Nitration processes of

acetaminophen in nitrifying
activated sludges
Serge Chiron, Marseille University
E.Gomez and H.Fenet, Montpellier University

An unexpected biotransformation pathway:

nitration
OH

OH
Nitration
with amonia oxidizing bacteria
HO

HO

17 -ethinylestradiol

Skotnicka-Pitak et al. ES&T 2009

O2N

Cl

Cl

Nitration
Cl

N
H

in CAS batch reactor

Cl

OH

N
H

O
OH

Diclofenac

Perez et al. Anal. Chem. 2008

Nitration

In soil/sludge mixture
HO
353-nonylphenol

HO
NO2
Telscher et al. ES&T 2005

Objectives
To investigate the nitration mechanisms
of phenolic compounds in nitrifying
activated sludge.
At different scales: field-, batch- and
molecular-scale experiments.
Acetaminophen (paracetamol) as a probe
compound.

Why paracetamol ?
NHCOCH3

pKa = 9.4
logKow = 0.45
Sw > 1g/L
OH

- Readily prone to nitration

- High occurrence in WWTPs (1-10 g/L range)

- Nitrated derivatives can be easily synthetized

Field studies: Targeted compounds

110

NHCOCH3

(+) ESP

(+) ESP

NHCOCH3

NHCOCH3

126

195

(-) ESP

1
6
5

2
3

NO2

OH
168

OH

OH

OH

Acetaminophen
(APAP)

3-OH-APAP
(I)

108

3-nitro-APAP
160
(II) 150

152
150
134

(+) ESP

144

NHCOCH3

(-) ESP

NHCOCH3

(-) ESP

NHCOCH3

229

195

240

Cl
OH

3-chloro-APAP
(III)
109

O2N

Cl

O2N
OH

3-chloro-5-nitroAPAP
(IV)

NO2
OH

184

3,5-dinitro-APAP
(V)
199

186
212

165
150

Analytical methodology
On-line SPE-LC-MS/MS

Compound
3-OH-APAP

Recoveries
(%)
55 ( 12)

APAP

75 ( 8)

3-nitro-APAP

78 ( 8)

3-chloro-APAP

86 ( 6)

3-chloro-5-nitroAPAP

93 ( 4)

3,5-dinitroAPAP

95 ( 5)

Aix-en-Provence WWTP effluent analysis

2,4-D d3
Representative MRM chromatogram obtained in (-) ESP LC-MS/MS (I.S)

Preconcentration volume: 50 mL
Grit removal
Influent

Primary
clarifier

Stage-2

Stage-1

X
Conventional
Activated
Sludge

Nitrifying
Activated
Sludge

Secondary
clarifier
Final
Effluent

X: sampling point

3-nitro-APAP
0.21 0.02 g/L

APAP
0.18 0.02 g/l

Time (min)

3-chloro-5-nitro-APAP
0.028 2x10-3 g/L

Field data (24 h composite samples)

Sampling
month
Stage-2
influent

Stage-2
effluent

APAP

3-OHAPAP

3-chloroAPAP

3-nitroAPAP

3-chloro-5nitro-APAP

Oct. 2008

3.45

0.21

0.96

0.11

0.24

0.02

n.d

n.d

Nov. 2008

5.35

0.32

1.44

0.17

0.85

0.04

n.d

n.d

Dec. 2008

6.75

0.41

1.86

0.22

0.76

0.04

n.d

n.d

Oct. 2008

0.19

0.02

n.d

n.d

0.18

0.02

0.03 1x10-3

Nov. 2008

0.35

0.03

n.d

n.d

0.26

0.03

0.11 5x10-3

Dec. 2008

0.64

0.05

n.d

n.d

0.32

0.03

0.09 3x10-3

Batch experiments
Experimental conditions:
10

[MLSS] = 2.5 g/L

T = 25 C
[O2] > 3 mg/L
[NH4+] = 10 mg/L
[APAP]0 = 100 g/L

[3-chloro-APAP]0 = 100
g/L

Concentration (mg/L)

pH = 7-7.5

NH4-N

NO3-N

8
6
4
NO2-N

2
0
0

50

100

Time (h)

150

200

Batch 1 experiment: no nitrification inhibition

3-chloro-APAP

Concentration (x10-7M)

[NH4-N]0 = 10 mg/L
3-OH-APAP [APAP]0 = 100 g/L
[3-chloro-APAP]0 =
100 g/L

Concentration (x10-8M)

APAP
3-nitro-APAP

3-chloro-5-nitroAPAP

0
0

50

100

Time (h)

