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Supplementary Materials

Occurrence and fate of most prescribed antibiotics in different water environments of Tehran, Iran

Roya Mirzaei1, Masoud Yunesian2, 3, Simin Nasseri1, 2, Mitra Gholami4, Esfandiyar Jalilzadeh5, Shahram Shoeibi6, 7, Alireza Mesdaghinia1, 2

1. Center for Water Quality Research (CWQR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran.2. Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.3. Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran.4. Research center for environmental health technology, Department of Environmental Health Engineering, School of Public Health, Iran University of Medical Sciences, Tehran, Iran.5. Water and Wastewater Company, Department of Water and Wastewater Quality Control Laboratory, Tehran, Iran.6. Food and Drug Laboratory Research Center, Food and Drug Organization, Ministry of Health & Medical Education, Tehran, Iran.7. Food and Drug Reference Control Laboratories Center, Food and Drug Organization, Ministry of Health & Medical Education, Tehran, Iran.

Correspondence to:

Alireza Mesdaghinia, Center for Water Quality Research (CWQR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, 8th floor, Gol Building, North Karegar St., Enghelab Sq., Tehran, Iran, Tel.:

+98 2188978399, Fax: +982188978398, E-mail: [email protected].

1

mailto:[email protected]

Table S.1

Target antibiotics organized by their therapeutical groups, structures, molecular weight (MW), PKa values and their assigned internal standard with their

characteristics

Chemical Groups

compound Chemical formula

MW PKa Corresponding internal standard

CAT number of internal standard

Chemical formula for internal standards

Β-lactam Amoxicillin C16H19N3O5S 365.4 g/mol

3.32 Cefuroxime-d3 sc-217864 C16H13D3N4O8S

Penicillin G C16H18N2O4S 334.4 g/mol


Cephalosporin Cephalexin C16H17N3O4S 347.39 g/mol


Ceftriaxone C18H18N8O7S3 554.58 g/mol


cefixime C16H15N5O7S2 453.452 g/mol


Fluoroquinolones Ciprofloxacin C17H18FN3O3 331.346 g/mol

5.76 Ciprofloxacin-d8

sc-217902 C17H10D8FN3O3•HCl

Macrolide Azithromycin C38H72N2O12 748.984 g/mol

8.74 Azithromycin-d3

sc-217686 C38H69D3N2O12

Erythromycin C37H67NO13 733.93 g/mol

8.88 Azithromycin-d3

sc-217686 C38H69D3N2O12

Nitro imidazole antibiotics

Metronidazole C6H9N3O3 171.15 g/mol

15.44 Metronidazole-d4

sc-217686 C6H5D4N3O3

MW: Molecular weight

2

Table S.2

Specification of studied water treatment plants

Name of drinking water treatment plants

The capacity of designing (m3/s)

Type of clarifier

Filters type Coagulant agent

Disinfection Water supply resources

Specification of water supply resources or catchment area

DWTP No.1 (Jalaiyah)

2.7 accelerator Gravity Rapid sand

Ferric Chloride

chlorination Karaj River and Taleghan Dam

no direct input of wastewater and no recreational use is permitted

DWTP No.2 (Kan)

8 Pulsator Gravity Rapid sand

Ferric Chloride

chlorination Karaj River and Taleghan Dam

no direct input of wastewater and no recreational use is permitted

DWTP No. 3 & 4 (Tehran Pars)

3 Pulsator Gravity Rapid sand

Ferric Chloride

chlorination Latian Dam Having wastewater discharge and recreational use is permitted

DWTP No. 5 (Sohanak)

7.5 Pulsator Gravity Rapid sand

Ferric Chloride

chlorination Lar Dam Having wastewater discharge and recreational use is permitted

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Table S.3

General information on the studied WWTPs

Name Ekbatan WWTP Tehran Southern WWTPPopulation served 100000 people 2100000 peopleFlows (m3/day) 10000 m3/day 450000 m3/dayType of wastewater Sanitary sewage Sanitary and Industrial sewage Type of treatment technology

