1

Biotic and abiotic degradation studies

of organic contaminants

Manuela Peschka

Ankara, 8th October 2007

Outline

• Organic pollutants

Selected compounds

• Simulation of abiotic/biotic degradation

Fixed bed bioreactor

Bottle test

Suntest

• Analysis of micopollutants and identification of transformation products

Sample pretreatment

Analytical Instrumentation

• Results:

Case study I: barbiturates

Case study I: photodegradation of bentazone

Case study II: biodegradation of a new fluorosurfactant

Case study IV: biotransformation of clotrimazole

2

pesticides

surfactants

pharmaceuticals

OrganicMicropollutants

HN SO2

N

O

bentazone

F

F

F

OS

O

O

O

10-(trifluoromethoxy) decane-1-sulfonate

C NH

N OO

O

R3

R1

R2

barbiturates

abiotic degradation

biodegradation

monitoring

OrganicMicropollutants

NN

Cl

clotrimazole

Degradation Tests:

3

sampling

10 L storage tank

aerator

fixed bed

pump

Fixed Bed Bioreaktor

5 L storage tankaerator

Bottle Test

4

quartz tubes

xenon arc lampE = h·ν

Suntest – simulation of abiotic photodegradation

Sample Pretreatment:

5

200mbar

sorbent

OAIS® HLB Reversed Phase material with HHydrophilic – LLipophilic BBalanceA = 810 m2/gpore diameter = 80 ÅV = 1,3 cm3/gparticle diameter = 60, 30, 25, 15 und 5 µm

NO

NO

hydrophilicmonomer

lipophilicmonomer

1 atm1 atm

6

SO3-

N

O

SO3-

O NH2

OH

OAIS® MCX

MMixed-Mode CCation eXXchange and Reversed Phase1,0 meq/g SO3H Ion exchange capacity

OAIS® MAX – Phasenchemie

MMixed-Mode AAnion eXXchange and Reversed Phase

COO-

CH3O

S

R+

NO

CH2

N+ CH3H3C

C4H9

7

A B

– typical solvents:

• methanol

– methanol with acid

– methanol with base

• acetonitrile

• acetic acid

• THF

• acetone

• dichlormethan, chloroform

• hexane

increasing elution potential

Elution (for non polar SPE)

Analysis

Analytical Instrumentation:

8

signalconvertion

vacuum system

result

injector ion source detectormassmass analyzeranalyzer

10-5 – 10-8 Torr

General setup of a mass spectrometer

chromatographic separation

GC-MS

9

Advantage: utilization of mass spectra librariesstrong fragmention gives structural information

Disadvantage: single MSEI only in combination with GC

LC-MS

10

Advantage: - analysis of polar, thermolabile compounds

- very robust for routine analysis

Disadvantage: - no spectral libraries

- matrix effects

Quadrupole Time of Flight Mass Spectrometer

UezvmEkin **²** ==21

Uezm **²** =⎟⎠⎞

⎜⎝⎛

21

21

²²

*)**(Lt

Uezm 2=

L = ca. 2m

11

Advantage: - high resolution, e.i. determination of up to the

fourth dezimal (e.g. 255.0432 amu)

- determination of the elemental composition

- MS²

Disadvantage: - not sensitive

QqLIT

Advantage: - structure elucidation of unknown compounds

- high sensitivity

Disadvantage: ?

- user can select various modi, e.g. Q1MI, MRM, product ion scan

- enhanced modi: Q3 acts as trap resulting in higher sensitivity

12

Ion Ion chromatographychromatography

CH3 CH3

S SO

O-

O OO

O-

A+

A+

E+

E+

flow direction

stationary phase

-determination of inorganic paramter, e.g. SO42-, Cl-, F-

- mineralization of a molecule

GC-MSLC-MS

QqTOF

QqLIT

monitoringmonitoring

structurestructureelucidationelucidation

(IC)

13

Case studies:

2,08

0,21 0,

49

0,28

0,09 0,12 0,30 0,39

0,30 0,40 0,51

0,98

2,48

1,41

0,86

4,55

0,31

0,29

0,28

0,19

0,22 0,27

0,88

0,10

0,10

1,5

0,3

0,6

5,45,3

0

1

2

3

4

5

2004

-04-

02

2004

-04-

12

2004

-04-

30

200

4-05

-30

2004

-09-

30

2005

-02-

02

2005

-02

mix

2005

-03-

28

2005

-04-

08

2005

-04-

09

2005

-04-

10

2005

-04-

11

2005

-04-

12

2005

-05-

07

2005

-07-

01

2005

-07-

02

2005

-07-

03

2005

-11-

13

conc

. [µg

/L] pentobarbital

butalbitalsecobarbitalphenobarbital

time [h]0 2 13 21

conc

entra

tion

[ng/

L]

