Does Long-term Irrigation with Untreated Wastewater

Supporting Information for

Does Long-term Irrigation with Untreated Wastewater Accelerate the Dissipation of Pharmaceuticals in Soil? Philipp Dalkmann†, Christina Siebe§, Wulf Amelung†, Michael Schloter#, Jan Siemens†* †

Institute of Crop Science and Resource Conservation – Soil Science and Soil Ecology, University of Bonn, Bonn, Germany

§

Instituto de Geología, Universidad Nacional Autónoma de México, México D.F., México #

Research Unit for Environmental Genomics, Helmholtz Zentrum München, Oberschleissheim, Germany

* Corresponding author, Phone: +49 228 73 2965, Fax: +49 228 732782, e-mail: [email protected]

The SI section contains thirteen pages including two text paragraphs, six tables, and one figure.

S1

Text S1. Extraction of pharmaceuticals For the determination of the pharmaceutical concentrations in fractions of different sorption strength, freeze-dried soil samples were extracted sequentially. Therefore, at each of the extraction days soil samples were transferred to borosilicate centrifuge glasses and extracted with 25 mL of a 0.01 M CaCl2 solution to determine the easy extractable or “bioaccessible” compound fraction (e.g., [1]). Afterwards, centrifuge glasses were shaken end-over-end in an overhead shaker for 24 h followed by centrifugation at 2500 × g for 20 min. Supernatants were decanted and acidified to a pH of 2.4 with 12 M hydrochloric acid (HCl). Afterwards, an aliquot of 950 µL of each sample was transferred to HPLC vials. Subsequently, 50 µL of the internal standard solution were added as a cocktail with a concentration of 1 µg mL-1 of each isotope-labeled pharmaceutical. HPLC vials were stored at -21°C until measurement. This first CaCl2 extraction step was followed by a second one with 0.01 M CaCl2 to determine the easily desorbable compound fraction by repeating the procedure mentioned above. To assess the strongly bound, sequestered fraction of pharmaceuticals in soil, the CaCl2extracted soil samples were freeze-dried again and extracted via accelerated solvent extraction (ASE). We combined two different solvents for the extraction to account for the different physico-chemical properties of the pharmaceuticals (Table 2 in the main article). Following Dalkmann et al. [2] we used an aqueous 50 mM phosphoric acid:acetonitrile solution (50:50, v/v; according to Golet et al. [3] and a methanol:water solution (50:50, v/v; according to Göbel et al. [4]). Details on the ASE-extraction procedure are given in Table S3. Aliquots of 950 µL were transferred to HPLC vials and spiked with the internal standard solution as already described for the CaCl2 samples. Extraction recoveries of the extraction method varied between 54-95%. Recovery rates for the individual compounds are presented in Table S2. All chemicals were of HPLC gradient grade quality. Text S2. LC-MS/MS analysis The analysis of pharmaceutical concentrations in soil extracts was performed with liquid chromatography tandem mass spectrometry (LC-MS/MS). A ThermoFinnigan system was used that was composed of a Surveyor autosampler plus, a Surveyor MS pump plus, and a TSQ Quantum Ultra tandem mass spectrometer equipped with an heated electrospray ionization ion source (HESI) operating in positive mode (Thermo Finnigan, Dreieich, Germany). The separation of pharmaceuticals was achieved with an XBridge C18 3.5 µm, S2

2.1x150 mm (Waters, Milford, MA, USA) HPLC column with guard column (Sentry 2.1x10 mm, Waters, Milford, MA, USA). All pharmaceuticals were analyzed in the same run. Eluents were methanol (A) and Millipore water (B) both acidified with 0.1% formic acid. The flow rate was 300 µL min-1. The gradient elution started with 5% A, increasing after 5 min to 60%, raising from 60 to 80% after 15 min, further raising from 80 to 95% after 16 min, maintaining 95% for 0.5 min, and then back to initial conditions in 0.5 min. These initial conditions were kept until method end after 25 min. For CaCl2- and ASE- extracts, 10 µL of sample were injected into the system using partial loop injection. Ionization parameters were: discharge current 4.0 kV, vaporizer temperature 390°C, and capillary temperature 217°C. Nitrogen served as sheath and auxiliary gas and helium was used as collision gas at a pressure of 1.5 mTorr. The MS was operated in selected reaction monitoring (SRM) mode with at least two transitions being measured for each compound.

