Supplemental Material. Fate and effect of antibiotics in beef and dairy manure during static and turned composting

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1 Supplemental Material Fate and effect of antibiotics in beef and dairy manure during static and turned composting Partha Ray *1, Chaoqi Chen 2, Katharine F. Knowlton 3, Amy Pruden 4, and Kang Xia 2 1 Division of Animal, Dairy & Food Chain Sciences, School of Agriculture, Policy and Development, University of Reading, Reading RG6 6AR, United Kingdom 2 Department of Crop and Soil Environmental Sciences, and 3 Department of Dairy Science, 4 Department of Civil & Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States Number of pages: 13 Number of figures: 1 Number of tables: 6 * Corresponding author (drray@vt.edu)

2 MATERIALS AND METHODS Sample analysis Compost properties One set of samples was dried at 55 C until constant weight was achieved, ground, and analyzed for total C and total N. The d 0 and d 42 fresh samples were extracted with water (1:10, w/v, for 15 min) and analyzed for ph (Accumet AB150, Fisher Scientific, Pittsburgh, PA) and EC (CDH-SD1, Omega Engineering Inc., Stamford, CT) (Michel et al., 1993). Ash content was determined by incinerating dried samples at 550 C for 5 h. Total C and N concentrations were quantified by combustion (1,800 C) using a C-N analyzer (Model: vario EL cube, Elementar Analysensysteme GmbH, Hanau, Germany). UPLC-MS/MS Quantification of Antibiotics Freeze-dried samples of dairy compost were analyzed for cephapirin and pirlimycin using the methods described previously (Ray et al., 2014a; b). These methods were modified to make them suitable for freeze-dried compost samples. Briefly, 0.5 g of freeze-dried samples was extracted using 15 ml of extractant [methanol (70%) and phosphate buffer (50 mm at ph 8)]. Extracts were cleaned with solid phase extraction (SPE) using OASIS HLB Plus Short Cartridges (250 mg sorbent; Waters, Milford, MA). Aliquots of 1 ml clean extracts were dried at 35 C to dryness under N 2 gas (Zipvap 20 evaporator, Glas-Col, Terre Haute, IN) and dissolved in 1 ml of methanol:water (30:70, v/v) with 0.1% formic acid. Dissolved extracts were filtered through 0.2 µm PVDF syringe filters (Fisher, Pittsburgh, PA) into 1.5 ml amber glass HPLC vials and analyzed for cephapirin and pirlimycin using UPLC-MS/MS (Agilent 1290 UPLC coupled with Agilent 6490 Triple Quad tandem mass spectrometry). A gradient elution program consisting of two mobile phases (mobile phase A: 0.1% formic acid in water; mobile phase B:

3 % formic acid in methanol) were used at a flow rate of 0.5 ml/min. The concentration of cephapirin and pirlimycin in compost samples was quantified using the calibration curve of 7 matrix-matched standards (0.5, 1, 2, 4, 5, 10, and 20 µg L -1 matrix solution). Matrix-match standards were prepared using the SPE cleaned extracts of blank compost samples. The recovery of cephapirin and pirlimycin were 87 to 114 and 88 to 93%, respectively. The method was sensitive for analysis of cephapirin and pirlimycin in compost with a limit of detection (LOD) of 0.07 and 0.03 ng g -1 for cephapirin and pirlimycin, respectively. Beef compost samples were analyzed for sulfamethazine, tylosin, chlortetracycline, and tetracycline using a method modified from published methods (Jacobsen et al., 2004). Mixtures of sulfamerazine, roxithromycin, and oxytetracycline (each at 1 ppm) were used as internal standards. A fixed volume (50 ml) of internal standards and 10 ml of extractant [McIlvaine buffer (ph 7.0): methanol, 50:50, v/v] were added into 1 g of freeze-dried samples, and samples were mixed using a vortex mixer for 30 s and sonicated for 20 min. Samples were centrifuged at 2,200 g for 10 min, diluted to 500 ml and pre-filtered using glass microfiber filters (GF/F grade, GE Healthcare Bio-Sciences). Samples were further purified and concentrated using solid phase extraction (Agilent 20-port vacuum manifold processing stations). Oasis HLB Cartridges (60 mg sorbent; Waters, Milford, MA) were pre-conditioned with 3 ml of methanol and 3 ml of MilliQ water. Supernatants were passed through the cartridges at an approximate rate of 5 ml min -1. After washing the cartridges with MilliQ water, 3 ml of methanol was used to elute compounds. Elutes were dried to dryness at 40 C under N 2 gas (RapidVap N 2 Evaporation Systems, Labconco Corp., MO) and reconstituted with 1 ml of methanol:water (50:50, v/v). Dissolved extracts were filtered through 0.2 µm PTFE syringe filters (Fisher, Pittsburgh, PA) into 1.5 ml amber glass HPLC vials, and then analyzed using UPLC-MS/MS (Agilent 1290