0

500

1000

1500

2000 aprobarbitalpentobarbitalbutalbitalsecobarbitalhexobarbitalphenobarbital

Fixed Bed BioreactorExposion to sunlightHydrolysis (pH 2 – 12)

groundwater in Berlin

Barbiturates

C NH

N OO

O

R3

R1

R2

14

Bentazone

HN SO2

N

O

• one of the most applied herbicides in the Ebro river delta • 31 µg/L in application time from May to August (mean value) (Ebro 0.27 µg/L)• comparable high concentrations river waters of the Tiber region in Italy

• not biodegradeable in a fixed bed bioreactor• passes through membrane bioractor• present in ground- and surface water

quartz tubes

xenon arc lampE = h·ν

0

500

1000

1500

2000

2500

3000

3500

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

0

20

40

60

80

100

120

140

Are

a (S

O3-

bent

azon

e)

bentazoneSO3-bentazone

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

0

50

100

150

200

250

Are

a (S

O3-

bent

azon

e)

bentazoneSO3-bentazone

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

0

20

40

60

80

100

120

140

160

180

200

Are

a (S

O3-

bent

azon

e)

bentazoneSO3-bentazone

B - moderately hard water

A – distilled water

C – moderately hard water plus 5 µg/mL humic acids

bentazone

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

bentazone

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

bentazone

0

500

1000

1500

2000

2500

3000

3500

0 2 4 7 9 11 13 14 16 18 20 23

Time [h]

Are

a (b

enta

zone

)

bentazone

15

observed mass(m/z)

elementalcompositionactual mass

∆ mDa

255.0432 ±0.0013 C10H11N2O4S

m/z = 255.0440 - 0.8

197.0044C7H5N2O3S

m/z = 197.0021 2.3

191.0826

C10H11N2O2m/z = 191.0821 0.5

132.0320C7H4N2O

m/z = 132.0324 - 0.4

HN SO2

N

O

OH

NH

HN

O

SO

OHO

observed mass (m/z)

elemental composition actual mass

∆ mDa

257.0584 ± 0.0034

C10H13N2O4S m/z = 257.0596 - 1.2

177.1024 ± 0.0003

C10H13N2O m/z = 177.1028 - 0.4

92.0497 C6H6N m/z = 92.0500 - 0.3

present in drainage channels in the Ebro delta

HN SO2

N

O

0

5

10

15

20

25

30

35

40

1999 2000 2001 2002 2003 2004 2005

An

zah

l d

er

Pu

blikati

on

en

(P

rim

ärl

itera

tur)

Fluorosurfactants

F

F

F

OS

O

O

O

properties:

- persistent to hydrolysis, oxidation, reduction and biodegradation- accumulate in liver, kidneys, mussel tissue and bind to blood proteins

blood

16

10-(trifluormethoxy)decane-1-sulfonate

β = 100 mg/LV = 5 L surface waterpH 7T = room temperature (20°C)

Biodegradation in a Biodegradation in a FixedFixed BedBed BioreaktorBioreaktor

AnalyticalAnalytical methodologiesmethodologies

Ion Ion ChromatographyChromatographyF-, SO4

2-, TOCQQ--ToFToF

high resolutionmass spectrometry

QQ--TrapTrapMS³-experiments

17

massmass spectrumspectrum of 10of 10--(trifluormethoxy)(trifluormethoxy)--

decanedecane--11--sulfonatesulfonate

F

F

F

O

S

O

O

O

m/z = 85

m/z = 80

F3CO

(CH2)10

SO3

[M-H]-

m/z = 305

SO42-

OSO3

F3C

F3C O O

F3C O COO

F3C O COOH

F3C O COOH

oxidation

beta-oxidation

- CO2

-CO2

F3C OH

- HF

CO2 F-

O

F F

H

H

SO42-

oxygenase

SO3-

(CH2)x(CH2)x

OF3C

O

- F-

HO SO3

HOOC SO3

HOOC SO3

- CO2

SO42-

beta-oxidation-CO2

18

MonitoringMonitoring of TOC, of TOC, FF-- and and SOSO4422--

0

5

10

15

20

25

30

35

40

0 10 20 30 40Time [d]

c (F

- , S

O4

2- ,

TO

C)

[mg

/L]

0,00

0,20

0,40

0,60

0,80

1,00

1,20

c/co

(Tri

flu

orm

eth

oxyd

eca

nsu

lfo

nat)

TOC F- SO42- primary degradation

metabolites (m/z = 85)

OSO3

F3C

after 28 days:

- primary degradation: 100%- > 90 % SO4

2- - concentrationtheoret.- 58 % F- - concentrationtheoret.