Anaerobic, Anoxic, Oxic Biological , tricking filter beds

Table S.4

The gradient elution of mobile phase

Step Total Time(min) Flow Rate(µl/min) A (%) B (%)0 0.00 800 95.0 5.01 1.00 800 95.0 5.02 5.00 800 12.0 88.03 12.00 800 0.0 100.04 20.00 800 95.0 5.0

solvent A: HPLC grade water acidified with Formic Acid at 0.1%, solvent B: methanol acidified at 0.1% with formic acid

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Table S.5

The optimized MRM (Multiple Reaction Monitoring) conditions and retention time for antibiotics by HPLC/MS/MS

raw

Antibiotic Time (msec)

Precursor ion(m/z)

Q3 (1) DP/EP/CEP/CE/CXP Q3 (2) DP/EP/CEP/CE/CXP RT(min)

1 Azithromycin-d3 100 753.076 [M+H]+ 83.2 71/7.5/64/75/4 158.2 71/7.5/64/51/4 5.0962 Amoxicillin 100 366.32 [M+H]+ 114.3 21/5/36/29/4 134.2 21/5/36/43/4 1.863 Cefixime 100 454.699 [M+H]+ 126.2 41/6/42/45/4 285.3 41/6/42/23/6 5.224 Ciprofloxacin 100 332.498 [M+H]+ 314.500 46/5.5/36/37/6 231.400 46/5.5/36/47/6 4.965 Ciprofloxacin-d8 100 340.578 [M+H]+ 322.500 46/7/38/35/6 235.300 46/7/38/51/6 4.96026 Erythromycin 100 735.086 [M+H]+ 158.200 46/6.5/64/39/4 83.100 46/6.5/64/73/4 5.46077 Penicillin-G 100 335.480 [M+H]+ 91.100 56/7.5/38/65/4 128.200 56/7.5/38/37/4 5.148 Cephalexin 100 348.497 [M+H]+ 158.1 21/4.5/34/19/4 106.200 21/4.5/34/37/4 4.919 Cefuroxime-d3 100 427.888 [M+H]+ 324.700 71/6.5/36/23/6 143.200 81/6/38/41/4 9.5510 Azithromycin 100 750.048 [M+H]+ 158.300 76/7.5/62/51/4 83.100 76/7.5/62/75/4 5.0967

DP: Declustering potentialEP: Entrance potentialCE: Collision energy CXP: Collision cell exit potentialRT: Retention Time[M+H] +1 : for each antibiotic [M+H] +1 was selected as the precursor ion in positive ion mode.Q3 (1) , Q3 (2) : for each compound, two MRM transitions were monitored, the most abundant fragment ion of which was used for quantification and the other one was used for identification (Gros et al., 2013).Time: Dwell time

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Table S.6

Calibration equation, instrumental detection limits (IDLs), linearity and repeatability (run-to-run analysis) determined for target antibiotics

No.

Antibiotic Calibration equation Regression coefficient

IDL (ng.L-1)(injected)

Repeatability %RSD(n = 5)

1 Metronidazole y=0. 172X+0.324 0.992 1 13.42 ceftriaxone y=5.32 e-4X- 4.7e-4 0.984 2.5 11.63 Penicillin y=0. 0067X- 0.058 0.991 2.5 8.14 Amoxicillin y=0. 028 X- 6.8e-4 0.956 1 5.35 Azithromycin y=0. 052X- 0.32 0.974 1 7.86 Cephalexin y=0. 003X- 0.024 0.991 2.5 5.17 ciprofloxacin y=0. 013X- 0.042 0.993 2.5 8.48 erythromycin y=0. 015X+0.36 0.987 1 6.79 cefixime y=5. 8e-4X+0.02 0.990 2.5 6.3

Table S.7

Method validation parameters including, recoveries obtained for target antibiotics, method detection limits (MDLs), and method quantitation limits (MQLs) in

treated water, ground water and river water matrices

No.

