a new insight into non-extractable residue formation

K. M. Nowak

, C. Girardi

, A. Miltner

, A. Schäffer

, M. Kästner

Biotransformation of ibuprofen in soil:

a new insight into non-extractable residue formation

1 Dept. Environmental Biology and Chemodynamics, Institute for Environmental Research, RWTH Aachen University, Germany

2Dept. Environmental Biotechnology, UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany

INTRODUCTION EXPERIMENTAL RESULTS CONCLUSIONS PLANS…

INTRODUCTION

MASS BALANCE OF A XENOBIOTIC IN SOIL

Microbial biomass

Incorporation

CO

Xenobiotic

compound

Xenobiotic metabolites

SOM NER

NER structure??

Risk??

Simple systems!

Soils: mostly quantification!

1. Sequestered NER

Clay particle xenobiotic

xenobiotic binding

2. Chemically bound

INTRODUCTION EXPERIMENTAL RESULTS CONCLUSIONS PLANS…

INTRODUCTION

BioNER FROM A XENOBIOTIC IN SOIL

Incorporation

CO

Microbial biomass Xenobiotic

compound

Xenobiotic metabolites

SOM NER

BioNER pathways?

Biomass residues

Fixation

Starvation

SOM

NER

Page 4

INTRODUCTION RESULTS CONCLUSIONS PLANS…

INTRODUCTION

IBUPROFEN (IBU)

EXPERIMENTAL

 Anti-inflammatory and analgesic drug

 Most commonly consumed drug

 Detected in effluents and sewage sludge

 Biodegraded in soil

 High NER content NER structure?? Risk??

13

C

-Ibuprofen

M

- 206.28 g/mol

H

O solubility - 21 mg/L

log K

- 3.5

INTRODUCTION RESULTS CONCLUSIONS PLANS…

INTRODUCTION

13 C ₆ -IBU EXPERIMENT

EXPERIMENTAL

• Living biomass:

(PLFA and bioAA)

• Total in soil:

(non-living + living biomass:

tFA and tAA)

Extractable compound residues (parent compound +

primary metabolites)

Non-extractable residues (NER) (EA-C-IRMS)

GC/MS;

GC-C-IRMS

13

C

-IBU RESIDUES

• Darkness, 20ºC; 60% of WHC

• ¹³C₆-IBU: 20 mg/kg

• 2, 4, 14, 28, 59 and 90 days

• 21% clay, 68% silt, 11% sand

• Abiotic, ¹³C-abundance controls

Labelled IBU

H₂O Safety NaOH traps

trap pump

CO

(TIC;

GC-C-IRMS)

Page 6

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

C-MASS BALANCE ( C ₆ -IBU)

EXPERIMENTAL RESULTS

incubation time (days)

0 20 40 60 80

% of applied 13 C (from ring-labeled ibuprofen) 0 20 40 60 80 100

mineralization extractable

ibuprofen and metabolites

non-extractable residues

extractable,

unknown composition

BIOTIC ABIOTIC

incubation time (days)

0 20 40 60 80

% of applied 13 C 6 ibuprofen 0 20 40 60 80 100

proteins mineralisation

extractable ibuprofen and metabolites

NER extractable,

unknown composition

• Mineralisation: high

• NER: high → bioNER?

•

C-IBU + metabolites:↓

• Mineralisation: low

• NER: low → time dependant

•

C-IBU + metabolites: high NER = microbial activity!

Nowak et al, 2013 Nowak et al, 2013

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

INCORPORATION OF ¹³ C INTO FA and AA

EXPERIMENTAL RESULTS

• PLFA: fast

• PLFA:↓

incubation time (days)

0 10 20 30 40 50 60 70 80 90

13 C in FA fraction (% of initial 13 C 6-IBU equivalents) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

1.6 _tFA

PLFA

FATTY ACIDS

Nowak et al, 2013

• bioAA: fast

• bioAA:↓

• tAA: 27%!

incubation time (days)

0 10 20 30 40 50 60 70 80 90

13 C in AA fraction (% of initial 13 C 6-IBU equivalents) 0 5 10 15 20 25 30

35 tAA

corrected biomass AA biomass AA

AMINO ACIDS

Nowak et al, 2013

• G

markers: initial degraders

• G

markers: later phase

• Starvation marker:↑over time

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

C INCORPORATION INTO BIOMASS

EXPERIMENTAL RESULTS

incubation time (days)

