Diana Álvarez Muñoz, Albert Serra Compte, Natàlia Corcoll, Belinda Huerta, Sara Rodríguez Mozaz, Sergi Sabater, Damià Barceló.

Diana Álvarez Muñoz, Albert Serra Compte, Natàlia Corcoll, Belinda Huerta, Sara Rodríguez Mozaz, Sergi Sabater, Damià Barceló.

INTRODUCTION MATERIAL AND METHODS Experimental Analytical determination Signal processing and multivariate analysis RESULTS AND DISCUSSION Metabolites identification Biofilm response to environmental stressors CONCLUSIONS ACKNOWLEDGEMENTS

INTRODUCTION Drought Pollution Landfill Animal waste Aquaculture Hospital waste Industrial domestic waste Pharmaceuticals mixtures Chronic exposure

INTRODUCTION Biofilm Primary producers Biogeochemical cycle of organic and inorganic matter Sensitive to river changes Have a rapid interaction with dissolved substances Short life cycle Bioaccumulation capacity BIOMARKERS OF EXPOSURE DISRUPTED METABOLIC PATHWAYS

GOALS 1.To identify biofilm biomarkers of drought stress and pharmaceutical exposure. 2.To elucidate metabolic pathways affected due to a dry period and/or pharmaceutical exposure. 3.To evaluate if the effects of a dry period on the metabolome of biofilm was influenced for the co occurrence of pharmaceutical exposure.

MATERIAL AND METHODS Fluvial mesocosm Experimental Treatments: 1. Control (C) 2. Pharmaceutical exposure (P) Compound Therapeutic family Nominal Concentration (ng/l) Ibuprofen Anti inflammatory 404 Diclofenac Anti inflammatory 366 Carbamazepine Psychiatric drug 124 Sulfamethoxazole Antibiotic 699 Erithromycin Antibiotic 169 Metoprolol β Blocker 1845 Atenolol β Blocker 117 Gemfibrozil Lipid regulator 140 Hydrochlorothiazide Diuretic 1135 Total duration of the experiment 42 d 21 d colonization 7 d drought 14 d flow rewetting 3. Dry exposure (7 days) (D) 4. Dry and pharmaceutical exposure (D+P)

MATERIAL AND METHODS Analytical determination Samples were collected at the end of the experiment lyophilized and grounded Pressurized liquid extraction (ASE 350): ACN:citric buffer (1:1) Tª 60º C, 1500 psi, 3 static cycles of 5 min each Samples were dry down and re dissolve in 100 ml of water prior solid phase extraction (SPE) on OASIS HLB cartridges The eluates were evaporated and reconstituted in 1 ml of methanol, 10 µl ISTDs mix were also added. HPLC HRMS (LC LTQ Orbitrap Velos) Equipped with electrospray ionization operating both in positive and negative mode.

MATERIAL AND METHODS Signal processing and multivariate analysis Mass spectra: 100 700 m/z, deconvoluted and aligned using Sieve software. The markers were exported to R software for multivariate analysis. Clear separation between the biofilm exposed and not exposed to drought (D) Slight separation between control (C) group and biofilm exposed only to pharmaceuticals (P) Not clear separation between D and drought + pharmaceuticals (D+P) Identification of the markers: Accurate mass Elemental composition. Biochemical data bases Match up MS/MS response. Confirmation by STDs. Fig 1.Principal component analysis (PCA) of significant metabolites of biofilm exposed to the treatments

RESULTS AND DISCUSSION Metabolites identification Table 1.Tentative identification of markers in biofilm after treatments by using LC LTQ Orbitrap (ESI+ and ESI ). M/z value of marker ion RT (min) ESI 251.2003 5.29 + 279.2319 5.58 + 297.2424 5.85 + 191.0193 0.71 187.0975 3.73 269.2119 5.15 275.2012 5.76 255.2323 6.12 339.3264 6.75 367.3575 6.94 409.2353 9.62 253.2170 5.93 Putative experimental molecular formula of ion Theoretical mass METABOLIC PATHWAYS Error (ppm) Putative Identity C 16 H 27 O 2 [M+H] + 251.2011 3.2 LPA (16:3) C 18 H 31 O 2 [M+H] + 279.2324 1.8 alfa linolenic acid C 18 H 33 O 3 [M+H] + 297.2429 1.7 A vernolate C 6 H 7 O 7 [M H] 191.0192 0.5 Citrate C 9 H 15 O 4 [M H] 187.0970 2.7 Azelaic acid C 16 H 29 O 3 [M H] 269.2117 0.7 16 Oxohexadecanoic acid C 18 H 27 O 2 [M H] 275.2011 0.3 Stearidonic acid C 16 H 31 O 2 [M H] 255.2324 0.4 Palmitic acid C 22 H 43 O 2 [M H] 339.3263 0.3 Behenic acid C 24 H 47 O 2 [M H] 367.3576 0.3 Lignoceric acid C 19 H 38 O 7 P [M H] 409.2355 0.4 LPA(0:0/16:0) C 16 H 29 O 2 [M H] 253.2168 0.8 Palmitoleic acid

RESULTS AND DISCUSSION Metabolites identification Fragmentation pattern of Lysophosphatidic acid (LPA (0:0/16:0). Fig.2. A) Total ion chromatogram from biofilm sample exposed to pharmaceuticals, B) Mass spectrum of this compound at 30 v CID.

RESULTS AND DISCUSSION Biofilm response to environmental stressors Table 2. Significant variation of markers levels (p<0.05) in biofilm after each treatment compared to the control group. It is represented as an increase ( ) or decrease ( ).

RESULTS AND DISCUSSION Biofilm response to environmental stressors: Drought Table 3. metabolite type and response to a dry period Lipids were the most affected metabolites. Both saturated and unsaturated fatty acids increased after a dry period: changes in membrane fluidity, increase of energy reservoirs and a growth phase of microorganisms after drought. The increase in carboxilic acids: signaling functions. Oxohexadecanoic acid it is involved in unsaturated fatty acids biosynthesis.

RESULTS AND DISCUSSION Biofilm response to environmental stressors: pharmaceuticals Table 3. metabolite type and response to a pharmaceutical exposure Lipids were the most affected metabolites. Saturated fatty acids decreased after pharmaceutical exposure: higher demand and consume of energy. The increase of unsaturated fatty acids: conversion of saturated into unsaturated by the organism. Glycerophospholipid: baseline toxicity of pharmaceutical and alteration of the membrane structure.

CONCLUSSIONS LPA (0:0/16:0) and palmitic acid have been proposed as specific biomarkers of pharmaceutical exposure in biofilm. In the case of drought: palmitoleic, LPA (16:3), alpha linoleic, stearidonic, 16 Oxohexadecanoic, azelaic acids and citrate have been pointed out. Behenic and lignoceric acids have been also proposed but as common biomarkers with different observed effect. The biosynthesis of fatty acids is the main endogenous metabolic pathway disrupted by both stressors but other biological functions can also be altered: membrane fluidity, signaling, energy reservoirs. When biofilm was simultaneously exposed to drought and pharmaceuticals the stressor that produced a higher alteration on the biofilm metabolome was the drought, although a slight alteration due to the co ocurrence of pharmaceuticals can not be discarded.

Acknowledgements Thanks to all the co authors specially to Albert Serra and Natalia Corcoll. This project was supported by the Scarce Consolider Ingenio 2010 (CSD2009 00065) Assessing and Predicting Effects On Water Quantity and Quality In Iberian Rivers Caused By Global Change (2009 2014) and the Spanish ministry of economy and competitiveness. To all of you for your attention. Thank you!