F3C O O

F3C O COO

F3C O COOH

F3C O COOH

oxidation

beta-oxidation

- CO2

-CO2

F3C OH

- HF

CO2, F-

O

F F

H

H

SO42-

oxygenase

SO3-

(CH2)x(CH2)x

OF3C

O

?

19

0,E+00

1,E+04

2,E+04

3,E+04

4,E+04

5,E+04

6,E+04

0 5 10 15 20 25 30

Time [d]

are

a

SO3-

(CH2)x(CH2)x

O

F3C

O

0

5

10

15

20

25

30

35

40

0 10 20 30 40Time [d]

c (F

- , S

O4

2- ,

TO

C)

[mg

/L]

0,00

0,20

0,40

0,60

0,80

1,00

1,20

F- SO42-

20

[M-H]-

* *

*

* * **

C11H18F3SO5-* 319.0833

SO3-

(CH2)x(CH2)x

O

F3C

O

F

F

F

OS

O

O

OO

-CO

SO3

oxidation in omega-position!

MSMS²² MSMS³³

85

233

205

233

CO

SO3

[M-H]-

319233

21

OSO3

F3C

oxygenase

SO3-

(CH2)x(CH2)x

OF3C

O

- F-

HO SO3

HOOC SO3

HOOC SO3

- CO2

SO42-

beta-oxidation-CO2

C6H11SO5-195.0333

SO3OOC

SO3HOOC

97.0058

195.0341

22

F3C O O

F3C O COO

F3C O COOH

F3C O COOH

oxidation

beta-oxidation

- CO2

-CO2

F3C OH

- HF

CO2, F-

O

F F

H

H

SO42-

OSO3

F3C

> 90%fast

oxygenase

SO3-

(CH2)x(CH2)x

OF3C

O

<10%slow

- F-

HO SO3

HOOC SO3

HOOC SO3

- CO2

SO42-

beta-Oxidation-CO2

494bp [°C]

141 – 145mp [°C]

3.31*10-7Vp [Pa]

0.49Swater [mg/L]

4.1logKOW

344.84MW [g/mol]

NN

Cl

Mode of action:Inhibitor of ergosterolsynthesis resulting in the damage of cell membrane.

23

approx. 2 ng/L

False positive signal in next four measured samples(solvent - acetone)!

0

1

2

3

4

5

6

0,00 2,00 4,00 6,00 8,00 10,0 12,0 14,0pH

log

KO

W

N

N

ClNH

N

Cl

Log KLog KOWOW versusversus pHpH

24

• Acidification to pH 2pH 2 (stirring 30 min) of samples before filtration• SPE-Material Oasis HLB 3cc (60 mg), Waters• conditioning: 1 x 2 mL n-hexan, 3 x 2 mL methanol

5 x 2 mL groundwater (pH 2pH 2) • SPE: 100 ng Atrazin D5 (internal standard)

flow approx. 20 mL/min• drying: 45 min (N2)• elution: 4 x 1 mL acetone• evaporation: to 150 µL• external standard: 100 ng Fluazifop-buthyl (ESTD) and fill up to

200µL final volume with acetone

adapted SPE-Method

Adsorption hinders standardized biodegradation tests

FBBR

0

0,2

0,4

0,6

0,8

1

1,2

0 2 4 6 8 10 12 14t [d]

c/c0

two hours after FBBR passage

25

OHphase I

CRz.B. glucoronic acid

SH

phase II

reaction with „electrophiles“

reaction with „nucleophiles“

5 L storage tankaerator

1 1 5 11 50 60 68 68 68 98

0,00E+00

5,00E+05

1,00E+06

1,50E+06

2,00E+06

2,50E+06

3,00E+06

3,50E+06

Are

a

Time [d]

CLT-metabolite clotrimazole

26

478.1478.1

EPI

- Cl

Cl

Cl

NH3

O

HO

NH3

O

O

NN

Cl

HONH3

O

O

86.1

118.1

165.1

199.1

242.2

277.1

478.1

27

SummarySummary::

bentazone

newfluorosurfactant

barbituratesclotrimazole

FateFate& &

DegradationDegradation

no biotic nor abioticdegradation⇒ persist in theaquatic enironmentfor several years

no bioticdegradation butundergoesphotolysis⇒ degradable in sunlight exessablecompartments

undergoes biodegradation⇒ potential substitute for the harmfulfluorosurfactants presently used

biotransformed⇒ furtherinvestigations

AcknowledgementAcknowledgement::

• AQUATERRA (Project number 505428 GOCE)

• Spanish Ministry of Education and Science

(Project number CTM2005-25168-E)

• Merck KGaA

ThankThank youyou forfor youryour attentionattention!!