Antibiotic Treated water Ground water River waterMean

recovery (%)

RSD(n=3

)

MDLng. L-1

(Spiked)

MQLng. L-1

(Spiked)

MeanRecovery

(%)

RSD(n=3)

MDLng. L-

1

(Spiked)

MQLng. L-1

(Spiked)

Mean recovery

(%)

RSD(n=3)

MDLng. L-1

(Spiked)

MQLng. L-1

(Spiked)

1 Metronidazole 43 7.3 5 10 45 6.4 5 10 41 8.1 10 252 ceftriaxone 108 3.8 10 25 113 9.8 10 25 107 8.6 10 253 Penicillin 107 5.6 5 10 101 4.6 5 10 90 3.6 5 104 Amoxicillin 96 6.1 2 5 109 7.4 2 5 95 6 2.5 55 Azithromycin 52 7.6 1 2.5 55 7.1 0.5 2 47 4.5 0.8 2.56 Cephalexin 117 4.9 5 25 120 5.5 5 25 111 7.3 5 257 ciprofloxacin 75 7.3 2.5 5 88 6.1 2 5 85 6.5 2.5 58 erythromycin 56 8.6 1 5 44 3.7 0.8 2 42 8.9 2.5 59 cefixime 55 9.3 10 25 57 7.4 25 50 53 8.5 25 50

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Table S.8

Method validation parameters including, mean recoveries, Regression coefficient (r2), method quantitation limits (MQLs) in influent and effluent of the studied

WWTPs

Groups Analytes r2Linearity range

(ng/L)

MQL Mean Recovery ± SD (%)

Influent(ng/L) (Spiked)

Effluent(ng/L)(Spiked)

Influent Effluent

β-lactamAmoxicillin 0.9967 0.1- 2000 25 10 116.4 ± 11.2 98.9 ± 6.2

Penicillin 0.9827 0.1-2000 20 10 87.3 ± 6.8 92.5 ± 9.5

CephalosporinCephalexin 0.9932 0.1-2000 10 5 104.2 ± 9.7 111.9 ± 10.4

Cefixime 0.9856 0.1-2000 50 25 57.3 ± 10.2 62.2 ± 8.9

Fluoroquinolone Ciprofloxacin 0.9986 0.1-2000 10 5 120.1 ± 12.6 114.7 ± 16.3

MacrolideAzithromycin 0.9838 0.1-2000 10 5 53.67 ± 14.8 72.5 ± 13.2

Erythromycin 0.9912 0.1-2000 10 5 55.1 ± 11.1 64.2 ± 8.9

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Table S.9

Average removal efficiencies obtained in different WWTPs for the selected antibiotics with respect to the operating SRT and HRT in the bioreactor and the

corresponding references.

Antibiotics Wastewater treatment process SRT [d] HRT[h] Removal efficiency [%] ReferencesAmoxicillin CAS+ MF/RO 12.5 11 84 (Watkinson et al., 2007)

CAS ---- ----- 100 (Zuccato et al., 2010)CAS 16.5 12.5 99* (Watkinson et al., 2009)

Azithromycin Anaerobic/anoxic/oxic (A2O) 15 8.9 Low removal efficiency (Yan et al., 2014)Anaerobic/anoxic/oxic (A2O) 11 17 Low removal efficiency (Yan et al., 2014)

CASS 25 16 (Batt et al., 2007)

Oxidation ditch + secondary clarifier and UV

7.5 12 (Zhou et al., 2013)

Aerated Lagoon Not Applicable 26 (Guerra et al., 2014)

Facultative Lagoon Not Applicable 82 (Guerra et al., 2014)

Conventional type Chemically assisted–

primary treatment

Not Applicable -78 (Guerra et al., 2014)

Activated SludgeSecondary treatment

4 5.3 -40 (Guerra et al., 2014)


6 11 54 (Guerra et al., 2014)

Advanced TreatmentBiological nutrient removal

9 6.7 62 (Guerra et al., 2014)