4 UPLC coupled with Agilent 6490 Triple Quad tandem mass spectrometry). A gradient elution program consisting of two mobile phases (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in 95% acetonitrile) were used at a flow rate of 0.5 ml min -1. The peak areas of antibiotics were normalized to the peak areas of internal standards and then quantified using a calibration curve. For quality assurance and quality control one laboratory blank sample was included in every batch. Also one spiked-sample (with addition of a known amount of antibiotics) was included and the recovery of antibiotics for each batch was determined. The recovery of chlortetracycline, tetracycline, sulfamethazine, and tylosin ranged from 57.2 to 73.7, 44.1 to 74.2, 137 to 160, and 94 to 149%, respectively. The LOD values for chlortetracycline, tetracycline, sulfamethazine, and tylosin were 1.30, 1.11, 0.31, and 0.44 ng g -1 dry compost, respectively. 73

5 REFERENCES Jacobsen, A.M., B. Halling-Sorensen, F. Ingerslev, and S.H. Hansen Simultaneous extraction of tetracycline, macrolide and sulfonamide antibiotics from agricultural soils using pressurised liquid extraction, followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A. 1038: Michel, F.C., C.A. Reddy, and L.J. Forney Yard waste composting: Studies using different mixes of leaves and grass in a laboratory scale system. Compost Sci. Util. 1: Ray, P., K.F. Knowlton, C. Shang, and K. Xia. 2014a. Development and validation of a UPLC- MS/MS method to monitor cephapirin excretion in dairy cows following intramammary infusion. PLoS ONE. 9:e Ray, P., K.F. Knowlton, C. Shang, and K. Xia. 2014b. Method development and validation: Solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry quantification of pirlimycin in bovine feces and urine. J. AOAC Int. 97:

6 Fig. S1 Dissipation kinetics of (A) tetracycline and (B) sulfamethazine during static and turned small-scale composting of beef manure. Manure was collected from steers fed chlortetracycline sulfamethazine each at 350 mg d -1.

7 94 95 Table S1. Effect of manure type, composting approach, and composting day on the concentrations of moisture, total carbon (C), nitrogen (N), phosphorus (P), and potassium (K) in beef and dairy manure compost Moisture Total C Total N Total P Total K % mg g -1 dry compost--- Manure Beef antibiotic 54.6 ± ± ± 0.03a 1.82 ± 0.05a 11.4 ± 0.14 Beef control 54.6 ± ± ± 0.03ab 2.13 ± 0.05b 10.9 ± 0.14 Dairy antibiotic 56.4 ± ± ± 0.03b 2.32 ± 0.05b 12.9 ± 0.14 Dairy control 58.1 ± ± ± 0.03ab 2.14 ± 0.06b 11.1 ± 0.14 Composting Static 51.6 ± ± ± ± ± 0.10 Turned 60.2 ± ± ± ± ± 0.10 Day ± ± 0.15a 1.99 ± ± 0.07a 7.79 ± ± ± 0.16ab 2.02 ± ± 0.07b 11.0 ± ± ± 0.15ab 2.11 ± ± 0.07b 12.1 ± ± ± 0.15bc 2.27 ± ± 0.07c 12.4 ± ± ± 0.15bc 2.23 ± ± 0.07cd 13.0 ± ± ± 0.15c 2.07 ± ± 0.07cd 13.2 ± ± ± 0.15d 2.02 ± ± 0.07d 14.2 ± 0.17 P value Manure < < <0.05 Composting < < <0.05

