MONITORING TRACE ORGANIC CONTAMINANTS IN WATER TREATMENT PROCESSES. FROM TARGET ANALYSIS, SUSPECT SCREENING TO NON TARGET ANALYSIS

Pra ćenje tragova organskih zaga đivala tijekom procesa obrade voda. Od ciljane i pretražne do sveobuhvatne analize

Mira Petrovic, 1,2 Yaroslav Verkh 1, Mira Celic 1, Meritxell Gros 1

1ICRA – Catalan Institute for Water Research, Girona, Spain 2ICREA – Catalan Institution for Research and Advanced Studies, Barcelona, Spain Outline

• Chemical cocktails: Regulated contaminants (priority contaminants) vs emerging contaminants

• LC-MS for monitoring of organic micropollutants (OMPs) in wastewater and receiving aquatic environment

XIC of +MRM (70 pairs): Exp 1, 372.0/72.0 amu from S... Max. 1.8e6 cps. +EPI (916.40) Charge (+0) CE (25) FT (20.3092): Exp ... Max. 6.4e6 cps.

TIC OF ALL MRM TRANSITIONS s p c , y t i s n e t n I EPI (MS/MS) SCAN at CE= 25 eV 17.88 916.1 1.8e6 RECORDED 6.0e6 1.6e6 5.0e6 4.0e6

s p c , y t i s n e t n I 1.4e6 3.0e6 1.2e6 • Target analysis 2.0e6 1.0e6 1.0e6 772.9 898.8 8.0e5 100 200 300 400 500 600 700 800 900 6.0e5 m/z, amu +EPI (916.40) Charge (+0) CE (40) FT (6.76457): Exp ... Max. 1.4e7 cps.

4.0e5 s p c , y t i s n e t n I EPI (MS/MS) SCAN at CE= 40 eV 916.3 • Suspect screening 2.0e5 1.4e7 1.2e7 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 1.0e7 Time, min 8.0e6 XIC of +MRM (70 pairs): Exp 1, 916.4/174.2 amu from ... Max. 5.5e4 cps. 6.0e6 XIC FOR 772.6 13.22 4.0e6 5.5e4 916>174 2.0e6 • Non-target screening TYLOSIN 174.4 5.0e4 318.2 598.6 4.5e4 100 200 300 400 500 600 700 800 900 m/z, amu s p c , y t i s n e t n I 4.0e4 +EPI (916.40) Charge (+0) CE (55) FT (9.48163): Exp ... Max. 2.0e6 cps.

3.5e4 s p c , y t i s n e t n I EPI (MS/MS) SCAN at CE= 55 eV 174.2 3.0e4 2.0e6 772.4 2.5e4 1.5e6 2.0e4 916.7 1.5e4 1.0e6 598.6 1.0e4 156.2 5.0e5 5000.0 12.66 13.59 98.2 389.3 754.3 0.0 • Conclusions 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 100 200 300 400 500 600 700 800 900 Time, min m/z, amu Growing use of chemicals by our technological society: CAS REGISTRY SM contains more than 106 million unique organic and inorganic chemical substances CHEMLIST ® (Regulated Chemicals Listing), produced by CAS, is an electronic collection of thousands of chemical substances that are regulated in key markets across the globe. CHEMLIST contains more than 345,000 substances REACH ( Registration, Evaluation, Authorization and Restriction of Chemicals) is a regulation of the European Union, adopted to improve the protection of human health and the environment from the risks that can be posed by chemicals, while enhancing the competitiveness of the EU chemicals industry. : ~30,000 compounds ( 10,000 already registered)

Muir and Howard, ES&T, 40(23),7157 (2006) Environmental pathways of organic micropollutants (OMPs)

H. Hamid. (2016)

Analytical methods have become more sensitive, therefore more compounds can be detected at lower concentrations

Not everything that can be measured is worth measuring, and not everything worth measuring is measurable. EU WFD List of priority substances (45)

Dicofol Dioxin and dioxin like compounds Heptachlor/Heptachlor epoxide Aclonifen HBCDD Bifenox PFOS Cybutryne Quinoxyfen Cypermethrin Terbutryn Dichlorvos Watch list

A mechanism designed to allow targeted EU-wide monitoring of substances of possible concern to support the prioritization process in future reviews of the priority substances list.

Name of substance/group of substances Class

17-Alpha-ethinylestradiol (EE2) Synthetic hormone

17-Beta-estradiol (E2), Estrone (E1) Natural hormone

Diclofenac Pharmaceutical 1st (2015) 2,6-Ditert-butyl-4-methylphenol Antioxidant

2-Ethylhexyl 4-methoxycinnamate UV filter

Macrolide antibiotics: Erythromycin, Pharmaceutical Clarithromycin, Azithromycin Methiocarb Pesticide

Neonicotinoids: Imidacloprid, Thiacloprid, Neuro-active insecticides Thiamethoxam, Clothianidin, Acetamiprid Oxadiazon Herbicides

Tri-allate Pesticide Watch list

A mechanism designed to allow targeted EU-wide monitoring of substances of possible concern to support the prioritization process in future reviews of the priority substances list.

