IONIZING RADIATION INDUCED DECOMPOSITION OF ANTIBIOTICS IN WASTEWATER

Erzsébet Takács, László Szabó, Tünde Tóth, Csilla Mohácsi-Farkas,

László Wojnárovits

Wide varieties of toxic organic compounds are entering the aquatic environment. The main sources of these impurities are the wastewater treatment plants for domestic sewage. The co-occurrence of sublethal antibiotic concentration and high dense of microbial population provides ideal condition for facilitating the selection and propagation of resistant bacteria in sewage treatment plants. Conventional methods in sewage treatment plants are not able to completely eliminate the antibiotics. The effluent emitted to the surface waters contains pharmaceuticals and bacteria.

Pharmaceuticals in the aquatic environment

Test compound Concentration

µg ml-1

Biodegradation

after 28 days, %

Biodegradation

after 40 days, %

Cefotiam

dihydrochloride 4.8 7 10

Ciprofloxacin 3.5 0 0 Metropenem 2.5 7 7 Penicillin G 3.0 27 36 Sulfamethoxazole 3.8 0 0

Biodegradation of antibiotics. Results of closed bottle test.a)

a) Al-Ahmad, A., Dashner, F.D., Kummerer, K., Biodegradability of cefotiam,

ciprofloxacin, meropenem, penicillin G and sulfamethoxazole and inhibition of waste bacteria. Archiv. Environ. Contam. Toxicol. 37, 158 (1999)

Pharmaceuticals in the aquatic environment

How to solve this problem?

Post treatment of the wastewater treatment plant effluent by ionizing radiation.

Elimination of the antimicrobial activity and deactivation of the

bacteria in one step.

H2O H, eaq, OH, H+, OH, H2O2, H2

G (H) = 0.06 µm J-1; G(OH) = 0.28 µm J-1; G(eaq) = 0.28 µm J-1

The purpose of our work is:

• To describe degradation mechanisms and determine rate constants for the reactions of the water radiolysis intermediates with the pollutant molecules.

• To suggest the dose necessary to eliminate the toxicity and biological (e.g. antimicrobial) activity of pharmaceuticals.

Radiolysis in aqueous solutions

Mechanism of free-radical induced oxidation

• Pulse radiolysis – Tesla Linac LPR-4 accelerator (4 MeV)

– Pulse width 800 ns

– Dose/pulse 20-40 Gy

• Final product analysis

– 60Co facility with 11.5 kGy h1 dose rate

• LC/ESI-MS analysis

– Agilent 1200 liquid chromatograph

– Agilent 6410 triple quadrupole MS/MS with electrospray ionization (ESI) interface

• CO2-release using a Shimadzu TOC-L equipment

Decomposition kinetics and mechanism of free-radical induced degradation

0 20 40 60

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0 2 4

-0.005

0.000

0.005

0.010

0.015

0 10 20 30

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0 400 800 1200

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

DC

B

MV.+

A

bso

rban

ce

Time (s)

A

= 415 nm

ABTS.+

= 600 nm

A

bso

rban

ce

Time (s)

= 415 nm

A

bso

rban

ce

Time (s)

k' = 1.78 x 10

5 s

-1

= 430 nm

Q.-

A

bso

rban

ceTime (s)

250 300 350 400 450 500 550

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.02 0.04 0.06 0.08 0.1000

1

2

3

4

5

0 2 4 6 8 10 12

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

A

bso

rban

ce

Wavelength (nm)

a

b

c0.0 0.5 1.0 1.5

-6

-5

-4

B

ln(

A)

Time (ms)

Fitting at 325 nm

k' (

s-1)

Concentration (10-3 mol dm

-3)

x 105

A

CKinetic trace at 485 nm

A

bso

rban

ceTime (s)

Decomposition kinetics of penicillin derivative (-lactam) antibiotics

Transient absorption spectra of intermediates in eaq

reaction in N2 saturated AMX solution

Reaction of eaq

10 μs

80 μs

580 μs

250 300 350 400 450 500

0.000

0.005

0.010

0.015

0.020

0.025

0 2 4 6 8 10 12-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.0 0.5 1.0 1.5 2.0 2.5 3.0-0.005

0.000

0.005

0.010

0.015

0.020

0 1 2 3 4 5 6-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

B

A

bso

rban

ce

Wavelength (nm)

a

b

250 300 350 400 450 500

0.000

0.005

0.010

0.015

0.020

0.025

d

c

A

bso

rban

ce

Wavelength (nm)

E

D

C

e290 nm

f260 nm

A

bso

rban

ce

Time (ms)

e

f

350 nm

A

bso

rban

ce

Time (ms)

405 nm

A

bso

rban

ce

Time (ms)

A

Decomposition kinetics of penicillin derivative (-lactam) antibiotics

Transient absorption spectra observed of 0.1 mM amoxicillin solution saturated with N2O (A), and in the same solution containing 0.1 mM K3Fe(CN)6.

