Presentation: Developing Science-Informed Policy Responses to Curb Endocrine Disruption in Freshwater, Ron von der Oost - Toxicologist, Waternet (water company for Amsterdam and surrounding area)

1. Effect-based SIMONI strategy
for risk assessment of
endocrine disrupters and other
micropollutants
OECD EDC workshop Paris, October 2022
Ron van der Oost

2. Classificatie: Intern
Waternet
Only Dutch Water Cycle Institute
Surface water, Waste water, Drinking water
Amsterdam area, The Netherlands
EOCD EDC workshop

3. Classificatie: Intern
• Introduction effect-based monitoring
• SIMONI strategy
• Results
• Conclusions & future developments
Outline
EOCD EDC workshop

4. Classificatie: Intern
Effects or substances..?
www.cas.org
EOCD EDC workshop

5. Classificatie: Intern
Bioanalytical tools:
 Limited amount of assays can give a cost-
effective and reliable risk assessment
 Low substance specificity
 Bioavailability included
 Mixture toxicity included
 Metabolites included
 Unknown substances included
 Chronic exposure is difficult and expensive
 No accepted classification available
 Biomagnification not included
 No effects  no worries
….
Chemical analyses:
 Search for a needle in a haystack: obligatory
analysis of more then 200 substances in
drinking water
 Many analyses are yet impossible (e.g. matrix
effects)
 Not enough toxicity data available for risk
assessment (ERA)
 No information on bioavailability
 No information on mixture toxicity
 Direct comparison to substance-directed legal
guidelines
 Low concentrations  still worries
 Surrogate security and accuracy
….
Monitoring effects or substances..?
Effect-based trigger values!
De Zwart et al. 1995 EOCD EDC workshop

6. Classificatie: Intern
Effect-based surface water quality monitoring
Biomarkers:
Biochemical changes
Bioassays
Passive sampling
Feral fish Caged fish
EOCD EDC workshop

7. Classificatie: Intern
• Introduction effect-based monitoring
• SIMONI strategy
• Results
• Conclusions & future developments
Outline
EOCD EDC workshop

8. Classificatie: Intern
msPAFmetals & NH3
Metals & NH3 in water
ACCEPTABLE
RISK
LOW RISK
INCREASED
RISK
Toxicology:
in vivo bioassays
EDA
Concentrated water
by passive or active
sampling
Toxicology
(bioassays)
Biology KRW+
(msPAF ecology)
SRI < 0.5
> Trigger values?
Advanced chemistry:
msPAF for more
new substances
SRI ≥ 1.0
SRI 0.5-1.0
SIMONI & EKF Toxicity
TIER 2:
CUSTOMIZED
RESEARCH
TIER 1:
HAZARD/RISK
IDENTIFICATION
HIGH RISK!
Van der Oost et al., 2017

9. Classificatie: Intern
Toxicological endpoints SIMONI
• In situ toxicity (water):
• Daphnids: mortality (1 week)
• General toxicity (concentrated extracts):
• Cell culture: cytotoxicity
• Bacteria: luminescence
• Algae: growth inhibition
• Daphnids: mortality (immobilisation)
• Specific toxicity (concentrated extracts):
• Endocrine disruption: ER, anti-AR, GR, anti-PR
• Xenobiotics metabolism (DR, PXR)
• PAH toxicity
• Lipid metabolism: PPAR
• Antibiotics activities (5 classes)
• Reactive toxicity (concentrated extracts):
• Genotoxicity
• Oxidative stress
CALUX gene
expression in vitro
assays
EOCD EDC workshop

10. Classificatie: Intern
Effect-based trigger values SIMONI
In vivo aquatic toxicity data: PNEC, NOEC, LOEC, EC50 and LC50
→ Conversion to BEQ (bioanaly cal equivalents in bioassay)
Field investigations
Realistic benchmark approach
Species sensitivity distribution
(SSD) analysis on BEQs
Safe approach
Lowest BEQ values divided by
assessment factor
Concentrations that will
negatively affect 5% of the
species
Bioassay results at sites with
good ecological status
Safe BEQ HC5 BEQ Background BEQ
EBT
EOCD EDC workshop

11. Classificatie: Intern
WWTP
undilluted
Greenhouse area
Moderately polluted:
Rivers Amstel & Vecht
References
SIMONI Risk Indication (overall risk)
<0.5: low risk 0,5-1,0: acceptable risk >1,0: increased risk
SRI = mean cumulative risk quotient: (∑bioassay effect/EBT)* weight factor
EOCD EDC workshop

12. Classificatie: Intern
TIER 2: Effect-Directed Analyses (HT-EDA)
Fractionation UPLC
Bioassays in all fractions
MS-TOF bioactive fractions
Extract
Identification
EOCD EDC workshop