Name of substance/group of substances Class

17-Alpha-ethinylestradiol (EE2) Synthetic hormone

17-Beta-estradiol (E2), Estrone (E1) Natural hormone

Macrolide antibiotics: Erythromycin, Pharmaceutical Clarithromycin, Azithromycin 2nd (2018) Methiocarb Pesticide

Neonicotinoids: Imidacloprid, Thiacloprid, Neuro-active insecticides Thiamethoxam, Clothianidin, Acetamiprid Metaflumizone Insecticide

Amoxicillin Pharmaceutical Ciprofloxacin Pharmaceutical Chemical analysis and monitoring of contaminants

C.G. Daughton U.S. EPA, 2013 Emerging contaminants

• Global Organic Contaminants (flame retardants, perfluorinated compounds, siloxanes…etc.)

• Pharmaceuticals

• Personal Care Products (preservatives, UV filters, biocides, insect repellents, fragrances, etc..)

• Endocrine Modulating Chemicals (hormones, industrial chemicals, pesticides)

• Nanoparticles

• Microplastics

• Industrial Chemicals (new and recently recognized)

• Biological Metabolites and Toxins

• Transformation products Chemical analysis output for a typical environmental sample

The spectrum of contaminants identified in a sample is just a portion of those present and their significance in term of risk is unknown

Adapted from: C.G. Daughton TIC – tentatively identified compounds U.S. EPA July 2002 Analytical challenges

Complex environmental sample

Number of target compounds

Cost of analysis Time Effort Analytical challenges

“What we see depends mainly on what we look for ” Sir John Lubbock

LC-Orbitrap (UHP)LC-MS/MS (QqQ) (UHP)LC-(Qq)TOF

Prioritize compounds of interest Source: EAWAG

Target analytes: 84 pharmacueticals (parent compounds + metabolites)

THERAPEUTIC GROUPS COMPOUNDS THERAPEUTIC GROUPS COMPOUNDS Ketoprofen Acridone Acetamonophen Diatrizoate Naproxen X-RAY CONTRAST AGENTS Iopromide Ibuprofen Iohexol Indomethacine ANALGESICS AND ANTI- Diclofenac Metformin hydrochloride * ANTIDIABETIC Glibenclamide INFLAMMATORIES 4’-OH-Diclofenac Propyphenazone Phenazone Furosemide Piroxicam DIURETIC Torasemide Meloxicam Hydrochlorothiazide Nimesulide Valsartan Gemfibrozil Irbesartan LIPID REGULATORS AND Bezafibrate ANTIHYPERTENSIVES Losartan CHOLESTEROL LOWERING Atorvastatin calcium STATIN DRUGS Fluvastatin Sodium Diltiazem Verapamil Fluoxetine hydrochloride CALCIUM CHANNEL BLOCKERS Norverapamil * Paroxetine hydrochloride Amlodipine Norfluoxetine * Carbamazepine Atenolol 10,11-epoxycarbamazepine * * Sotalol hydrochloride PSYCHIATRIC DRUGS 2’-OH-Carbamazepine β-BLOCKERS Metoprolol tartrate Citalopram Propranolol hydrochloride Sertraline Nadolol Olanzapine Venlafaxine α1-SELECTIVE ALPHA BLOCKER Tamsulosin Trazodone Clopidogrel Loratadine ANTIPLATELET AGENT Desloratadine Warfarin HISTAMINE H1 AND H2 Famotidine ANTICOAGULANT RECEPTOR ANTAGONISTS Ranitidine Phosphodiesterase type V inhibitors Sildenafil Cimetidine hydrochloride Vardenafil Tetracycline hydrochloride ANTIHYPERTENSIVES Enalapril maleate Erithromycin Enalaprilat * Clarithromycin Azithromycin VETERINARY PHARMACEUTICALs Dimetridazole Trimethoprim Ronidazole Metronidazole Thiabendazole Metronidazole-OH * Albendazole ANTIBIOTICS Sulfamethoxazole Triclabendazole Ciprofloxacin Levamisol Ofloxacin Xylazine Amoxicillin Carazolol Ampicillin Salbutamol Cefalexin Azaperone Cefuroxime Azaperol *Pharmaceutical metabolites Pharmaceuticals selected due to their high consumption in Spain (National Health System) and their classification as priority pharmaceuticals according to the USEPA and EMEA Comparison of CAS and MBR performances – elimination of pharmaceutical residues

