Effect-directed analysis (EDA) : a case study applied to endocrine disruptor identification in a river impacted by industrial discharges

Transcription

1 Effect-directed analysis (EDA) : a case study applied to endocrine disruptor identification in a river impacted by industrial discharges Budzinski Hélène 1 Gardia-Parège Caroline 1, Creusot Nicolas 2, Dévier Marie-Hélène 1, Aït- Aïssa Selim 2, Balaguer Patrick 3 1- Université Bordeaux 1/CNRS, LPTC/EPOC 2- INERIS 3- INSERM ECOT U896

2 CHEMICAL PRODUCTS CHEMICAL CONTAMINANT Human activities = thousands of compounds released in the environment MORE THAN 18 MILLIONS OF MOLECULES REGISTERED AT CAS MONDIAL PRODUCTION RISE FROM 1 MILLION OF TONS IN 1930 TO 400 MILLIONS OF TONS NOWADAYS MORE THAN 9O % CONTAIN CARBON MORE THAN SUBSTANCES ARE COMMONLY USED AT HIGH QUANTITIES HOW MANY REALLY REPRESENT A RISK FOR HUMAN HEALTH AND/OR THE ENVIRONMENT REACH (Europe) Water Framework Directive (WFD) (Europe)

3 Sources and origins of contaminants Natural Origin Chloroalcanes Metals Alkylphenols Urban Origin PAHs Exposure from media (air, water ) Trophic exposure from food Pharmaceuticals PAHs Alkylphenols PAHs Chloroalcanes Nitrates Industrial Origin COV Pesticides Agricultural Origin Metals Alkylphnols

4 Thousands of molecules Use - spill Uncomplete degradation - spill Presence - Impact?

5 Emerging Contaminants hydrophilic compounds Focazio et al. (2008) We analyze well what we know well : problems of coincidences Impossible to analyze everything New compounds Unknown compounds (transformations, ) What compounds and why? - Link Presence - Effect?

6 Analyzed Compounds Polycyclic Aromatic Hydrocarbons (PAHs) Organo-Chlorine Compounds Pesticides, Polychlorobiphenyls, Dioxins, Furans Flame retardants (PolyBorminatedDiphenylEhers (PBDE), ) Pharmaceutical Substances, Cosmetics PFOS, PFOAS Alkylphenols polyethoxylates (detergents) Pesticides Phtalates Bisphenol A BTEX, Chlorinated Solvents, MTBE/ETBE,.

7 Environmental Contaminations Low levels (ex : ng/l) (sensitivity traces and ultra-traces) Case of hormones : toxic effects at ng/l EQN : endosulfan 0.5 ng/l!!! Complex Matrices Multi-contaminants - Mixtures (specificity / multi-residues) Link presence - effects? Variability / Representativeness (spatial / temporal)?

8 CONTAMINATION? Chemical approach Biological approach In vitro Tests Detection of targeted compounds Sensitive, selective Expensive Unknown compounds, transformation products, mixture effect? Detection of active compounds Sensitive, specific/integrative, rapid, quantitative Identification of active compounds in relation with the observed responses Contaminants Bio-analytical Approach? Effects

9 To find active compounds remaining unknow Effect Directed Analysis (EDA) approach (from Brack 2003)

10 Context Sanchez, W. et al. Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges. Environ. Int. 37, (2011) River with Pharmaceutical industry Waste Water Treatment Plant and an Urban Waste Water Treatment Plant (WWTP) Gonadal hypertrophy, spawning insufficiency, intersexuality and VGT introduction Endocrine disruptor contamination Research of the compounds responsible for the observed effects

11 Context Water targeted chemical analyses No molecules found Concentrations < LD Wrong targeted molecules Passive Sampler = POCIS In vitro tests

12 Preliminary study Concentration in POCIS (Thèse, N. Creusot 2011) Pharmaceutical Industry WWTP Upstream X X Urban WWTP X Downstream Sampling campaign for the water column contamination 3 sampling locations : Upstream, Midstream and Downstream Passive sampling: POCIS Stainless POCIS steel ring PES membrane (polyethersulfone) Adsorbant phase OASIS-HLB Phase [Poly(divinylbenzene)-co-N-vinylpyrrolidone PES membrane Stainless steel ring Linear uptake Equilibrium Time

