Belinda Huerta, Victoria Osorio, Marina Gorga, Anna Jakimska, Nuria de Castro, Lidia Ponsati, Isabel Muñoz, Sandra Pérez, Mira Petrovic, Sara

Transcription

1 Uptake and Bioaccumulation of Endocrine Disruptors and Pharmaceutical compounds in Biofilm, Macroinvertebrates, and Fish in four Mediterranean Rivers Belinda Huerta, Victoria Osorio, Marina Gorga, Anna Jakimska, Nuria de Castro, Lidia Ponsati, Isabel Muñoz, Sandra Pérez, Mira Petrovic, Sara Rodríguez-Mozaz, Damià Barceló

2 INTRODUCTION OBJECTIVES ANALYTICAL METHODOLOGIES PRESENCE OF PhACs & EDCs IN BIOTA CONCLUSIONS

3 INTRODUCTION

4 Introduction WHO-State of the Science of Endocrine Disrupting Chemicals

5 Introduction WHO-State of the Science of Endocrine Disrupting Chemicals

6 Introduction EDCs Disruption of endocrine system PhACs Biologically active Highly potent at low levels acute & chronic toxicity antibiotic resistance genotoxicity endocrine disruption UNPREDICTED ENVIRONMENTAL IMPLICATIONS At environmental concentrations has been seen that synthetic hormone 17α-ethinylestradiol (EE2) has induction of estrogenic effects in fish diclofenac led to tissue damage in fish behavioural alterations in fish after exposure to sertraline

7 Introduction Few studies related to uptake of pharmaceuticals in freshwater organisms Organisms such as invertebrates or biofilm possess the capacity to integrate environmental variations. They are also at the very bottom of the trophic chain. Fish considered to be a feasible organisms for pollution monitoring, as their biological particularities make them potentially susceptible to pharmaceutical bioaccumulation (ubiquitous, role as carrier of energy, lifespan)

8 Introduction PREVIOUS FIELD STUDIES Ramirez et al Chu & Metcalfe Diphenydramine, 2007 diltiazem Fluoxetine carbamazepine In fish In fish Brodin et al Oxazepam in fish Li et al Antibiotics in Mollusks Brooks et al Fluoxetine and sertraline in fish brain tissue (max 30ng/g) Coogan et al Triclosan in algae Rüdel et al Triclosan in fish Ramashwamy et al TBEP & Triclosan in fish bile Pharmaceutical Active Compounds PhACs Endocrine Disrupting Compounds EDCs

9 OBJECTIVES

10 Objectives To develope an analytical methodology for the analysis of PhACs & EDCs in: -fish - macroinvertebrates -biofilm To characterize the presence and bioaccumulation of PhACs and EDCs in aquatic organisms from different trophic levels biofilm, two macroinvertebrates: Hydropsyche sp, (collector-filterer insect larvae) and Dreissena polymorpha (zebra mussel), and fish collected from four rivers basins in Spain: Ebro, Llobregat, Júcar and Guadalquivir.

11 ANALYTICAL METHODOLOGIES

12 Analytical Methodology: Fish PhACs EDCs PLE Extraction GPC Clean-up Analysis by UPLC-MS/MS 1 g of freeze-dried fish or 0.5 g of mussel extracted using PLE Extracts purified in a GPC system for lipid removal Final extracts analyzed by UPLC/QTRAP 5500 MS QuEChERS Extraction & Purification Analysis by UPLC-MS/MS 0.5 g of freeze-dried fish & mussel extracted in ACN/H2O) after addition of extraction salts Extracts purified using dspe Final extracts analyzed by UPLC/QTRAP 5500 MS Huerta et al. J of Chrom. (2013) Jakimska et al. J of Chrom. (2013)

13 Analytical Methodology: Macroinvertebrates Extraction by sonication Purification by Ostro 96-well plate Analysis by UPLC-MS/MS PhACs & EDCs 100 mg of freeze-dried sample placed in a 50- ml Falcon tube + 3 ml of MeOH Sonication applied in 3 cycles of 120 s, with amplitude (intensity) of 30 % Samples centrifuged at rpm, for 5 min and supernatant collected Extracts redissolved in H2O/ACN (1:3, 1 % HCOOH, v/v) Extracts added to an OstroTM 96-well plate After extraction by vacuum, purified extracts were collected Final extracts analyzed by UPLC/QTRAP 5500 MS

14 Analytical Methodology: Biofilm PLE Extraction PhACs & EDCs 200 mg of freeze-dried sample placed in 10 ml ASE 350 cells SPE Purification Extraction applied in 3 cycles of 5 min, with a mixture of citric buffer (ph 4)/ACN (1:1) as solvent and a Tª=60 ºC Extracts were diluted to 100 ml and purified by SPE Oasis HLB (6ml, 200 mg) Analysis by UPLC-MS/MS Eluates in MeOH collected and evaporated Final extracts analyzed by UPLC/QTRAP 5500 MS

15 Analytical Methodology: Summary Fish:PhACs Fish:EDCs Macr: EDCs & PhACs Biofilm:EDCs & PhACs PLE Extraction Extraction by sonication PLE Extraction GPC Clean-up QuEChERS Extraction & Purification Purification by Ostro 96-well plate SPE Purification Analysis by UPLC- MS/MS Analysis by UPLC-MS/MS Analysis by UPLC- MS/MS Analysis by UPLC- MS/MS Nº compounds: 20 PhACs Main challenges: - lipid content - trace levels Nº compounds: 19 EDCs Main challenges: - lipid content - trace levels Nº compounds: 41 PhACs + 21 EDCs Main challenges: - sample size - variety Nº compounds: 43 PhACs + 14 EDCs Main challenges: - sample size