S3

Table S1. Consumption data, excretion rate, Predicted Environmental Concentrations (PEC), and measured wastewater concentrations of the target pharmaceuticals.

Compound

Consumptiona

Excreted fractionb

min. PECc

max. PECd

Concentrations of pharmaceuticals measured in wastewater Gibson et al. [6]

Siemens et al. [7]

Chávez et al. [8]

2011-2012e

[kg yr-1]

[%]

trimethoprim

14,115

80

0.17

1.13

carbamazepine

9,395

3

0.01

0.04

sulfamethoxazole

70,164

30

0.42

2.82

naproxen

84,752

70

1.19

7.94

bezafibrate

2,138

70

0.02

0.14

diclofenac

11,033

37

0.08

0.55

ciprofloxacin

16,045

20

0.06

0.43

0.47 ± 0.30

enrofloxacin

n.a.f

n.a.

-

-

0.04 ± 0.03

clarithromycin

4,082

25

0.07

0.46

a

b

[µg L-1] 0.28-0.32

0.13 ± 0.11 0.20-0.28

0.26 ± 0.14 1.21 ± 0.76

15.22-16.65

2.84-5.60

1.61-16.34

0.07-0.1 1.72-6.36

14.02 ± 15.87 3.64 ± 2.54

0.42-0.55

1.22-3.77

0.40-1.40

1.45 ± 0.87

0.02 ± 0.03 c

average consumption in 2003 and 2004 in Mexico [5]; parent compound including conjugates, data are from Siemens et al. [7] and Verlicchi et al. [9]; minimum Predicted Environmental Concentration (considering a wastewater volume of 25.9x109 L/day for the Mexico City Metropolitan area); d maximum PEC considering a wastewater volume of 3.89x109 L/day) e Measured wastewater concentrations at La Licuadora (wastewater channel; mean of three sampling campaigns: 03/2011, 09/2011, 05/2012 ± STDDEV); f not available

S4

Table S2: General chemical characteristics of untreated Mexico City wastewater used for irrigation in the Mezquital Valley. Parameter pH -1

el. cond. [µS cm ]

SARH 1985 [10, 11]a

CNA et al. [12]b

Jiménez and Landa [13]

Chávez et al. [8]

2011-2012c

7.2-7.4

6.97-7.64

7.33-7.84

-

7.26-8.07

605-1483

1114-1673

830-2110

734-3000

1013-1559

-1

-

78-131

-

-

-

-1

-

294-315

262-358

113-577

-

682-1488

-

95-185

49-383

-

18-29

23-23

31-40

-

-

NH4-N [mg L ]

7.51-17.5

8.5-18.5

18-29

-

3.6-4.0

NO3-N [mg L-1]

0.02-0.03

0.2-0.3

-

0.02-0.2

0

Total P [mg L-1]

2.5-3.7

1.9-2.1

8-11

-

-

Ortho-P [mg L-1]

-

1.6-1.7

1.5-1.9

-

-

Ca2+ [mg L-1]

38-53

-

-

36-52

33-47

Mg2+ [mg L-1]

18-25

-

-

-

15-30

K+ [mg L-1]

16-43

18-37

-

-

21-37

Na+ [mg L-1]

59-205

78-337

-

-

101-203

Cl- [mg L-1]

66-225

77-243

-

70-176

81-162

HCO3- [mg L-1]

189-295

233-597

-

270-504

400-720

SO42- [mg L-1]

40-43

187-346

-

53-2492

31-103

BOD [mg L ] COD [mg L ] -1

TSS [mg L ] -1

Total N [mg L ] -1

a

b

data from Officinas Centrales del Distrito de Riego 03, Mixquiahuala, Hidalgo, Mexico for 1985; samples from 1993; Measured wastewater concentrations at La Licuadora (wastewater channel; three sampling campaigns: 03/2011, 09/2011, 05/2012).