Reaction of OH radical

sulfuranyl radical

10 s

400 s

2 s

10 s

K3Fe(CN)6

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 150

1

2

3

4

Ab

un

da

nce

Time (min)

x 106

Mechanism of free-radical induced oxidation of AMX LC-MS/MS

100 300 500 700200 400 600 800

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0

Abundance

m/z

366

731

349

208160114

+ Scan (6.279 min) x10

6

A

100 300 500 700200 400 600 800

0.4

0.8

1.2

1.6

2.0

2.4

2.8

0

B

- Scan (6.311 min) x105

Abundance

m/z

729

364

320

223

Total ion chromatogram

Mass spectra of AMX in positive mode (A) and in negative mode (B)

Final products of

amoxicillin (P9) exposed

to oxidative stress.

Structures suggested based

on LC-MS/MS analysis

Mechanism of free-radical

induced oxidation

Elimination of the toxicity and antibacterial activity

Microbiology

• Biological assays • Acute and chronic effects toward bacteria

• Vibrio fischeri bioluminescence inhibition assay • Vibrio fischeri growth inhibition assay

• Bacterial susceptibility tests • Agar diffusion test

• Staphylococcus aureus • Bacillus subtilis • Eschericia coli

• Broth macrodilution test • Staphylococcus aureus • Eschericia coli

Elimination of the antibacterial activity Tracking the elimination of the pharmacophore

0 2 4 6 80

20

40

60

80

100

Gro

wth

inh

ibit

ion

(%

)

Dose (kGy)

Growth inhibition of Vibrio fischeri in amoxicillin (■), cloxacillin (●) and ampicillin (▲) samples as a function of absorbed dose.

2200 2000 1800 1600 1400 1200 1000 800 600

B

2240 M .OH

280 M .OH

Ab

sorb

ance

Wavenumber (cm-1)

168 M .OH

(d)

1768 cm-12067 cm

-1

(c)

(b)

(a)

A

0 2 4 6 8

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

-l

acta

m c

on

ten

t (m

M)

Am

ou

nt

of

. OH

in

ject

ed (

M)

Dose (kGy)

0

500

1000

1500

2000

2500

(A) FTIR spectra of amoxicillin, (B) Quantitative FTIR analysis of the β-lactam content in the case of amoxicillin (■), cloxacillin (●) and ampicillin (▲) as a function of absorbed dose.

0.6 kGy

1 kGy

0 kGy

Elimination of the antibacterial activity of penicillin derivative (β-lactam) antibiotics

8 kGy

Agar diffusion is a routine method in clinical microbiology to assess bacterial susceptibility to certain antibiotics. It is based on the formation of inhibition zones in the vicinity of the antibiotic solution diffusing in agar media seeded with a certain microorganism.

Agar diffusion test of amoxicillin solutions, treated with increasing dose, on agar plates seeded with B. subtilis (A) and E. coli (B).

Elimination of the antibacterial activity in purified water

Agar diffusion test using S. aureus B.01755 for erythromycin dissolved in the synthetic effluent wastewater matrix received increasing absorbed dose.

Elimination of the antibacterial activity of erythromycin in a complex synthetic wastewater matrix by EB treatment

Sample 0 kGy 4 kGy 8 kGy 12

kGy

16

kGy

20

kGy

24

kGy

28

kGy

1. 21.5 16.0 14.0 12.0 12.5 11.5 - 10.0

2. 21.0 16.0 15.0 14.0 12.5 12.0 11.0 -

Sample 32

kGy 36 kGy

40

kGy

60

kGy

80

kGy

100

kGy

200

kGy

1. 12.0 10.0 - - - - -

2. 10.0 10.0 - - - - -

The absorbed doses should be divided by 500.

80 Gy

humic acid, inorganic salts

SUMMARY

• Based on pulse radiolysis combined with final products analysis degradation mechanism was suggested

• It is important to follow the change in toxicity and in antibacterial activity

• A mild treatment (80 Gy) was just enough to eliminate antimicrobial activity of a complex matrix.

THANK YOU FOR YOUR ATTENTION