0 10 20 30 40 50 60 70 80 90

13 C-PLFA (% of initial 13 C 6-ibuprofen)

0.0 0.2 0.4 0.6 0.8 1.0

normal

methyl branched monounsaturated polyunsaturated cyclopropyl

PLFA

• Aspartate: initially → CO

fixation

• diverse bioAA: later phase

incubation time (days)

0 10 20 30 40 50 60 70 80 90

13 C-bioAA (% of initial 13 C 6-IBU equivalents) 0 1 2 3 4

ala gly thr val

-ala leu ile pro asp glu phe lys

bioAA

Nowak et al, 2013 Nowak et al, 2013

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

CALCULATION OF TOTAL BioNER

EXPERIMENTAL RESULTS

Incorporation of

C into the biomass of C. necator grown on

C

-2,4-D

tFA instable (Nowak et al, 2011)! THUS tAA → calculation

Conversion factor of ~ 2 for tAA (proteins) Name Incubation time (days) [% of

C

-2,4-D]

2 3 7 14

Biomass 9.2 ( ± 1.5) 10.0 ( ± 1.5) 14.5 ( ± 1.8) 17.4 ( ± 0.06) PLFA 0.5 ( ± 0.02) 0.6 ( ± 0.01) 0.8 ( ± 0.05) 0.6 ( ± 0.04) bioAA 4.7 ( ± 0.2) 6.1 ( ± 0.3) 7.3 ( ± 0.2) 8.1 ( ± 0.4)

Biomass/PLFA 18.4 17 18 29

Biomass/AA 1.9 1.6 2 2.1

13C₆-2,4-D Cupriavidus necator JMP 134

GC/MS;

EA-C-IRMS GC-C-IRMS

• ¹³

C in PLFA and bioAA

• Total ¹³

C in biomass

Nowak et al, 2011

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

C-MASS BALANCE ( C ₆ -IBU)

EXPERIMENTAL RESULTS

General mass balance New mass balance incl. BioNER

incubation time (days)

0 20 40 60 80

% of applied 13 C (from ring-labeled ibuprofen) 0 20 40 60 80 100

mineralization extractable

ibuprofen and metabolites

non-extractable residues

extractable,

unknown composition

incubation time (days)

0 20 40 60 80

% of applied 13 C (from ring-labeled ibuprofen) 0 20 40 60 80 100

proteins biogenic residues

mineralization extractable

ibuprofen and metabolites

non-extractable residues

extractable, unknown composition

Nearly all NER biogenic!

• extractable (unknown composition): bioNER?

Nowak et al, 2013 Nowak et al, 2013

INTRODUCTION EXPERIMENTAL RESULTS CONCLUSIONS PLANS…

INTRODUCTION

BioNER FROM CO ₂ FIXATION

Incorporation

CO

Microbial biomass Xenobiotic

compound

Xenobiotic metabolites

SOM NER

BioNER pathways?

Biomass residues

Fixation

Starvation

SOM

NER

INTRODUCTION RESULTS CONCLUSIONS PLANS…

INTRODUCTION

CO ₂ FIXATION EXPERIMENT

EXPERIMENTAL

• Living biomass:

(PLFA)

GC/MS;

GC-C-IRMS

13

C-LABEL ANALYSES

CO₂ CO₂

CO₂

HCL

Na₂CO₃ Unlabelled 2,4-D

(20 mg/kg) H₂O

Labelled 2,4-D Safety NaOH traps trap

pump

13

CO

experiment

C

-2,4-D experiment

INCUBATION

•

CO

fixation (day 16)

• PLFA: decline

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

INCORPORATION OF ¹³ C INTO PLFA

EXPERIMENTAL RESULTS

incubation time (days)

0 10 20 30 40 50 60

13 C-label distribution in soil (%)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

13CO₂ fixation

13C₆-2,4-D 13

C-incorporation into PLFA

(

C

-2,4-D and

CO

experiments)

incubation time (days)

0 10 20 30 40 50 60

% of applied 13 C (from ring-labeled 2,4-D) 0 20 40 60 80 100

mineralization

extractable, 2,4-D and meta- bolites

non-extractable residues extractable,

unknown composition

13

C-mass balance in

C

-2,4-D experiment

Nowak et al, 2011

Nowak et al, 2011

INTRODUCTION CONCLUSIONS PLANS…

INTRODUCTION

C IN PLFA CLASSES

EXPERIMENTAL RESULTS

• G

markers: initially

incubation time (days)

