investigating the potential of transformation products of
TRANSCRIPT
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
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
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