S5

Table S3. Recoveries of ASE-extractions Ciprofloxacin Enrofloxacin Sulfamethoxazole Trimethoprim Clarithromycin Carbamazepine Naproxen Diclofenac Bezafibrate [%] [%] [%] [%] [%] [%] [%] [%] [%] 89

78

54

83

93

77

95

85

96

Table S4. Extraction parameters of the Accelerated Solvent Extraction (ASE) Method 1 (after Göbel et al.a) methanol : Millipore-water (1:1) (v/v) 100

Method 2 (after Golet et al.b) 50 mM aqueous H3PO4 : acetonitrile (1:1) (v/v) 100

Temperature [°C]

100

100

Heating time [min] Static extraction [min] Extraction cycles

5

5

5

10

2

3

Flush volume [%]

60

90

Flush with N2 [sec]

60

180

Parameter Solvent Pressure [bar]

a

[3]; b [4]

S6

Table S5. Total extracted soil concentrations of the unspiked control samples incubated for 0.1 and 150 days, respectively (means, standard deviation in brackets) Irrigation time

Nonsterile/ sterilea

TRI

CAR

SMX

NAP

BEZ

DIC

CIP

ENR

CLA

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

day 0.1

day 150

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

[µg kg-1]

0 years

n

0.16

0.03 (0.05)

< RLOQb

0.01 (0.02)

0.26

< RLOQ

0.16

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

< RLOQ

14 years

n

0.44 (0.07)

0.49 (0.19)

4.87 (1.16)

5.33 (0.55)

3.64 (1.37)

2.15 (0.22)

2.74 1.07

0.49 (0.45)

< RLOQ

< RLOQ

0.02 0.03

< RLOQ

1.99 (0.40)

1.38 (0.11)

0.79 0.13

0.42 (0.18)

0.80 (0.51)

0.17 (0.12)

100 years

n

1.75

3.19 (0.56)

7.02

8.75 (1.35)

3.91

4.03 (0.93)

2.97

1.02 (1.02)

< RLOQ

< RLOQ

< RLOQ

< RLOQ

2.22

1.46 (0.11)

1.90

0.76 (0.28)

1.80

1.36 (1.55)

100 years

s

3.35 (0.81)

3.62 (0.65)

8.69 (1.78)

9.16 (0.69)

6.44 (1.48)

4.56 (0.29)

< RLOQ

0.11 (0.20)

0.89 (0.50)

0.60 (0.13)

0.33 (0.13)

0.29 (0.18)

5.34 (0.36)

n.m.c

n.m.

n.m.

1.66 (1.36)

1.65 (1.28)

TRI = trimethoprim; CAR = carbamazepine; SMX = sulfamethoxazole; NAP = naproxen; BEZ = bezafibrate; DIC = diclofenac; CIP = ciprofloxacin; ENR = enrofloxacin; CLA = clarithromycin a n = non-sterile, s = sterile incubation; b Routine Limit Of Quantification; c not measured

S7

Table S6. Estimated initial values of the easily extractable (EAS) fraction (C0(EAS)) and the residual (RES) fraction (C0(RES)), as well as rate coefficients kEAS (transfer rate of pharmaceuticals from the EAS fraction into the RES fraction), kRES (transfer rate of pharmaceuticals from the RES fraction into the EAS fraction), and qEAS (pooled rate of dissipation due to mineralization, transformation, and formation of non-extractable residues); n.c. = not calculated. compound

soil

C0(EAS)

C0(RES)

[µg kg-1]

[µg kg-1]

kEAS

kRES

qEAS

kEAS/kRES

R2

trimethoprim

Santiago Tezontlale El Tigre 1 El Tigre 2 Casco de Hacienda 1 Casco de Hacienda 2 Tlaxcoapan Ulapa 1 Ulapa 2 Juandhó 1 Ulapa 1 (sterile) Ulapa 2 (sterile) Juandhó 1 (sterile)