16 PRESENCE OF PHACS & EDCS IN BIOTA

17 Sampling Campaign

18 Results: Fish PhACs 8 compounds detected Families: Psychiatric drugs, non-steroidal anti-inflammatory drugs (NSAID) and β- blockers Diclofenac found in 4 rivers Most polluted river basin: Llobregat Highest conc. diclofenac in LLO6, after the entrance of Anoia (highly contaminanted by industrial WW) Ebro: no clear pollution gradient in the 5 sites PhACs detected in 4 sites Jucar & Guadalquivir: PhACs conc. Only detected in fish in 1 site (GUA4 and JUC5) of each river No difference among species, according to their feeding habits, age or niche EDCs 11 compounds detected Families: Parabens, triazoles, flame retardants, antibacterial, plasticizer The most ubiquitous contaminants in fish samples was the flame retardant TBEP, found inthe 75% of all samples analyzed at values up to ng/g. Llobregat higher levels of EDCs (in river and fish homogenates) TBEP levels and extremely high levels of BPA ( ± 11.51) in in LLO5 Jucar and Guadalquivir river samples were comparatively less polluted, except by the sampling point GUA4, where high level of BPA (59.09 ± 8.12) was determined

19 Results: Macroinvertebrates PhACs Two species: Trichoptera & Zebra mussels (only in Ebro) 4 compounds detected: 4 (citalopram, carbamazepine, diclofenac, levamisole) in mussel and 1 (diclofenac) in trichoptera Families: Psychiatric drugs, nonsteroidal anti-inflammatory drugs (NSAID) and anthelminthic Conc. in mussel < 3 ng/g Conc. in trichoptera higher than fish (50 ng/g) Diclofenac was found in samples from all the river basins EDCs Two species: Trichoptera & Zebra mussels (only in Ebro) 4 compounds detected: 2 in mussel (TBEP, methylparaben) and 3 in trichoptera (TBEP, benzylparaben, nonylphenol) Families: Parabens, alkylphenols and flame retardants Conc. parabens < 3 ng/g Mean conc. of TBEP and nonylphenol ng/g (d.w.) Detection frequency of TBEP: 100%

20 Results: Biofilm PhACs EDCs Samples from Ebro & Llobregat only No compounds in Ebro 15 compounds (4 metabolites) detected in Llobregat Clear pollution gradient: 0 compounds detected in LLO3 to 15 compounds in LLO7 Families: Psychiatric drugs, nonsteroidal anti-inflammatory drugs (NSAID), antibiotics, diuretics, lipid regulators and β-blockers Max. conc. for sertraline (230 ng/g, dw) Samples from Ebro & Llobregat only 5 compounds detected Detection frequency: % Families: Parabens, flame retardants, plasticizer Similar levels in both river basins Conc. range between nd 270 ng/g (dw) (case of Bisphenol A in Ebro) Average conc. 50 ng/g (dw)

21 RESULTS: SUMMARY PhACs Psychiatric drugs, non-steroidal anti-inflammatory drugs as the most common families Concentration: Biofilm> Macroinv. > fish EBRO No clear pollution gradient in the 5 sites Non-detected in biofilm 4 compounds in macroinv. & fish GUADALQUIVIR Lower levels in water &sediment biota No data for biofilm Occurrence in GUA4 after WWTP in fish and macroinv. JUCAR LLOBREGAT Evident pollution gradient from LLO3 to LLO7 Biofilm as the species that accumulates > nº compounds (15) Fish & Macroinv: 3 compounds Metabolites of carbamazepine and β-blockers detected in biofilm Lower levels in water &sediment biota No data for biofilm Occurrence in JUC5 in fish (no data for macroinv. In JUC5 & JUC6)

22 RESULTS: SUMMARY EDCs Parabens, flame retardants, and plasticizer as the most common families Concentration: Biofilm ~ Macroinv. ~ fish EBRO No clear pollution gradient in the 5 sites BPA in biofilm TBEP detection frequency in all the organisms: 100% Highest nº compounds detected in fish (6) GUADALQUIVIR Lower levels in water &sediment biota No data for biofilm GUA4: BPA in fish TBEP detected in 100% of samples in fish and biofilm JUCAR LLOBREGAT Higher levels of EDCs TBEP detection frequency in all the organisms: % BPA in fish and biofilm Parabens detected in 100 % of biofilm and fish samples Lower levels in water &sediment biota No data for biofilm TBEP detected % of fish and macroinv. samples

23 CONCLUSIONS

24 CONCLUSIONS Methodologies for the analysis of PhACs (20-43) & EDCs (17-21) were developed for 3 aquatic organisms: fish, macroinvertebrates and biofilm. A total of 80 biota samples were analyzed in the four rivers Up to 15 PhACs and 11 EDCs were detected in biota Psychiatric drugs, non-steroidal anti-inflammatory drugs for PhACs and parabens, flame retardants, and plasticizer for EDCs as the most common families Highest concentrations of PhACs found in biofilm, while for EDCs differences among species are not so obvious Pollution gradient: Llobregat > Ebro > Jucar > Guadalquivir NEXT STEPS: Establish relations between concentration in biota with that in water and sediment Investigate the presence of metabolites and transformation products of target pollutants in the samples

25 Assessing and predicting effects on water quantity and quality in Iberian rivers caused by global change (SCARCE) Consolider Ingenio 2010 CSD SCARCE Spanish Ministry of Economy and Competitiveness Thank you all for your attention