Benzotriazoles & benzothiazoles in a WWTP in Greece: Occurrence and removal efficiencies Alexandros G. Asimakopoulos1,2, Kurunthachalam Kannan2, Nikolaos S. Thomaidis1 1Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, Athens, Greece. 2Wadsworth Center, New York State, Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Albany, NY, USA.

Benzotriazoles (BTRs) – Benzothiazoles (BTHs): Uses & Applications BTRs contain the 1,2,3-benzotriazole skeleton in their structure. : Flame and corrosion inhibitors. Ultraviolet (UV) light stabilizers in plastics & antifogging agents. Found in pigments, dishwasher detergents, de-icing fluids. R: chemical group or atom BTHs contain the 1,3-benzothiazole skeleton in their structure. Corrosion inhibitors, are used as herbicides, slimicides, fungicides, photosensitizers. Constituents of azo dyes, de-icing fluids, drugs, food flavors, rubber. Some occur naturally in the environment, i.e., tobacco smoke and tea leaves. Btrs high production volume chemicals (> 9000 tons only in the U.S.) R: chemical group or atom

Target chemicals assessed in the WWTP BTRs: BTHs: BTRs : 1H-benzotriazole (1H-BTR) 2. 1-hydroxy-benzotriazole (1-OH-BTR) 3. Tolyltriazole (TTR, a mixture of isomers of 4-methyl-1H-benzotriazole [4-Me-1H-BTR] and 5-methyl-1H-benzotriazole [5-Me-1H-BTR]) Xylyltriazole (XTR, or 5,6-dimethyl-1H-benzotriazole [5,6-diMe-1H-BTR]) BTHs: benzothiazole (BTH) 2. 2-hydroxy-benzothiazole (2-OH-BTH) 2-methylthio-benzothiazole (2-Me-S-BTH) 2-amino-benzothiazole (2-amino-BTH) Polar compounds. BTRs are weak bases. BTHs present both acidic and basic properties.

Schematic diagram of Treatment Process

Sample collection Wastewater and sludge samples were collected from a WWTP that serves a population of 3.700.000, in Athens. Samples were collected in April 2012; the average flow rate of sewage was approx. 743.000 m3/day, and the average production of dewatered sludge was approx. 112.000 kg/day. Influent and effluent samples (50 mL), primary sludge (5 mL), secondary sludge (20 mL), and mixed liquor samples from the bioreactors (20 mL) were immediately filtered after collection. The filtered wastewater samples were acidified to pH 2.50±0.10, and stored in the dark at 4 °C until analysis. All solid samples were stored at −20 °C until analysis.

Schematic diagram of the analytical method Evaporation to near-dryness with N2 gas, reconstituted in 1 mL CH3OH/ACN (1:1 v/v) Adjustment pH to 3.0±0.1 Filtration: pre-ashed GF/F filter SPE: Strata-X RP (200mg, 6cc) Conditioning: 10 mL CH3OH Equilibration: 10 mL acidified H2O Washing: 2×5 mL acidified H2O Elution: 10 mL CH3OH/ACN (1:1 v/v) Liquid samples: 50/20/5 mL Supernatant dilution to 50 mL Centrifugation: 4000 rpm/10 min (×1,×2,×3) SLE: Vortex mixing for 1 min with 5 mL MeOH/Milli-Q water (1:1 v/v) (adjusted to pH 3.00±0.10). Sonication for 45 min (37 oC). Dried Sludge: 100 mg Filter Filtrate 3 DIAFANEIA THA LEEI PROVLIMATA LC-ESI(+)MS/MS