40.5 15.3 23.2 42.2 26.1 16.9 27.5 51.3 0.0 0.0 31.5 23.0

964.1 723.8 750.6 955.8 688.8 964.0 852.6 824.5 941.2 874.9 1123.5 1026.2

0.741 0.676 0.199 0.161 0.103 0.714 0.751 0.383 0.511 0.194 0.488 0.565

0.057 0.092 0.026 0.024 0.021 0.067 0.022 0.022 0.033 0.005 0.020 0.019

0.072 0.193 0.451 0.336 0.544 0.559 0.261 0.261 0.256 0.020 0.044 0.035

13.04 7.33 7.73 6.74 4.87 10.62 34.79 17.75 15.49 40.73 24.25 29.02

0.98 0.85 0.95 0.98 0.98 0.99 0.94 0.99 0.98 0.97 0.99 0.99

carbamazepine

Santiago Tezontlale El Tigre 1 El Tigre 2 Casco de Hacienda 1 Casco de Hacienda 2 Tlaxcoapan Ulapa 1

768.5 676.5 565.2 562.4 522.7 604.4 462.6

299.0 366.8 461.9 486.7 499.5 445.7 600.7

0.027 0.020 0.033 0.030 0.036 0.029 0.188

0.030 0.016 0.021 0.014 0.020 0.022 0.082

0.001 0.002 0.005 0.001 0.002 0.002 0.003

0.92 1.21 1.59 2.17 1.83 1.34 2.28

0.93 0.94 0.95 0.91 0.96 0.94 0.84

S8

Table S6 (continued). compound

soil

C0(EAS)

C0(RES)

[µg kg-1]

[µg kg-1]

kEAS

kRES

qEAS

kEAS/kRES

R2

carbamazepine

Ulapa 2 Juandhó 1 Ulapa 1 (sterile) Ulapa 2 (sterile) Juandhó 1 (sterile)

474.7 431.2 435.8 523.7 450.8

527.4 576.9 594.2 532.3 603.4

0.017 0.014 0.027 0.072 0.026

0.008 0.006 0.013 0.043 0.012

0.000 0.001 0.002 0.002 0.002

2.04 2.32 2.18 1.67 2.12

0.96 0.97 0.97 0.92 0.98

sulfamethoxazole


6483.5 6180.5 4883.7 5301.8 5548.2 5982.8 4260.9 4999.4 5005.2 4177.0 5176.5 4674.8

-133.9 105.1 454.3 337.8 309.5 208.7 789.3 401.4 475.6 609.8 299.9 560.5

0.014 0.015 0.033 0.031 0.028 0.025 0.030 0.028 0.034 0.032 0.040 0.033

0.040 0.010 0.011 0.010 0.013 0.011 0.013 0.010 0.009 0.014 0.008 0.008

0.020 0.070 0.188 0.104 0.114 0.093 0.072 0.109 0.145 0.055 0.155 0.179

0.35 1.48 3.10 3.28 2.15 2.23 2.32 2.85 3.88 2.25 5.16 4.43

0.99 0.99 0.98 0.99 1.00 1.00 0.95 0.96 0.98 0.96 0.98 0.96

naproxen

Santiago Tezontlale El Tigre 1 El Tigre 2 Casco de Hacienda 1 Casco de Hacienda 2 Tlaxcoapan

8715.6 8208.8 6968.4 7478.1 6852.1 8103.0

1109.2 1225.8 1831.2 1390.3 1502.6 1309.7

0.007 0.001 0.009 0.003 0.015 0.000

0.034 0.017 0.057 0.014 0.139 0.025

0.031 0.042 0.078 0.044 0.104 0.053

0.22 0.09 0.16 0.21 0.11 0.00

1.00 0.99 0.99 1.00 0.99 0.99

S9


soil

C0(EAS)