0 10 20 30 40 50 60

13 C-PLFA (% of initial 13 CO2)

0.0 0.1 0.2 0.3

0.4 ^normal

methyl branched monounsaturated polyunsaturated cyclopropyl

incubation time (days)

0 10 20 30 40 50 60

13 C-PLFA (% of initial 13 C 6-2,4-D)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

normal

methyl branched monounsaturated polyunsaturated cyclopropyl 13

CO

experiment

C

-2,4-D experiment

• G

markers: initially

Nowak et al, 2011 Nowak et al, 2011

INTRODUCTION PLANS…

INTRODUCTION

FINAL REMARKS

EXPERIMENTAL RESULTS CONCLUSIONS

 NER from

C

-IBU biogenic = no risk!

 NER in abiotic soil low

 tAA high

 bioNER from xenobiotic and CO

HOWEVER:

 no biodegradation → xenobiotic NER

 bioNER → biodegradation (↑CO

) → SOM formation

INTRODUCTION PLANS…

INTRODUCTION

SOM FORMATION

EXPERIMENTAL RESULTS CONCLUSIONS

Decay

Cell wall fragments

Cell wall fragments formation cycle

Growth

Plant

material Patchy fragments on

mineral surfaces

SOM

Starvation

Xenobiotic compound

Miltner et al, 2012

INTRODUCTION PLANS…

INTRODUCTION

FINAL REMARKS

EXPERIMENTAL RESULTS CONCLUSIONS

AND:

 Biotic vs abiotic NER formations (3 types of NER)!

 NER from

C

-IBU biogenic = no risk!

 NER in abiotic soil low

 tAA high

 bioNER from xenobiotic and CO

HOWEVER:

 no biodegradation → xenobiotic NER

 bioNER → biodegradation (↑CO

) → SOM formation

INTRODUCTION PLANS…

INTRODUCTION

NER CLASSIFICATION

EXPERIMENTAL RESULTS CONCLUSIONS

 type I: sequestered NER:

- reversible

- remobilisation → risk for the environment

Clay particle

 type III: bioNER

- biomolecules (amino acids, fatty acids) → SOM - no risk

 type II: chemically bound (covalent bonding)

- irreversible

- low risk for environment

BIOTIC AND ABIOTIC NER FORMATION

INTRODUCTION PLANS…

INTRODUCTION

ABIOTIC vs BIOTIC NER FORMATION

EXPERIMENTAL RESULTS CONCLUSIONS

ABIOTIC

Covalent binding entrapment

RISK

SOM xenobiotic NER

(type I and II) Xenobiotic

compound

Xenobiotic metabolites

Incorporation

Living biomass

Starvation

Biomass residues

Stabilisation

BIOTIC

CO

Fixation

=

SOM BioNER (type III)

low

high no

Kästner et al, in press

 BioNER from other contaminants

(different structure, slower degradation)

 AA: 50%, FA: 5% of BioNER: other components?

 New risk assessment including bioNER formation

INTRODUCTION INTRODUCTION

FURTHER RESEARCH

EXPERIMENTAL RESULTS CONCLUSIONS PLANS…

THANK YOU FOR

YOUR ATTENTION!

EXTRACTION OF BIONER

• Total in soil:

(non-living + living biomass:

tFA and tAA)

FATTY ACIDS AMINO ACIDS

• Living biomass:

(PLFA)

• Living biomass:

(bioAA)

Purification: silica gel  PLFA (CH₃Cl, ACN, MeOH)

• Total in soil:

(non-living + living biomass:

tFA and tAA)

• Phospholipids (PLFA)

• Neutral lipids

• Glycolipids

Extraction:

PB/MeOH/CH₃Cl (0.8/2/1, v:v)

Derivatization:

MeOH/TMCS; 9:1, v:v

GC/MS;

GC-C-IRMS Derivatization:

(MeOH/TMCS; 9:1, v:v )

Purification: silica gel (diethyl ether)

Biomass extraction:

chelating cation exchange resin+

sodium deoxycholate/Polyethylenglycol 600 (0.1%/2.5%)

Hydrolysis: 6M HCl, 110ºC

Purification: DOWEX 50W-X8 (oxalic acid, 0.01M HCl, H₂O, 2.5M NH₄OH)

Derivatization:

(Isopropanol/acetylchloride;

DCM/Trifluoroacetic acid anhydride)

Purification:

(PB:CH₃Cl)