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

HO

OH

HO

Nitration

17 -ethinylestradiol Skotnicka-Pitak et al. ES&T 2009

Cl

Cl

NH

OH

O

Nitration

Cl

Cl

NH

OH

O

Diclofenac Perez et al. Anal. Chem. 2008

O2N

HO

Nitration

HO

NO2353-nonylphenol Telscher et al. ES&T 2005

with amonia oxidizing bacteria

in CAS batch reactor

In soil/sludge mixture

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 ?

OH

NHCOCH3

- Readily prone to nitration

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

- Nitrated derivatives can be easily synthetized

pKa = 9.4

logKow = 0.45

Sw > 1g/L

Field studies: Targeted compounds195

160150

NHCOCH3

OH

NO2

3-nitro-APAP

(II)

NHCOCH3

OH

Cl

3-chloro-APAP

(III)

229

212

199

184

NHCOCH3

OH

ClO2N

3-chloro-5-nitro-

APAP

(IV)

186

144

109

240

195

165

150

NHCOCH3

OH

NO2O2N

3,5-dinitro-APAP

(V)

152

134

110NHCOCH3

OH

Acetaminophen

(APAP)

168

126

150

108

NHCOCH3

OH

OH

3-OH-APAP

(I)

(+) ESP (-) ESP (-) ESP

(+) ESP

(+) ESP (-) ESP

1

2

34

5

6

Analytical methodology

Compound Recoveries

(%)

3-OH-APAP 55 ( 12)

APAP 75 ( 8)

3-nitro-APAP 78 ( 8)

3-chloro-APAP 86 ( 6)

3-chloro-5-nitro-

APAP

93 ( 4)

3,5-dinitro-

APAP

95 ( 5)

On-line SPE-LC-MS/MS

Aix-en-Provence WWTP effluent analysis2,4-D d3

(I.S)

3-nitro-APAP

0.21 0.02 g/L

3-chloro-5-nitro-APAP

0.028 2x10-3 g/L

APAP

0.18 0.02 g/l

Representative MRM chromatogram obtained in (-) ESP LC-MS/MS

Preconcentration volume: 50 mL

Grit removalPrimary

clarifierStage-1 Stage-2

Secondary

clarifierX X

X: sampling point

Conventional

Activated

Sludge

Nitrifying

Activated

Sludge

Final

EffluentInfluent

Time (min)

Field data (24 h composite samples)

Sampling

month

APAP 3-OH-

APAP

3-chloro-

APAP

3-nitro-

APAP

3-chloro-5-

nitro-APAP

Stage-2

influent

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

Stage-2

effluent

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

Time (h)

0 50 100 150 200

Co

nc

en

tra

tio

n (

mg

/L)

0

2

4

6

8

10

NH4-NNO3-N

NO2-N

Experimental conditions:

[MLSS] = 2.5 g/L

pH = 7-7.5

T = 25 C

[O2] > 3 mg/L

[NH4+] = 10 mg/L

[APAP]0 = 100 g/L

[3-chloro-APAP]0 = 100

g/L

Batch 1 experiment: no nitrification inhibition

Time (h)0 50 100 150 200

Co

ncen

trati

on

(x10-7

M)

0

2

4

6

Co

ncen

trati

on

(x10-8

M)

0

2

4

6

8

3-chloro-APAP

APAP

3-OH-APAP

3-nitro-APAP3-chloro-5-nitro-

APAP

[NH4-N]0 = 10 mg/L

[APAP]0 = 100 g/L

[3-chloro-APAP]0 =

100 g/L

Batch 2 experiment : ammonia oxygenase inhibition

Time (h)

0 50 100 150 200

Co

ncen

trati

on

(x10-7

M)

0

1

2

3

4

5

6

7

Co

ncen

trati

on

(x10-8

M)

0

2

4

6

8

10

APAP

3-chloro-

APAP

3-OH-APAP

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

[Allylthiourea] = 5 mg/L

Reaction of APAP with HNO2

NHCOCH3

OH

NO2

2-nitro-APAP

195

178

165

148

123

(-ESP)-LC/MS

[APAP] = 70 M

[NO2-] = 0.1 mM

pH = 5

APAP

Dimer

Trimer

Time (min)

2-nitro-APAP

Proposed mechanism of 2-nitro-APAP formation

NHCOCH3

OH

NCOCH3

O

HNO2

pH < 5.5

1,4 Michael addition

NO2-

APAP

NHCOCH3

OH

NO2

2-nitro-APAPN-Acetyl-p-benzoquinone-imine

Matsumo et al. Chem. Pharm. Bull. 1989

pKa (HNO2/ NO2- ) = 3.4

Reactivity of APAP with horseradish peroxidase

Time (min)

Dimer

Trimer

3-nitro-

APAP

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

Reactivity of APAP with peroxynitrite

Time (min)

3-OH-APAP

APAP

Dimer

Trimer

3-nitro-APAP(-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

Proposed mechanism of 3-nitro-APAP formation

NCOCH3

O.

Polymerization

NHCOCH3

OH

APAP

High selectivity

CO3.-

High reactivty

NHCOCH3

OH

3-nitro-APAP

.NO2

NO2

ONOO- + H+ ONOOH pKa = 6.8

.NO2 +

.OH k = 5.3 x 10

9 M-1s-1

ONOO- + CO2.NO2 + CO3

.- k = 1 x104 M-1s-1

ONOOH

k = 2.109 M-1s-1

Where is peroxynitrite coming from?

_ First step

NH3 + O2 + 2H+ 2e-

Ammonia mono-oxygenase (AMO) NH2OH + H2O

.NO (side-product)

- Second step

NH2OH + H2OHydroxylamine oxidoreductase

HNO2 + 4H+ + 4e-

- Third step

NH4+ oxidation by ammonia oxidizing bacteria (AOB)

.NO + O2

.- ONOO- k = 1.9 x 1010 M-1s-1

ONOO- + H+ ONOOH pKa = 6.8

O2 + e- O2

.-

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

PhotonitrationNO3 + h + H2O OH + NO2 + OH [ 1 = 0.01]

NO2 + h + H2O OH + NO + OH [ 2 = 0.02-0.07]

NO2 + OH NO2 + OH [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

NH2

OO

OH

NH

OH

OO

OH

NH

Hydrolysis

Atenolol

Cl

Cl

NH

OH

O

Hydroxylation

Cl

Cl

NH

OH

O

HO

Diclofenac

Radjenovic et al. J. Chromatogr. A. 2008

Perez et al. Anal. Chem. 2008

NO OH

OH

II

I

HN

O

OH

OH

NH

O

O

NO OH

OH

II

I

HN

O

OH

OH

NH

O

O

Oxidation

O

O

Iopromide Schulz et al. ES&T 2008