C0(RES)

[µg kg-1]

[µg kg-1]

kEAS

kRES

qEAS

kEAS/kRES

R2

naproxen

Ulapa 1 Ulapa 2 Juandhó 1 Ulapa 1 (sterile) Ulapa 2 (sterile) Juandhó 1 (sterile)

6373.7 7003.8 6614.4 6500.2 7692.5 7101.2

2157.0 1535.3 1752.4 1177.2 726.2 1144.9

0.041 0.000 0.002 0.096 0.070 0.051

0.101 0.014 0.012 0.190 0.183 0.111

0.068 0.040 0.033 0.002 0.003 0.003

0.41 0.01 0.14 0.50 0.38 0.46

0.98 0.99 0.99 0.93 0.96 0.95

bezafibrate


215.4 167.7 142.7 139.7 114.4 157.0 109.0 127.5 116.3 175.2 191.5 170.9

42.8 29.8 53.1 51.6 25.4 36.0 46.3 24.9 55.9 75.0 56.6 82.6

0.032 0.044 0.015 0.018 0.024 0.040 0.149 0.224 -0.004 0.121 0.110 0.043

0.041 0.036 0.034 0.021 0.037 0.062 0.097 0.271 0.012 0.106 0.105 0.036

0.133 0.367 0.373 0.164 0.546 0.240 0.125 0.106 0.110 0.014 0.037 0.017

0.78 1.22 0.43 0.86 0.65 0.64 1.54 0.83 n.c. 1.14 1.04 1.21

0.98 0.99 0.99 0.98 0.99 0.99 0.94 0.93 0.96 0.88 0.94 0.88

diclofenac

Santiago Tezontlale El Tigre 1 El Tigre 2 Casco de Hacienda 1 Casco de Hacienda 2

925.9 656.6 371.2 277.3 283.3

50.9 82.1 88.4 101.3 59.5

0.001 -0.006 0.111 -0.086 0.193

0.055 0.074 0.438 0.062 0.792

0.339 0.426 0.848 0.765 1.256

0.02 n.c. 0.25 n.c. 0.24

0.99 0.99 0.99 0.98 1.00

S10


soil

C0(EAS)

C0(RES)

[µg kg-1]

[µg kg-1]

kEAS

kRES

qEAS

kEAS/kRES

R2

diclofenac

Tlaxcoapan Ulapa 1 Ulapa 2 Juandhó 1 Ulapa 1 (sterile) Ulapa 2 (sterile) Juandhó 1 (sterile)

386.6 198.3 189.9 191.8 462.6 511.6 361.2

93.0 35.8 53.1 135.4 229.9 167.1 223.5

-0.034 0.297 -0.261 -0.414 -0.311 -0.166 -0.959

0.144 0.341 0.033 0.012 0.004 0.006 0.006

0.803 0.503 1.807 1.188 1.251 0.978 2.929

n.c. 0.87 n.c. n.c. n.c. n.c. n.c.

1.00 0.98 0.99 0.98 0.82 0.95 0.96

ciprofloxacin


8.7 0.7 1.6 1.5 1.2 0.4 1.9 3.0 1.3 n.c. n.c. n.c.

61.9 38.3 58.4 59.1 27.6 44.2 68.8 43.8 59.1 n.c. n.c. n.c.

0.437 0.009 0.033 0.057 0.202 0.023 0.289 0.019 0.074 n.c. n.c. n.c.

0.097 0.025 0.034 0.019 0.046 0.032 0.034 0.018 0.019 n.c. n.c. n.c.

0.147 0.069 0.529 0.340 0.448 0.564 0.554 0.444 0.290 n.c. n.c. n.c.

4.49 0.36 0.96 2.98 4.43 0.73 8.43 1.08 3.81 n.c. n.c. n.c.

0.90 0.92 0.97 0.98 0.95 0.97 0.90 0.94 0.98 n.c. n.c. n.c.

