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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].
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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
2.74 Cefuroxime-d3 sc-217864 C16H13D3N4O8S
Cephalosporin Cephalexin C16H17N3O4S 347.39 g/mol
4.5 Cefuroxime-d3 sc-217864 C16H13D3N4O8S
Ceftriaxone C18H18N8O7S3 554.58 g/mol
3.19 Cefuroxime-d3 sc-217864 C16H13D3N4O8S
cefixime C16H15N5O7S2 453.452 g/mol
3.45 Cefuroxime-d3 sc-217864 C16H13D3N4O8S
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)
Activated SludgeSecondary treatment
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)
Activated SludgeSecondary treatment
4 5.3 32 (Guerra et al., 2014)
Activated SludgeSecondary treatment
6 11 88 (Guerra et al., 2014)
Advanced TreatmentBiological nutrient removal
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)
Activated SludgeSecondary treatment
4 5.3 -10 (Guerra et al., 2014)
Activated SludgeSecondary treatment
6 11 -18 (Guerra et al., 2014)
Advanced TreatmentBiological nutrient removal
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)
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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
DWTP 2 influent 2 2 2 12effluent 2 2 2
DWTP 3 influent 2 2 2 12effluent 2 2 2
DWTP 4 influent 2 2 2 12effluent 2 2 2
DWTP 5 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
sampling eventSampling location
The number of Sampling sites the 13th of June, 2016
the 9th of July, 2016
the 2nd of August, 2016
Total samples collected in each sampling event.
Drinking Water wells
13 1 1 1 39
Total samples Collected during the sampling campaign 39
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.
WWTP 1 influent 2 2 2 12effluent 2 2 2
WWTP 2 influent 2 2 2 12effluent 2 2 2
Total samples Collected during the sampling campaign 24
12
Sampling protocol for river water samplesThe number of collected sample in each
sampling eventSampling location
Sampling sites in each river the 13th of June, 2016
the 9th of July, 2016
the 2nd of August, 2016
Total samples collected in each sampling event.
Kan River Site A 1 1 1 6Site B 1 1 1
Firozabad ditch Site A 1 1 1 6Site B 1 1 1
Total samples Collected during the sampling campaign 12
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.
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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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