error: -3.39ppm
NP+Cl2+I-
I2-Nonylphenol
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Oxidation with Chlorine
Dihalogenated DBPs:
Cl,I-Nonylphenol
I2-Nonylphenol
Cl,Br-Nonylphenol
Cl2-Nonylphenol
NP+Cl2+Br-+I-
Br2-Nonylphenol
Nonylphenol
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Identified DBPs after Chlorination
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ç
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Oxidation with Chlorine
Halogenated Phenol DBPs:
Br3-Phenol
Br,Cl2-Phenol
NP+Cl2+Br-+I-
Cl,Br2-Phenol
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Oxidation with Chlorine Dioxide
Nonylphenol
Br-Cl-I-benzene
DBP formation after oxidation with chlorine dioxide:
- 10min
- 30min
- 1h
- 2h
- 5h
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Oxidation with Chlorine Dioxide
Cl,I-Nonylphenol
I2-Nonylphenol
I-Nonylphenol
NP+Cl2+Br-+I-
Nonylphenol
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Identified DBPs after Oxidation with
Chlorine Dioxide
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Oxidation with Monochloramine
Nonylphenol
Br-Cl-I-benzene
DBP formation after oxidation with monochloramine:
- 10min
- 30min
- 1h
- 2h
- 5h
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Oxidation with Monochloramine
Cl,I-Nonylphenol
I2-NonylphenolCl,Br-Nonylphenol
Nonylphenol
NP+NH2Cl+Br-+I-
Br-Nonylphenol
I-Nonylphenol
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Identified DBPs after
Monochloramine
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Nonylphenol (technical mixture)
0.04M Phosphate buffer,
3ppm Nonylphenol
30 min
12ppm Chlorine
✖ Br- ✖ I-
✔ Br- ✖ I-
✖Br- ✔ I-
✔Br- ✔ I-
12ppm Chlorine Dioxide
✖ Br- ✖ I-
✔ Br- ✖ I-
✖Br- ✔ I-
✔Br- ✔ I-
12ppm Monochloramine
✖ Br- ✖ I-
✔ Br- ✖ I-
✖Br- ✔ I-
✔Br- ✔ I-
12ppm Bromine
✖ Br- ✖ I-
✖ Br- ✔ I-
12ppm Monobromamine
✖ Br- ✖ I-
✖Br- ✔ I-
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Nonylphenol – technical mixture
Nonylphenol
Cl2
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Nonylphenol – technical mixture
Cl2
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Nonylphenol – technical mixture
Cl,Br-Nonylphenol
Cl2-Nonylphenol
Br2-NonylphenolI-Nonylphenol
Br-Nonylphenol
Cl-Nonylphenol
ChlorinationCl2
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Nonylphenol – technical mixture
ClO2 + Br- + I-
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Nonylphenol – technical mixture
NH2Cl + Br- + I-
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Nonylphenol – technical mixture
NH2Cl + Br- + I-
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Identified Mono- and Dihalogenated
DBPs after Chemical Oxidation
Chlorine + Br- + I-
Chlorine dioxide + Br- + I-
Monochloramine + Br- + I-
Most abundant
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p53 in vitro Bioassay
• Uses CellSensor™ p53-
bla HTC-116 cell based
assay (Life
Technologies)
• This is a cell line that
has a fluorescence
based reporter gene
spliced into the cell to
allow the identification
of agonists/antagonists
of the p53 pathway.
• This assay can be used
as an indicator of DNA
damage.
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Linear NP – p53 bioassay
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Estrogen Receptor alpha (ERalpha) in vitro Bioassay
Estradiol
Cytoplasm
nucleus
hsp90
Binding to XRE
ER
ER target
genes
Reporter
gene
Fluorescence
• Uses ER alpha-UAS-blaGripTite cell based assay (Life Technologies)
• Measures the relative estrogenic response of a compound or mixture through binding of the estrogen receptor alpha
• Increase affinity to the receptor and/or increased concentration of estrogenic compounds lead to increase in fluorescence
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Tech. NP – ER bioassay
Estrogenicity decreased proportionally with the
decrease of the NP concentration
DBPs are not as potent as NP
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Summary/Next Steps
• Chlorinated, brominated and iodinated DBPs were identified in batch experiments using Cl2.
• I2-NP was the most prominent DBP in experiments using ClO2 or NH2Cl.
• Smaller amounts of brominated DBPs occurred in NH2Cl experiments.
• P53 assay showed – no activity for NH2Cl or NH2Br
– Toxicity in samples containing I- when using Cl2 and Br2
– Toxicity in all samples when using ClO2 in the presence of Br- and I-
• Estrogenicity of all samples decreased proportionally with decreasing NP concentration.
• Measuring NP-DBPs in wastewater effluents– ppt level found in tertiary effluent (NP-Cl,NP-Cl2, NP-Br, NP-Br2)
– Combining results from other analyses (GC-ICP-MS, LC-qTOF) to verify and identify other potential DBPs.
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Questions?
Thank you for your attention!