10-80% <10% >80% 100 9 3 1 6 12 20 10 1-naproxen, 90 22 19 2 2-ketoprofen, 4 3-ibuprofen, 80 4-diclofenac, 7 21 5-indomethacin, Analysis of +80 70 14 6-acetaminophen, compounds by Lc- 15 8 17 7-mefenamic acid, 8-propyphenazone, QqLIT-MS 60 16 13 9-ranitidine, 50 10- paroxetine, 5 18 11-carbamazepine, 12- ofloxacin, 40 13- sulfamethoxazole, 14- erythromycin, 30 15- atenolol, 16- metoprolol, MBR elimination, % MBR elimination, 20 17- hydrochlorothiazide, 18- glibenclamide, 10 19- gemfibrozil, 11 20- bezafibrate, 0 21- clofibric acid, 22- pravastatin 0 20 40 60 80 100 CAS elimination, %

Database of suspects Suspect screening using an exact mass compound database from list developed by the Catalan Water Agency and NORMAN Suspect List which includes more than 400 compounds, of major consumption in Catalonia, such as pharmaceuticals, personal care products, drugs of abuse, pesticides and biocides, industrial chemicals, complemented with a large number of relevant TPs (~100) 1. Data acquisition

SPE LC-LTQ Orbitrap Velos The SPE cartridges are prepared in house from four different adsorbents. The sorbent materials: Sepra ZT (Strata-X), Isolute ENV+ Sepra ZT-WCX (Strata-X-CW) Sepra ZT-WAX (Strata-X-AW).

Data processing

Gago-Ferrero et al., 2015 Environmental Science & Technology 2. Data processing Mass spectra data processing for suspect screening

HRMS-Orbitrap

Extraction of features Compound Discoverer Software 2.0 3. Data Mining - Results

Molecul Compound Formula ar H+ RT [min] Weight NI mode PI mode Pharmaceuticals Metformin C4 H11 N5 129.101 130.109 3.02 Carbamazepine C15 H12 N2 O 236.094 237.102 9.66 Molecul Compounds Formula ar H- RT [min] Telmisartan C33 H30 N4 O2 514.236 515.244 11.44 Weight Valsartan C24 H29 N5 O3 435.226 436.234 10.71 Tramadol C16 H25 N O2 263.188 264.196 7.36 Pharmaceuticals Citalopram C20 H21 F N2 O 324.163 325.171 8.88 Valsartan C24 H29 N5 O3 435.228 434.220 7.03 Venlafaxine C17 H27 N O2 277.204 278.211 8.21 Telmisartan C33 H30 N4 O2 514.237 513.230 8.23 C7 H8 Cl N3 O4 296.965 7.72 Lidocaine C14 H22 N2 O 234.173 235.180 7.55 Hydrochlorothiazide Lamotrigine C9 H7 Cl2 N5 255.007 256.015 7.74 S2 295.957 Paracetamol C8 H9 N O2 151.063 152.071 4.06 Paracetamol C8 H9 N O2 151.064 150.056 6.69 Mesalamine C7 H7 N O3 153.042 154.050 5.42 Theophylline C7 H8 N4 O2 180.065 179.057 6.51 Pesticides Prostaglandin E2 C20 H32 O5 352.226 351.218 7.66 Tebuconazole C16 H22 Cl N3 O 307.145 308.152 12.48 Insecticide C15 H17 Cl2 N3 Propiconazole 341.069 342.077 13.12 Tebufenozide C22 H28 N2 O2 352.216 351.208 12.94 O2 Food additive C15 H16 Cl3 N3 Prochloraz 375.030 376.038 13.10 Succinic acid C4 H6 O4 118.027 117.019 2.70 O2 Surfactants Metalaxyl C15 H21 N O4 279.147 280.154 10.86 230.152 6.27 Azoxystrobin C22 H17 N3 O5 403.116 404.124 11.89 Dodecanedioic acid C12 H22 O4 229.145 Metolachlor C15 H22 Cl N O2 283.133 284.141 13.14 Other industrial chemicals and Terbuthylazine C9 H16 Cl N5 229.109 230.117 12.04 chemical products Terbutryn C10 H19 N5 S 241.136 242.143 12.62 Panthenol C9 H19 N O4 205.132 204.124 6.23 Other industrial chemicals and Azelaic acid C9 H16 O4 188.105 187.098 3.42 chemical products 2-Naphthalenesulfonic acid C10 H8 O3 S 208.020 207.012 7.00 Triethanolamine (TEOA) C6 H15 N O3 149.105 150.112 2.81 Metabolites DEET C12 H17 N O 191.130 192.138 10.96 Hydrocinnamic acid C9 H10 O2 150.069 149.061 6.97 Oleamide C18 H35 N O 281.271 282.279 14.86 Ethylmalonic acid C5 H8 O4 132.043 131.035 2.65 Panthenol C9 H19 N O4 205.131 206.139 6.12 Xanthine C5 H4 N4 O2 152.034 151.026 3.64 Abused substances 1-Methylxanthine C6 H6 N4 O2 166.050 165.042 5.82 Nicotine C10 H14 N2 162.115 163.123 4.54 Tetradecanedioic acid C14 H26 O4 258.184 257.176 9.41 Caffeine C8 H10 N4 O2 194.080 195.088 7.03 1,7-Dimethyluric acid C7 H8 N4 O3 196.060 3.96 Cocaine C17 H21 N O4 303.147 304.154 8.13 195.052 Metabolites 3,7-Dimethyluric acid C7 H8 N4 O3 196.060 195.052 3.07 2'-Deoxyadenosine C10 H13 N5 O3 251.101 252.109 5.76 3-Hydroxyphenylacetic acid C8 H8 O3 152.048 151.040 6.08 4-Acetamidoantipyrine C13 H15 N3 O2 245.116 246.124 7.00 3-Phenyllactic acid C9 H10 O3 166.064 165.056 5.91 4-Formylaminoantipyrine C12 H13 N3 O2 231.100 232.108 7.04 N,N-Didesmethylvenlafaxine C15 H23 N O2 249.172 250.180 7.02 Atenolol acid C14 H21 N O4 267.147 268.154 6.78 Galaxolidone C18 H24 O2 272.177 273.185 14.81 Desethylterbuthylazine C7 H12 Cl N5 201.078 202.085 10.05 Cotinine C10 H12 N2 O 176.094 177.102 5.61 4-Aminonicotinic acid C6 H6 N2 O2 138.042 139.050 4.01 6ß-Hydroxytestosterone C19 H28 O3 304.203 305.211 12.93 >60 compounds Sampling map Peak area