13. Classificatie: Intern
water sample
bioassay test of
non-fractionated extract
extraction
bioassay chromatogram
HT-EDA
confirmation
with analytical
standard
1. Bioassay
total extract
3. Bioassay
chromatogram
4. Identification
2000 targets
5. Identification
unknowns
SIMONI TIER 2 workflow with HT-EDA platform
Houtman et al., 2020
2. Target analyses:
Mainly pesticides
& pharmaceuticals

14. Classificatie: Intern
Human & environmental SIMONI strategy
TIER 2:
CUSTOMIZED
RESEARCH
TIER 1:
HAZARD/RISK
IDENTIFICATION
Environment Human health
• Bioanalytical screening
• Metals & ammonium
• Bioanalytical screening
• Broad spectrum chemistry
• Combined EBT
• SRI: SIMONI Risk Indication
• Individual EBT
• DW guidelines
Assessment
• Broad spectrum chemistry
• In vivo bioassays (ZET)
• Effect-directed analysis
• Non-target analyses
• Advanced bioassays
• Effect-directed analyses
Measures to remove identified hazardous substances
EOCD EDC workshop

15. Classificatie: Intern
• Introduction effect-based monitoring
• SIMONI strategy
• Results
• Conclusions & future developments
Outline
EOCD EDC workshop

16. Classificatie: Intern
Results Waternet 2011-2021
Greenhouse area Landfill runoff
Sewage overflows
WWTP effluent
EOCD EDC workshop

17. Classificatie: Intern
SRI rivers Amstel & Vecht and greenhouse areas
EOCD EDC workshop

18. Classificatie: Intern
SIMONI surface waters 2021
landfill
sewage overflow
agriculture
sewage overflow
EDA
• Estrogenic
• Anti-androgenic
• Pregnane X
• Cytotoxicity
EOCD EDC workshop

19. Classificatie: Intern
Estrogenic activity (ER CALUX) POCIS extracts
0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14 16
Activity
in
CALUX
bioassay
(%
of
max.
light
induction
)
Retention time (min)
HAP007 Landfill runoff ER
0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14 16
Activity
in
CALUX
bioassay
(%
of
max.
light
induction
)
Retention time (min)
SBI034 sewage overflow ER
1
6
5
4
3
2
6
5
4
3
1. Unknown (40 compounds detected)
2. Dimethyl phtalate + ..?
3. Estriol (E3) & bisphenol A
4. Unknown (10 compounds detected)
5. Estradiol (E2), Ethinyl estradiol
(EE2), Estrone (E1)
6. Unknown (10 compounds detected)
EOCD EDC workshop

20. Classificatie: Intern
Endocrine bioassay chromatograms river and WWTP
Fold
induction
0
5
10
15
Fold
induction
0
1
2
3
4
5
Time (min)
Fold
induction
0 5 10 15 20 25
0
50
100
150
WWTP Effluent
WWTP Influent
Meuse
A.
B.
C.
River Meuse
WWTP influent
WWTP effluent
Bioassay
effects
Androgenic activity
Glucocorticoid activity
Estrogenic activity estriol
estradiol
estrone
/EE2
BPA
octyl phenol
dihydroepiandrosterone
sulfate
androsterone
sulfate
androstenedione
testosterone
DH-testosterone
dihydrodexamethasone
beclomethasone-
monopropionate
Retention time
Zwart et al., 2020 EOCD EDC workshop

21. Classificatie: Intern
• Introduction effect-based monitoring
• SIMONI strategy
• Results
• Conclusions & future developments
Outline
EOCD EDC workshop

22. Classificatie: Intern
Conclusions
• TIER 1 SIMONI is able to identify the risks of a wide range of
micropollutants with effect-based methods & ‘simple’ chemistry
• Highest micropollutants risks were observed at sites polluted by
greenhouse emission, sewage overflows, WWTPs and landfill runoff
• EDA was able to identify bioactive substances using reporter-gene
bioassays
• Identification of unknown compounds is still time-consuming, and
thus quite expensive…
EOCD EDC workshop

23. Classificatie: Intern
Perspectives
• Key Factor Toxicity 2.0 has been developed as a follow-up SIMONI
strategy (Milo de Baat)
• Continuous evaluation of effect-based trigger values and overall risk
indication (SRI)
• Extended number of bioanalytical endpoints for HT-EDA platform by
Water Laboratory Haarlem and VU University Amsterdam
• Cost and time reduction for identification of unknown compounds
by automation of web-based searching protocols
EOCD EDC workshop

24. Classificatie: Intern
24
More info: ron.van.der.oost@waternet.nl
EOCD EDC workshop
Bianca, Giulia, Maria, Laura, Corine, Sofia, Mai Thao