8 Manure Composting < <0.05 Day <0.05 <0.05 <0.05 <0.05 <0.05 Manure Day < Composting Day < < <0.05 Manure Composting Day <0.05 Mean separation is not provided if Manure Composting or Manure Day or both are significant. Mean separation is not provided if Manure Composting or Composting Day or both are significant. Mean separation is not provided if Manure Day or Composting Day or both are significant. Within variable, means followed by different letters are significantly different (P < 0.05)

9 Table S2. Effect of manure type, composting approach, and composting day on ph, electrical conductivity (EC), and concentration of ash in beef and dairy manure compost Manure ph EC, ds cm -1 Ash, % DM Beef antibiotic 7.26 ± ± ± 0.31 Beef control 7.22 ± ± ± 0.31 Dairy antibiotic 6.79 ± ± ± 0.31 Dairy control 6.99 ± ± ± 0.31 Composting Day Static 7.24 ± ± ± 0.22 Turned 6.89 ± ± ± ± ± ± 0.22a ± ± ± 0.22b P value Manure Composting < Manure Composting Day <0.05 <0.05 <0.05 Manure Day < Composting Day <0.05 < Manure Composting Day Mean separation is not provided if Manure Composting or Manure Day or both are significant. Mean separation is not provided if Manure Composting or Composting Day or both are significant. Mean separation is not provided if Manure Day or Composting Day or both are significant. Within variable, means followed by different letters are significantly different (P < 0.05). S9

10 Table S3. Reduction in concentrations of tetracycline and sulfamethazine antibiotics during static and turned small scale-composting of beef and dairy manure Chlortetracycline Tetracycline Sulfamethazine Day Static Turned Static Turned Static Turned Reduction, % of initial concentration P value Composting Day <0.05 <0.05 <0.05 Composting Day Static and turned are static and turned composting approaches. SE for mean reduction in chlortetracycline during static and turned composting are 9.81 and 12.0, respectively. SE for mean reduction in tetracycline during static and turned composting are 11.4 and 10.2, respectively. SE for mean reduction in sulfamethazine during static and turned composting of antibiotic beef manure is S10

11 Table S4. Reduction in concentrations of macrolide and lincosamide antibiotics during static and turned small scale-composting of beef and dairy manure Tylosin Pirlimycin Day Static Turned Static Turned Reduction, % of initial concentration P value Composting < Day <0.05 <0.05 Composting Day Static and turned are static and turned composting approaches. SE for mean reduction in tylosin during static and turned composting are 11.4 and 10.2, respectively. SE for mean reduction in pirlimycin during static and turned composting are 3.38 and 4.13, respectively. S11

12 Table S5. Dissipation rate constants of different antibiotics during static and turned small- scale composting of beef and dairy manure Static Turned Phase 1 Phase 2 Phase 1 Phase d Chlortetracycline ± 0.017ab ± 0.017c ± 0.017a ± 0.017bc Tetracycline ± 0.008a ± 0.008b ± 0.008b ± 0.008b Sulfamethazine ± 0.044a ± 0.044c ± 0.044ab ± 0.044bc Tylosin ± ± 0.026a ± 0.026b Pirlimycin ± ± Static and turned are static and turned composting approaches. Within a row, means followed by different letters are significantly different (P < 0.05). Tylosin dissipation followed single phase and bi-phasic first-order kinetics during static and turned composting, respectively. Pirlimycin dissipation followed single phase first-order kinetics. S12

13 Table S6. Half-life of different antibiotics during static and turned small-scale composting of beef and dairy manure Static Turned Phase 1 Phase 2 Phase 1 Phase d Chlortetracycline 8.68 ± ± ± ± 35.0 Tetracycline 12.4 ± 5.47a 41.2 ± 5.47ab 17.5 ± 5.47ab 37.8 ± 5.47b Sulfamethazine 2.03 ± 6.10a 73.7 ± 6.10b 2.78 ± 6.10a 21.5 ± 6.10a Tylosin 18.0 ± ± ± 23.2 Pirlimycin 4.51 ± ± 0.13 Static and turned are static and turned composting approaches. Within a row, means followed by different letters are significantly different (P < 0.05). Tylosin dissipation followed single phase and bi-phasic first-order kinetics during static and turned composting, respectively. Pirlimycin dissipation followed single phase first-order kinetics. S13