Occurrence in 24-h composite flow proportional Influent and Effluent samples . Target analytes 24-h composite flow proportional influent wastewater 24-h composite flow proportional effluent wastewater Removal efficiency (%) Dissolved phase (ng/L) D1/D2 Particulate matter (ng/L) D1/D2 Dissolved phase (ng/L) D1/D2 Particulate matter (ng/L) D1/D2 D1/D2 1H-BTR 587/580 154/146 325/385 143/162 37/25 1-OH-BTR 217/243 ND/ND 100/100 TTR 15841/7735 131/106 5156/5737 19/37 68/26 XTR ND/ ND ND/<1.1 -/- BTH 1119/1160 108/210 <55/<55 132/89 87/91 2-OH-BTH 481/640 54/80 151/172 16/86 69/64 2-Me-S-BTH 346/331 39/51 50/46 18/20 82/83 2-Amino-BTH <13/ND

Occurrence in primary and secondary sludge & influent bioreactor samples . Target analytes Primary sludge (grab samples) Influent bioreactor Secondary sludge Dissolved phase (ng/L) D1/ D2 Particulate matter (ng/g) Dissolved phase (ng/L) Particulate matter (ng/g) 1H-BTR 2621/4514 17/10 141/125 38/39 531/665 27/26 1-OH-BTR 173/ND ND/ND 31/35 TTR 27109/210883 16/71 6123/4639 22/71 4964/6383 23/32 XTR 1/ND 1/1 BTH 10543/14010 <41/<41 ND/172 ND/<41 288/ND <41.3/ND 2-OH-BTH 11545/30224 102/93 207/102 35/44 175/216 21/29 2-Me-S-BTH 1250/1887 26/33 29/21 25/28 2-Amino-BTH 108/248 <34/<34 <34/ND A major portion is biotransformed during the activated sludge treatment process.

Occurrence in Sewage sludge Target analytes Sewage Sludge (ng/g) D1/D2 1H-BTR 81/84 1-OH-BTR ND/ND TTR 116/107 XTR BTH 174/88 2-OH-BTH 74/33 2-Me-S-BTH 61/61 2-Amino-BTH . Removal by sorption onto sludge seems insignificant.

Distribution of BTRs and BTHs between aqueous phase and suspended solids in influent and effluent wastewater The presence of exocellular biopolymers on the surfaces of biosolids following secondary treatment creates favorable conditions for higher sorption onto particulates present in bioreactors and secondary sludge.

Solid-Liquid Distribution Coefficients, Log Kd of BTRs & BTHs Target Analytes Primary sludge Influent bioreactor Secondary 1H-BTR 0.58 1.65 2.46 TTR 0.35 0.68 0.86 BTH 0.32 1.99 2.17 2-OH-BTH 0.72 2.11 2.43 2-Me-S-BTH 1.28 -* 2-Amino-BTH 0.41 *Concentrations were not detectable Both BTRs & BTHs have low affinity for primary sludge and this changed gradually in biosolids from the bioreactor and the secondary sludge. However, log Kd values were lower than 2.5.

Average Daily Loadings & Discharges

Summary (1/2) Biotransformation of some BTHs had been reported, and this contributes significantly to their removal during wastewater treatment. A similar mechanism is expected for BTRs. The removal efficiencies for BTHs calculated agree with the results from previous studies. The concentrations of 1H-BTR and TTR in sludge from Athens were similar to those reported previously. Only one study had reported BTH concentrations in sludge, where BTH, 2-Me-S-BTH and 2-OH-BTH were found approximately three times higher than the values reported here. a principle of toxicology, expressed by Paracelsus 13

Summary (2/2) Based on the results of the influent and effluent wastewater samples, most of the target analytes were determined in all samples at concentrations similar to or lower than those reported for other WWTP samples. This is the first environmental study that analyzed the particulate matter of wastewater for these compounds. Distribution between aqueous phase and suspended solids in influent and effluent wastewater, and calculation of solid-liquid distribution coefficients were reported for the first time for these compounds. a principle of toxicology, expressed by Paracelsus

Acknowledgements This project was implemented under the Operational Program “Education and Lifelong Learning” and funded by the European Union (European Social Fund) and National Resources — THALIS.

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