Peak area

2. Data processing Mass spectra data processing for non-target analysis

Mass A Extraction of data

Retention Time a Mzmine 2.23

Formula CxHyOz “R” Relative Intensity 0-1

The sensibility of the found atomic ranges in PI and NI mode was confirmed using experimental data for 89 pharmaceutical compounds 3. Data Mining Analytical and statistical tools

van Krevelen plot Mass A

Retention Time a

Formula CxHyOz Kendrick Mass Relative Intensity 0-1 Defect analyses

Information relevant for the evaluation of performance of wastewater treatment process

- changes in the number of compounds, - change in averaged mass spectral parameters (O/C, H/C, DBE), - changes in the molecular weight of the substances, - changes in the intensity of the signals, - changes in the functional groups, - how many TPs have been generated during the treatment. DOM in Biological treatment

The number of the total detected features

42% of the features present in influent disappeared -23%

1100 new features appeared in effluent (24% of the features in the effluent are TPs)

3470 features were persistent (detected in both influent and effluent, but varying intensity) DOM in Nanofiltration treatment

The number of the total detected features

72% of the features present in influent disappeared

228 new features appeared in effluent -64%

789 features were persistent (detected in both influent and effluent, but varying intensity) Mass changes

Size restriction effects of the NF membrane start to limit the removal at the threshold of ≈ 500 Da,

The mean mass in NF significantly decreases by 46 Da after the treatment. Chemical profile

A mean DBE drop of 1 double bond equivalents after NF treatment shows that unsaturated compounds are preferentially removed compared to the saturated ones. Chemical profile

A van Krevelen plot of the influent and effluent NF DOM features with assigned elemental formulas Chemical profile

Kendrick mass defect

Evident disappearance of longer homologues and appearance of shorter homologues Scope of the Methods (limitations)

Total dissolved organic matter (OMPs) (DOM) DOM extracted by SPE : 50–60 %

Real concentration (removal) DOM after extraction + detected in 120 LC-ESI-MS (medium polar) Measured concentration (removal) 100

80 Ion suppression 60%

60 Ion suppression 17% 40 40% 20

0 -25% -20 Relative concentration and removal and concentration Relative -40 WWTP influent WWTP effluent Removal Conclusions

Despite many methodologies being developed to detect organic micropollutants (OMPs) in wastewater, their detection and detection of their transformation products in wastewater treatment remains a challenge .

Mass spectrometry (MS) has distinctive ability to identify small amounts of organic chemicals from increasingly complex mixtures providing a large amount of information on wastewater composition and OMPs of interest within.

The issue of OMPs is closely related to analytical capabilities . Increased sensitivity in MS, as a result of more efficient ionization techniques and better detectors, has enabled detection of virtually any new and potentially harmful contaminant at a very low level.

Target methods using LC-MS/MS method of choice for quantitative determination specific compounds and classes

However, non-targeted analysis drives the exploration of WW composition and transformations, as a result of wastewater treatment gradually displacing the interest in targeted methods in the researcher community. Many thanks for your attention Hvala na pažnji

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