S11

-1

extracted concentration of CAR [µg kg ]

800

1st CaCl2 extraction 2nd CaCl2 extraction

700

ASE extraction

600 500 400 300 200 100 0 0

20

40

60

80

100

120

140

160

days of incubation

Figure S1. Soil concentrations of carbamazepine (CAR) extracted with 0.01 M CaCl2 solution (1st and 2nd step) followed by Accelerated Solvent Extraction (ASE). REFERENCES 1. Rosendahl, I.; Siemens, J.; Groeneweg, J.; Linzbach, E.; Laabs, V.; Herrmann, C.; Vereecken, H.; Amelung, W. Dissipation and sequestration of the veterinary antibiotic sulfadiazine and its metabolites under field conditions. Environ. Sci. Technol. 2011, 45 (12), 5216–5222. 2. Dalkmann, P.; Broszat, M.; Siebe, C.; Willaschek, E.; Sakinc, T.; Huebner, J.; Amelung, W.; Grohmann, E.; Siemens, J. Accumulation of pharmaceuticals, Enterococcus, and resistance genes in soils irrigated with wastewater for zero to 100 years in Central Mexico. PLoS ONE 2012, 7 (9), e45397. 3. Golet, E. M.; Strehler, A.; Alder, A. C.; Giger, W. Determination of fluoroquinolone antibacterial agents in sewage sludge and sludge-treated soil using accelerated solvent extraction followed by solid-phase extraction. Anal. Chem. 2002, 74 (21), 5455–5462. 4. Göbel, A.; Thomsen, A.; McArdell, C. S.; Alder, A. C.; Giger, W.; Theiss, N.; Löffler, D.; Ternes, T. A. Extraction and determination of sulfonamides, macrolides, and trimethoprim in sewage sludge. J. Chromatogr. A 2005, 1085 (2), 179–189. 5. IMS IMS chemical country profile Mexico; IMS Health Incorporated: London, 2008. S12

6. Gibson, R.; Becerril-Bravo, E.; Silva-Castro, V.; Jiménez, B., Determination of acidic pharmaceuticals and potential endocrine, disrupting compounds in wastewaters and spring waters by selective elution and analysis by gas chromatography-mass spectrometry. J. Chromatogr. A 2007, 1169, 31–39. 7. Siemens, J.; Huschek, G.; Siebe, C.; Kaupenjohann, M., Concentrations and mobility of human pharmaceuticals in the world’s largest wastewater irrigation system, Mexico City– Mezquital Valley. Water Res. 2008, 42, 2124–2134. 8. Chávez, A.; Maya, C.; Gibson, G.; Jiménez, B, The removal of microorganisms and organic micropollutants from wastewater during infiltration to aquifers after irrigation of farmland in the Tula Valley, Mexico. Environ. Pollut. 2011, 159, 1354–1362. 9. Verlicchi, P.; Galletti, A.; Petrovic, M.; Barcelo, D., Hospital effluents as a source of emerging pollutants: An overview of micropollutants and sustainable treatment options. J. Hydrol. 2010, 389 (3-4), 416–428. 10. Siebe, C. Akkumulation, Mobilität und Verfügbarkeit von Schwermetallen in langjährig mit städtischen Abwässern bewässerten Böden in Zentralmexiko. Hohenheimer Bodenkundliche Hefte 1994, 17, p. 19. 11. Gutiérez-Ruiz, M. E.; Siebe, Ch.; Sommer, I. Effects of land application of wate water from Mexico city on soil fertility and heavy metal accumulation: a bibliographical review. Environ. Rev. 1995, 3, 318–330. 12. Comisión Nacional del Agua, British Geological Survey, London School of Hygiene and Tropical Medicine and University of Birmingham, Effects of wastewater reuse on groundwater in the Mezquital Valley, Hidalgo State, Mexico- Final Report November 1998, BGS Technical Report WC/98/42 p. 76. 13. Jiménez, C. B.; Landa, V. H. Physico-chemical and bacteriological characterization of wastewater from Mexico City. Water Sci. Technol. 1998, 37 (1), 1–8.

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