22 October 2019

ICRA, Girona, Spain

Investigating the potential of transformation products

of antibiotics formed during advanced wastewater treatment

to induce adverse biological effects and antibiotic resistance

Vasiliki Beretsou

Suspect

screeningNon-target

screening

Biological

effects?

Hazard?

Antibiotic

resistance?

Risk?

Target

screening

Exposure?

Academic background

B.Sc. in Chemistry (2013)Department of Chemistry

University of Athens (Greece)

M.Sc. in Analytical Chemistry (2015)Department of Chemistry

University of Athens (Greece)

Ph.D. in Environmental Engineering (2016-ongoing)Department of Civil and Environmental Engineering

Nireas-International Water Research Center (Nireas-IWRC)

University of Cyprus (Cyprus)

ESR14Vasiliki Beretsou

March 2013 - June 2013:

Internship at Pesticides Residues’ Laboratory

General Chemical State Laboratory of Greece (Athens, Greece)

Food analysis (pesticide residues by LC–MS/MS and GC–MS/MS)

Validation of analytical methods

October 2014 - December 2015:

Analytical Chemist at Laboratory of Analytical Chemistry

Department of Chemistry, University of Athens (Greece)

Food analysis (vitamins, aminoacids, pharmaceutical and veterinary residues

and aflatoxin M1 by LC-MS/MS and HPLC (UV/FLD))

Environmental analysis

Development of analytical methods

Working experience

Analytical chemistry (contaminants of emerging concern) –

Food and environmental analysis

Development and validation of multi-residue analytical methods

High-Resolution Mass Spectrometry (HR-MS)

Sample preparation techniques for chromatographic analysis

Identification of transformation products (TPs) of pharmaceuticals

through the use of target, suspect and non-target screening approaches

Scientific expertise

Title: Investigating the potential of transformation products (TPs) of

antibiotics (A) formed during advanced wastewater treatment to

induce biological adverse effects and antibiotic resistance

Lead Beneficiary: UCY, Nireas-IWRC

Advisor: Dr. Despo Fatta-Kassinos

Duration: April 2016 – March 2019

1. elucidate the structures of TPs of selected A formed during light-driven (solar,

UV, LED) + H2O2 oxidation

2. evaluate their potential of being active after their release in the environment and

to contribute to the development of resistance in soil bacteria

3. evaluate the potential of the A/TPs to induce toxicity,

cyto/muta/estro/genogenicity, thyroid and glucocorticoid disruption, etc and

understand the implications that this may have on ecological and human health

4. establish correlation factors between TPs, biological effects and resistance

development

Objectives according to the GA

Objectives of the ESR14 project

Wastewater reuse is considered

the most suitable and reliable alternative

for sustainable water management and

agricultural developmentChemical and biological

contaminants of emerging concern

Antibiotics and TPs

Antibiotic-resistant bacteria

Antibiotic resistance genes

Advanced Oxidation Processes

Free hydroxyl radicals

Highly reactive

Non-selective

Mineralization

down to CO2, H2O

and inorganics

Background

Krauss et al. 2010, Analytical and Bioanalytical Chemistry 397(3), 943-951

High Resolution Mass

Spectrometry

(HR-MS)

Determination of all present

compounds in full scan mode

without preselection

Accurate mass measurements

Allows differentiation of

isobaric compounds

Elemental composition of both

parent and fragment ions

Data-dependent & independent

MS/MS acquisition

Retrospective analysis

Approaches for the identification of TPs

Target screening

Non-target screening

The increasing knowledge in cell pathways led to the

development of chemically-induced toxicity novel bio-based

assays alternative to animal testing

For bioassays to be used as monitoring tools it is important

to include sensitive early warning endpoints, such as the

onset of cellular repair and defense mechanisms, to

complement effects that are based on the ultimate

manifestation of effect, such as DNA damage and formation

of reactive oxygen species

Relevant early warning signs can be proposed to be the

p53 protein damage and the induction of the Nrf2 pathway-

Relevant to UV-C/H2O2 oxidation!

Effect-based analysis

Compound Structure Molecular formula MW pKa logKow

Azithromycin

C38H72N2O12 748.9 8.7 4.02

Ofloxacin

C18H20FN3O4 361.4 5.7/7.9 -2.00

Trimethoprim

C14H18N4O3 290.3 6.9 0.91

Sulfamethoxazole

C10H11N3O3S 253.3 1.6/5.7 0.7

Target antibiotics

Ofloxacin

Can be regarded as ubiquitous in urban wastewater and is frequently detected

in surface water - Prior knowledge in the scientific literature regarding the

identification of its TPs with various treatment processes

Quinolones are derivatives of nalidixic acid, which was discovered as a

byproduct of the synthesis of chloroquine (a quinine) and function primarily by

inhibiting the DNA-gyrase or topoisomerase activity of bacteria

piperazine ring

fluoroquinolone core

Fluoroquinolone

core structure

DNA synthesis, mRNA transcription and cell division require the modulation of

chromosomal supercoiling through topoisomerase-catalysed strand breakage and

rejoining reactions

Case-study compound: ofloxacin

Integrating chemical and effect-based analysis to assess the target

antibiotics and their transformation products during UV-C and UV-

C/H2O2 oxidation

[A]0=100 μg/L

Addition of H2O2

Conventional Activated Sludge (CAS)

Wastewater

effluents

Matrix

Initial

concentration

Ultrapure

water

&[A]0=2 mg/L

&

Concentration of

oxidant

Experimental set-up

Compilation of a suspect list:

in silico prediction tools (Eawag PPS & MetPredict) &literature

Screening all time interval samples both in RP &

HILIC chromatography in bbCID mode (+ESI/-ESI)

Evaluation of candidate TPs:

Criteria, Presence in treated samples,

Absence from blank & control samples & t0 sample

Acquisition of MS/MS spectra in AutoMS mode with both RP & HILIC:

Interpretation of fragmentation pathway

Confirmation (if possible), with reference standard (tR & MS/MS spectra match)

1. Identification of TPs: Suspect screening workflow

Background subtraction (Bruker’s MetaboliteDetect 2.0)

Generation of mass list of unknown

Treat further as “suspect peaks”

Metabolite

Detect

Blank

sample

Background

subtraction

Treated

sample

Generation of mass

list of “unknown”

Background Subtraction

Parameters

Difference

eXpose mode, Ratio: 3-5

Detect -Mass spectrum

Parameters

Int. threshold: 30%

Max No. of peaks: 20

1. Identification of TPs: Non-target screening workflow

Schymanski et al. 2015, Environmental Science & Technology, 48(4), 2097-2098

1. Identification confidence levels of TPs

Until now…

Now…

WHO is there?

WHAT are they doing?

CALUX® (Chemically Activated LUciferase eXpression) assays exhibiting

good sensitivity, robustness and reproducibility are utilized as screening

tools for the effect-based analysis in the studied water matrices

2. Biological effects: CALUX assay principle

Kern et al. 2009 Environmental Science and Technology, 43(18), 7039–7046

Chemical analysis of the wastewater of Cyprus for the determination of

antibiotics and their transformation products

Sampling and Sample Preparation

24-hour composite

influent & effluent

wastewater samples

enrichment of the extract with a

broad range of compounds conditioning

loading

drying

elution4 different extraction sorbents

Oasis HLB → for neutral & aromatic compounds

Strata-X-AW → for acidic compounds with pKa<5

Strata-X-CW → for basic compounds with pKa>8

Isolute ENV+ → for polar compounds

100 mL

basic solution

acidic solution

evaporation

reconstitution

(500 μL MeOH/H2O 50/50)

Future Work: Cyprus - The real case-study of WW reuse

bbCID

fragments:

molecular

formula &

exact mass

Antibiotic: name, CAS number, category

molecular formula

retention time

exact mass of precursor

ion

also:

adducts &

in-source

fragmentsDetection

Confirmation

Target screening

Future Work: Cyprus - The real case-study of WW reuse

Biotransformation

Degradation

Recalcitrant

Preliminary results influent

effluent

Future Work: Cyprus - The real case-study of WW reuse

• Summer School, 13-23 June 2016, Barcelona, Spain

• Training Event B, 7-8 December 2016, Amsterdam,

The Netherlands

“Sample preparation and effect-based monitoring in water

quality assessment”

Training experiences within the Network

• Training Event C, 5-10 March 2017, Rehovot, Israel

“Microcontaminants in the aquatic water cycle-wastewater reuse:

The Cypriot/Israeli experience”

• Training Event E, 4 September 2017, Fisciano, Italy

“WW treatment by advanced technologies and risk assessment framework”

• 1st ANSWER Workshop, 5-6 September 2017, Fisciano, Italy

“Risk prognosis of environmental and public health aspects of antibiotics and

antibiotic-resistant bacteria and antibiotic resistance genes (A&ARB&ARGs)”

Training experiences within the Network

• Training Event F, 23-26 January 2018, Bela, Slovakia

“Practical exercise on computer tools for identification and structure elucidation

of antibiotics, their metabolites and TPs”

• Training Event H, 20-21 June 2018, Amsterdam, The Netherlands

“Environmental and human health risk assessment of antibiotics”

• 2nd ANSWER Workshop, 18-19 June 2018, Amsterdam, The Netherlands

“Modelling and risk assessment tools towards sustainable WW reuse”

Training experiences within the Network

Secondments

0.5 m at EI

0.75 m at HighChem

4.5 m at BDS

2.25 m at ARO

1 m at ISS

to get training on mass spectra

reading

to evaluate the potential of A and TPs

to contribute to antibiotic resistance

to structurally elucidate TPs

to work on advanced bioassays for

TPs testing

to obtain knowledge on

human risk assessment

according to the GA

Secondments

Dissemination and Outreach activities

• Participation in International Conferences

• Visit to Schools

• Radio Talk

• Articles in newspapers

• Participation in Cyprus Researchers' Nights

• Participation in a documentary of the ANSWER project as a success

story from the Net4Mobility (a Network of MSCA NCPs)

Impact of MSCA fellowship on my career

Intersectoral and multinational mobility has increased my own

flexibility and adaptability in different working environments

(both in research and industrial environments)

ANSWER has promoted not only my research skills but also

complementary and transferrable skills like preparation of

research proposals, project management and the development of

strong written and oral communication skills

Through networking and cooperation, I have managed to expand

my knowledge in other scientific fields and areas

A valuable asset both in the case you want to pursue a position

within or outside the academia

Dr. Emanuela Testai

Dr. Robert Mistrik

Miroslav Smrek

Dr. Michal Gramlicka

Dr. Harrie Besselink

Matthijs Naderman

Leon Schweisshelm

Dr. Jaroslav Slobodnik

Dr. Eddie Cytryn

Prof. Nikolaos Thomaidis

Dr. Maria-Christina Nika

ANSWER Network and Collaborators

Acknowledgements: The ANSWER project has received funding from the European Union’s

Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant

agreement No 675530.

Disclaimer: The content of this presentation is only the author’s view and the Agency is not

responsible for any use that may be made of the information it contains.

Prof. Despo Fatta-Kassinos

Dr. Irene Michael-Kordatou

Dr. Lida Ioannou-Ttofa

Dr. Costas Michael

Dr. Popi Karaolia

Dr. Magda Psichoudaki

Mr. Iakovos Iakovides

Ms. Stella Michael

Ms. Anna Korelidou