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
INTRODUCTIONDrought
Pharmaceuticalsmixtures
Landfill Animal waste Aquaculture Hospital waste Industrial domestic waste
Pollution
Chronic exposure
INTRODUCTION
Primary producersBiogeochemical cycle of organic
and inorganic matterSensitive to river changesHave a rapid interaction with
dissolved substancesShort life cycleBioaccumulation capacity
Biofilm
BIOMARKERS OF EXPOSUREDISRUPTED 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.
Experimental
MATERIAL AND METHODS
Fluvial mesocosm 2. Pharmaceutical exposure (P)
Compound Therapeutic familyNominal Concentration (ng/L)
Ibuprofen Anti‐inflammatory 404Diclofenac Anti‐inflammatory 366Carbamazepine Psychiatric drug 124Sulfamethoxazole Antibiotic 699Erithromycin Antibiotic 169Metoprolol β‐Blocker 1845Atenolol β‐Blocker 117Gemfibrozil Lipid regulator 140Hydrochlorothiazide Diuretic 1135
3. Dry exposure (7 days) (D)
4. Dry and pharmaceutical exposure (D+P)
1. Control (C)
Treatments:
21 d colonization
7 d drought
14 d flow rewetting
Total duration of the experiment 42 d
Analytical determination
MATERIAL AND METHODS
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 positiveand negative mode.
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.
MATERIAL AND METHODS
Identification of the markers:Accurate massElemental composition.Biochemical data basesMatch up MS/MS response.Confirmation by STDs.
Fig 1.Principal component analysis (PCA) of significant metabolites of biofilm exposed to the treatments
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)
RESULTS AND DISCUSSIONMetabolites identification
Table 1.Tentative identification of markers in biofilm after treatments by using LC‐LTQ‐Orbitrap (ESI+ and ESI‐).
METABOLIC PATHWAYS
M/z value ofmarker ion
RT(min)
ESIPutative experimental
molecular formula of ionTheoretical
massError(ppm)
Putative Identity
251.2003 5.29 +C16H27O2[M+H]+ 251.2011 3.2 LPA (16:3)
279.2319 5.58 +C18H31O2[M+H]+ 279.2324 1.8 alfa linolenic acid
297.2424 5.85 +C18H33O3[M+H]+ 297.2429 1.7 A vernolate
191.0193 0.71 ‐C6H7O7[M‐H]‐ 191.0192 ‐0.5 Citrate
187.0975 3.73 ‐C9H15O4[M‐H]‐ 187.0970 ‐2.7 Azelaic acid
269.2119 5.15 ‐C16H29O3[M‐H]‐ 269.2117 ‐0.7 16‐Oxohexadecanoic acid
275.2012 5.76 ‐C18H27O2[M‐H]‐ 275.2011 ‐0.3 Stearidonic acid
255.2323 6.12 ‐C16H31O2[M‐H]‐ 255.2324 0.4 Palmitic acid
339.3264 6.75 ‐C22H43O2[M‐H]‐ 339.3263 ‐0.3 Behenic acid
367.3575 6.94 ‐C24H47O2[M‐H]‐ 367.3576 0.3 Lignoceric acid
409.2353 9.62 ‐C19H38O7P[M‐H]‐ 409.2355 0.4 LPA(0:0/16:0)
253.2170 5.93 ‐C16H29O2[M‐H]‐ 253.2168 ‐0.8 Palmitoleic acid
RESULTS AND DISCUSSIONMetabolites 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.
Biofilm response to environmental stressors
RESULTS AND DISCUSSION
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 (↓).
Biofilm response to environmental stressors: Drought
RESULTS AND DISCUSSION
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.
Biofilm response to environmental stressors: pharmaceuticals
RESULTS AND DISCUSSION
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.
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