Cephalexin CASS 25 16 N.D (Zhou et al., 2013)Oxidation ditch + secondary clarifier

and UV7.5 12 100 (Zhou et al., 2013)

CAS 16.5 12.5 99* (Watkinson et al., 2009)

Ciprofloxacin Activated sludge 6 1 88.7 (Zhou et al., 2013)Extended aeration 17 28-31 86.1 (Zhou et al., 2013)

Rotating biological contactors ------ 4 59 (Batt et al., 2007)

Rotating biological contactors 15 1 64 (Batt et al., 2007)

CASS 25 16 75 (Batt et al., 2007)

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Antibiotics Wastewater treatment process SRT [d] HRT[h] Removal efficiency [%] References

Oxidation ditch + secondary clarifier and UV

7.5 12 70 (Batt et al., 2007)

Aerated Lagoon Not Applicable -582 (Guerra et al., 2014)

Facultative Lagoon Not Applicable 98 (Guerra et al., 2014)

Conventional typeChemically assisted–

primary treatment

Not Applicable 56 (Guerra et al., 2014)


4 5.3 32 (Guerra et al., 2014)


6 11 88 (Guerra et al., 2014)


9 6.7 75 (Guerra et al., 2014)

CAS (coupled with subsequent Ultrafiltration and Ozone oxidation

system)

---- ----- 78 (Li et al., 2013)

CAS ( anaerobic/anoxic/oxic) 20-25 15.3 98 (Li et al., 2013)

Erythromycin 12.5 28.9 (Zhou et al., 2013)

77.9 (Zhou et al., 2013)CAS (coupled with subsequent

Ultrafiltration and Ozone oxidation system)

---- ----- 20 (Li et al., 2013)

CAS ( anaerobic/anoxic/oxic) 20-25 15.3 93 (Li et al., 2013)

Erythromycin- H2O

Aerated Lagoon Not Applicable -16 (Guerra et al., 2014)Facultative Lagoon Not Applicable 69 (Guerra et al., 2014)Conventional type

Chemically assisted–primary treatment

Not Applicable -15 (Guerra et al., 2014)


4 5.3 -10 (Guerra et al., 2014)


6 11 -18 (Guerra et al., 2014)


9 6.7 21 (Guerra et al., 2014)

Penicillin G CAS+ MF/RO 12.5 11 N.D (Watkinson et al., 2007)CAS 16.5 12.5 -29* (Watkinson et al., 2009)

9

CAS: Conventional Activated SludgeN.D: Not Detected* Removal efficiencies were not reported by the authors in the cited study, and the removal efficiencies were calculated fromreported concentration data for influent and effluent in the study

Table S.10

Statistical results (Wilcoxon test) for significant differences between antibiotics concentration in influent and effluent of Tehran Southern WWTP.

C (ng/L)Effluent

C (ng/L)Influentp-valueAntibiotics

Median (P 0.25, P 0.75)Median (P 0.25, P 0.75)80.57(67.75 , 88.52)229.75(186.12,307.12)0.028Amoxicillin20.04(9.87,30.14)36.35(34.72,45.10)0.028Penicillin107.72(59.30,228.54)515.80(398.98,704.70)0.028Ciprofloxacin350.66(297.91,416.10)561.43(441.02,714.55)0.075Cefixime4.66(0.00,9.37)898.20(762.74,926.32)0.028Cephalexin166.62(141.42,180.11)45.57(28.56,87.94)0.028Erythromycin

P0.25: percentile 0.25P0.75: percentile 0.75C: concentration

Table S.11

Statistical results (Wilcoxon test) for significant differences between antibiotics concentration in influent and effluent of Ekbatan WWTP.