150

200

Batch 2 experiment : ammonia oxygenase inhibition

7

10

Concentration (x10-7M)

3-OH-APAP

5
6

4
3-chloroAPAP

2
2

3-nitro-APAP and 3-chloro-5-nitro-APAP

1
0

50

100

Time (h)

150

200

Concentration (x10-8M)

[Allylthiourea] = 5 mg/L

APAP

Reaction of APAP with HNO2

2-nitro-APAP
NHCOCH3

148

(-ESP)-LC/MS
[APAP] = 70 M
[NO2-] = 0.1 mM
pH = 5

NO2

165
123
OH

2-nitro-APAP

195

Dimer
178

Trimer
APAP

Time (min)

Proposed mechanism of 2-nitro-APAP formation

NHCOCH3

NCOCH3

HNO2
pH < 5.5

OH pKa (HNO2/ NO2- ) = 3.4

APAP

1,4 Michael addition

NHCOCH3
NO2

NO2-

O
N-Acetyl-p-benzoquinone-imine

OH
2-nitro-APAP

Matsumo et al. Chem. Pharm. Bull. 1989

Reactivity of APAP with horseradish peroxidase

3,5-dinitro-APAP

(-ESP)-LC/MS
[APAP] = 70 M
[peroxidase] = 80 nM
[NO2-] = 0.1 mM
[H2O2] = 0.2 mM
Phosphase buffer 100 mM / pH 7.5

Dimer

3-nitroAPAP

Time (min)

Trimer

Reactivity of APAP with peroxynitrite

(-ESP)-LC/MS
[APAP] = 70 M
[ONOOH] = 5 mM
[HCO3-] = 5 mM
[Cl-] = 5 mM
[DOM] = 10 mg/L
Phosphate buffer 100 mM / pH 7.5

3-nitro-APAP
Dimer

APAP
3-OH-APAP

Time (min)

Trimer

Proposed mechanism of 3-nitro-APAP formation

ONOO- + H+

.
.
NO2 + OH
.
.-

ONOOH
ONOO- + CO2

NO2 + CO3

NHCOCH3

OH

k = 5.3 x 109 M-1s-1

k = 1 x104 M-1s-1

NHCOCH3

NCOCH3

High selectivity

CO3

pKa = 6.8

ONOOH

High reactivty

.-

NO2

k=

APAP
Polymerization

2.109 M-1s-1

NO2
OH
3-nitro-APAP

Where is peroxynitrite coming from?

NH4+ oxidation by ammonia oxidizing bacteria (AOB)
_ First

step

NH3 + O2 + 2H+ 2e-

Ammonia mono-oxygenase
(AMO)

NH2OH + H2O

NO (side-product)

- Second step
NH2OH + H2O

Hydroxylamine oxidoreductase

- Third step

O2 + eNO + O2

.-

ONOO- + H+

O2

HNO2 + 4H+ + 4e-

.-

ONOO-

k = 1.9 x 1010 M-1s-1

ONOOH

pKa = 6.8

Conclusions
Formation of nitro-APAP derivatives with a
environmental profile more worrisome than
APAP.
Nitration is probably linked to .NO generation
by nitrifying bacteria.
This result must be validated with other
phenolic pollutants (i.e. bisphenol A,
nonylphenol).

Nitration processes in the environment

Photonitration
NO3 + h + H2O
OH + NO2 + OH
NO2 + h + H2O
OH + NO + OH
NO2 + OH

NO2 + OH

= 0.01]
[ 2 = 0.02-0.07]
1

[k3 = 1.0 1010 M 1 s 1]

Thermal nitration by HNO2 (in the dark)

pKa (HNO2/ NO2- ) = 3.4 relevant at pH< 5.5

Bionitration by peroxidases in presence of

NO2- and H2O2
Bionitration by ONOOH (peroxynitrite)
.
.
NO + O2 -

ONOO-

k = 1.9 x1010 M-1s-1

ONOO- + H+

ONOOH

pKa = 6.8

ONOO- + H+

.
NO2 + OH-

k = 5.3 x 109 M-1s-1

Expected biotransformation pathways

OH

NH2
Hydrolysis

O
N
H

N
H

O
OH
Atenolol

O
OH

Radjenovic et al. J. Chromatogr. A. 2008

Cl

HO

Cl

Hydroxylation
N
H

Cl

N
H

Cl

OH

OH

Diclofenac

Perez et al. Anal. Chem. 2008

OH

OH
O

O
O

N
H

OH

H
N
I

Iopromide

O
Oxidation

OH
OH

N
H

OH
O
H
N

Schulz et al. ES&T 2008

OH
OH
O