13 Enzymatic activity (%) Preliminary study Steroids receptors Xenobiotic receptors Molecule Natural cellular response In vitro bioassays based on reporter cell lines Endogenous gene Gene activation Receptors Line Environmental ligands References Reporter gene Luminescent or fluorescent signal Estrogen (ER) Androgen (AR) Glucocorticoid (GR) MELN MDA-kb2 E1,E2, EE2, alkylphenols, BPA, pharmaceuticals DHT, BPA, vinclozoline pharmaceuticals Balaguer et al Wilson et al Standard range Sample Progesterone (PR) HG5LN-hMR Steroids pharmaceuticals Molina-Molina et al.2006 Mineralocorticoid (MR) HG5LN-hPR Steroids pharmaceuticals Molina-Molina et al.2006 Dioxin (AhR) PLHC-1 / H4IIE HAP, PCB, PCDD/F... Louiz et al Pregnan (PXR) HG5LN-hPXR E2, alkylphenols, pharmaceuticals Lemaire et al Cette étude CE 20 Reference compound TEQ-Bio = CE 20 of the extract

14 Preliminary study In vitro activitiesum (µg EQ-standard/g de phase) (Thèse, N. Creusot 2011) Pharmaceutical Industry WWTP Upstream X X WWTP X Downstream Upstream Amont Midstream Entre Downstream Aval

15 Preliminary study TEQ-Bio (µg Standard/ g HLB) In vitro activity of POCIS extract (Thèse, N. Creusot 2011) ER PR GR anti-mr PXR Steroids and pharmaceutical compounds suspected Targeted chemical analyses

16 Preliminary study TEQ-Chem/TEQ-Bio (%) (Thèse, N. Creusot 2011) Targeted chemical analyses (LC MS/MS) Research of 113 compounds 61 compounds detected: 16 steroids 45 pharmaceutical compounds 120 TEQ-Chem= ΣCi x TEFi Dexamethasone CE20 detected compounds TEF = CE 20 reference compound Canrenone 6α methyl Prednisolone 0 ER PR GR anti-mr PXR EDA for research of active and no identified compounds

17 Experimental approach Activité enzymatique (%) RP-HPLC Fractionement 2 Characterization 3 Gross Sample 1 Biological analyses Confirmation Identification Chemical analyses (inspiré Brack 2003) Targeted analyses (LC MS/MS) Non targeted analyses (LC QTOF) 40 collected fractions List of compounds

18 Experimental approach 2 Fractionement Characterization 3 Gross Sample 1 Biological analyses Identification Chemical analyses Confirmation (inspiré Brack 2003) List of compound s

19 Activity (% relative to positive control) Results 140 Profil biologique Biological profile MR ER PXR PR a.mr GR Focus on some activities for compound search : glucocorticoid like mineralocorticoid receptor antagonist GR a.mr PR PXR ER MR

20 Activité Activity (% par (% relative rapport to au poistive contrôle control) positif) Results Targeted Analyses Analyses ciblées ? a.mr GR a.mr GR 6α methyl Prednisolone 34% GR 124% a.mr Dexamethasone + 6α méthyl Prednisolone 35% GR 63% a.mr Canrenone 30% a.mr 21

21 Non targeted chemical analysis using Agilent LC- QTOF accurate mass system Sample F15 Screening: (MS result) > 250 compounds Targeted MS/MS analysis to confirm the identity of n candidates identification of several corticosteroid-like chemicals in the F15

22 Abondance en % Dore F15 Riluzole amyotrophic lateral sclerosis therapeutic Database : Metlin MS- Diff Diff (abs. Diff Formula Mass Mass (DB) RT Score (ppm) ppm) (mda) C8 H5 F3 N2 O S Extract ion chromatogram MSMS-Score MS-Score 98.55

23 Results Non Targeted Analyses HPLC Fraction F15 F20 Nb of compounds Nb of MSMS spectra Agilent LC-QTOF Formyldienolone 344,1989 Da diff -0,39ppm Score MS: 99,36% Score MSMS: 79,13% Pregn-4-ene-3,20-dione, 6a,17-dihydroxy-6-methyl- 360,2301 Da diff -0,09ppm Score MS: 98,43% Score MSMS: 71,62% Simvastatin-6'-carboxylic acid Da diff -0,2ppm Score MS: 96,53% Score MSMS: 94,17% 2-hydroxymethylprogesterone 342,2197Da diff -0,57ppm Score MS: 98,18% Score MSMS: 79,01% Cicaprost Da diff -1,68ppm Score MS: 98,42% Score MSMS: 93,25%

24 Conclusion and perspectives Association of passive samplers, in vitro tests and effect directed analysis (EDA) for the detection of active compounds Considerable benefit of sensitive high resolution mass spectrometry techniques Effect Directed Analysis (EDA) to reduce the complexity of samples in order to identify compounds not detected by targeted analysis Link presence - effect Next step : Validation of identified compound in vitro activities But : Very time consuming and complex strategy, Loss of synergistic or antagonist effects : needs of go and return back (balances at each step) Even after fine fractionation, fractions remain sometimes too complex

25 Aknowledgment French Ministry for Ecology, Sustainable Development and Energy The European Regional Development Fund (FEDER) European Union (CPER A2E project ) The Regional Council of Aquitaine

26 Thank you for your attention