C (ng/L)Effluent

C (ng/L)Influentp-valueAntibiotics

Median (P 0.25, P 0.75)Median (P 0.25, P 0.75)67.83(36.92,83.99)280.00(185.00 , 427.45)Amoxicillin7.50(3.74,16.41)27.45(16.81,34.68)0.028Penicillin158.90(141.85,214.95)711.15(657.45,747.75)0.028Ciprofloxacin

10

63.77(52.00,87.49)168.90(0.00,299.62)0.173Cefixime14.75(10.27,20.81)297.10(177.77,438.25)0.028Cephalexin11.77(9.68,14.08)12.78(0.00,40.05)0.345Azithromycin40.95(33.33,50.51)111.95(69.66,131.37)0.028Erythromycin

P0.25: percentile 0.25P0.75: percentile 0.75C: concentration

Table S.12

Date of the sampling and number of the samples

Sampling protocol for DWTPsThe number of collected sample in each

sampling eventSampling location

Sampling sites in each location

the 13th of June, 2016

the 9th of July, 2016

the 2nd of August, 2016

Total samples collected in each sampling event.

DWTP 1 influent 2 2 2 12effluent 2 2 2





Total samples Collected during the sampling campaign 60

Sampling protocol for Wastewater Treatment Plants (WWTPs)The number of collected sample in each

11

Sampling protocol for drinking water wellsThe number of collected sample in each


The number of Sampling sites the 13th of June, 2016




Drinking Water wells

13 1 1 1 39



Sampling sites in each location

the 13th of June, 2016




WWTP 1 influent 2 2 2 12effluent 2 2 2

WWTP 2 influent 2 2 2 12effluent 2 2 2


12

Sampling protocol for river water samplesThe number of collected sample in each


Sampling sites in each river the 13th of June, 2016




Kan River Site A 1 1 1 6Site B 1 1 1

Firozabad ditch Site A 1 1 1 6Site B 1 1 1


Fig. S.1Box plots for concentration ranges (Min, P 0.25, Median, P 0.75 and Max) of target antibiotics individually in wastewater influent and effluent of studied WWTPs.

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Fig. S.2. Box plots for concentration ranges (Min, P 0.25, Median, P 0.75 and Max) of target antibiotics individually in Kan River and Firozabad ditch.

15

Reference

Batt AL, Kim S, Aga DS. Comparison of the occurrence of antibiotics in four full-scale wastewater treatment plants with varying designs and operations. Chemosphere 2007; 68: 428-435.

Gros M, Rodríguez-Mozaz S, Barceló D. Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-high-performance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry. Journal of Chromatography A 2013; 1292: 173-188.

Guerra P, Kim M, Shah A, Alaee M, Smyth S. Occurrence and fate of antibiotic, analgesic/anti-inflammatory, and antifungal compounds in five wastewater treatment processes. Science of the Total Environment 2014; 473: 235-243.

Li W, Shi Y, Gao L, Liu J, Cai Y. Occurrence and removal of antibiotics in a municipal wastewater reclamation plant in Beijing, China. Chemosphere 2013; 92: 435-444.

Watkinson A, Murby E, Costanzo S. Removal of antibiotics in conventional and advanced wastewater treatment: implications for environmental discharge and wastewater recycling. Water research 2007; 41: 4164-4176.

Watkinson A, Murby E, Kolpin D, Costanzo S. The occurrence of antibiotics in an urban watershed: from wastewater to drinking water. Science of the total environment 2009; 407: 2711-2723.

Yan Q, Gao X, Chen Y-P, Peng X-Y, Zhang Y-X, Gan X-M, et al. Occurrence, fate and ecotoxicological assessment of pharmaceutically active compounds in wastewater and sludge from wastewater treatment plants in Chongqing, the Three Gorges Reservoir Area. Science of The Total Environment 2014; 470–471: 618-630.

Zhou L-J, Ying G-G, Liu S, Zhao J-L, Yang B, Chen Z-F, et al. Occurrence and fate of eleven classes of antibiotics in two typical wastewater treatment plants in South China. Science of the total environment 2013; 452: 365-376.

Zuccato E, Castiglioni S, Bagnati R, Melis M, Fanelli R. Source, occurrence and fate of antibiotics in the Italian aquatic environment. Journal of hazardous materials 2010; 179: 1042-1048.

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