Supplemental Information for Occurrence and Transformation of Veterinary Antibiotics and Antibiotic Resistance Genes in Dairy Manure treated by Advanced Anaerobic Digester and by Conventional Treatment Methods
Authors: Joshua S. Wallace,1 Emily Garner,2 Amy Pruden,2 Diana S. Aga1*
1
Department of Chemistry, University at Buffalo-The State University of New York, Buffalo,
NY USA 2
Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State
University, Blacksburg, VA USA
*Corresponding author contact: email:
[email protected] phone: (716) 645-4220
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Supplemental Figure S1. Schematic representation of the Advanced Anaerobic Digester (AAD) in this study. Samples of raw manure (RM) were taken upstream of the manure receiving tank, post pasteurization (PP) from directly before anaerobic digestion, and post digestion (PD) immediately after discharge from the digester.
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Supplemental Figure S2. Schematic representation of the sampled Natural Aeration (NA) treatment system.
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Supplemental Figure S3. Schematic representation the of Liquid Solid Separation (LSS) treatment system
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Table S1. Physico-chemical properties of the collected manures from each treatment system. Sampling Location
Physical Parameters pH Total Solids (TS, %) Volatile Solids (VSS, %) CODa (g kg-1) Nutrient Parameters Total Nitrogen (TKN, mg kg-1) Ammonia (mg kg-1) Organic Nitrogen (mg kg -1) Phosphorus, P2O5 (mg kg-1) Potassium, K2O (mg kg-1)
Natural Aeration Raw Manure
Raw Influent
Liquid Effluent
Solid Effluent
Raw Influent
Pasteurized Manure
Post Digestion
7.2 7.7 6.16 76.1
7.7 8.3 6.28 61.7
7.8 4.9 3.29 42.4
8.8 37.4 33.4 127.4
7.3 7.9 6.20 72.9
6.8 7.7 6.41 100.0
7.7 4.3 3.0 34.8
4,610 2,446 2,153 623 3,819
4,029 2,414 1,616 508 2,435
3,931 2,450 1,481 471 2,435
4,444 2,180 2,265 892 2,241
3,840 1,593 2,247 465 4,210
2,917 717 2,200 519 1,578
3,281 1,609 1,589 220 1,185
Liquid-Solid Separation
Advanced Anaerobic Digester
a
COD: Chemical oxygen demand determined by EPA Standard method 5220B
Supplemental Table S2. Average density and total solids in liquid manure fractions for samples collected from all three treatment systems. (n = 9, Advanced digester; n = 3, Natural Aeration, Liquid-Solid Separation System)
Manure Sample
Parameter, average (SD) Liquid Density Total Solids -1 (g mL ) (g mL-1)
Advanced Digester Raw Manure Post Pasteurization Post Digestion
1.017 (0.009) 1.018 (0.002) 1.012 (0.006)
0.024 (0.001) 0.026 (0.002) 0.0140 (0.0002)
Natural Aeration
1.017 (0.001)
0.0240 (0.0006)
Separation System Raw Manure Separated Liquids
1.03 (0.02) 1.016 (0.006)
0.0282 (0.0005) 0.0220 (0.0001)
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Sample Processing Samples were centrifuged at approximately 4000 x g for 4 hours at 4°C. The supernatant was decanted into clean HDPE bottles and stored at -40°C prior to analysis. Remaining solids were frozen and lyophilized for 48 hours. Dried solids were pulverized and homogenized in a mortar and pestle, sieved to 0.5 mm and stored at -40°C prior to analysis.
Chemicals and Reagents Demeclocycline (DMC, surrogate), minocycline (MIN, internal standard), oxytetracycline (OTC), sulfachloropyridazine (SCP), sulfadiazine (SPD), sulfadimethoxine (SDM), sulfamerazine (SMR), sulfameter (SMT), sulfamethazine (SMZ), sulfamethiazole (SMI), sulfamethoxazole (SMX), tetracycline (TC), and were obtained from Sigma Aldrich (St. Louis, MO). Sulfathiazole (STZ) was obtained from ICN Biomedicals, Inc. (Aurora, OH). 4-epichlorotetracycline (ECTC), anhydrochlorotetracycline (ACTC), and anhydrotetracycline (ATC), and chlorotetracycline (CTC), were obtained from Acros Organics (Morris Plains, NJ). 4-epitetracycline (ETC) was obtained from Spectrum Chemical Mfg. Corp (Gardena, CA). Stable isotopes of phenyl-13C6sulfamethazine (13C6-SMZ) and d10-carbamazepine (d10-CBZ) were obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA) and d4-sulfamethoxazole (d4-SMX) from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). Methanol, acetonitrile, and ethyl acetate were of HPLC or LC-MS grade, obtained through Fisher Chemical (Fairlawn, NJ). Glacial acetic acid, phosphoric acid, and ammonium hydroxide were of ACS grade and obtained through J.T. Baker (Phillipsburg, NJ). Disodium ethylenediamine tetraacetate (EDTA) was obtained from Fisher Chemical (Fairlawn, NJ). Citric acid monohydrate and anhydrous dibasic sodium phosphate (Na2HPO4) were obtained from Sigma Aldrich (St.
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Louis, MO). NANOpure TM water was used in all experiments from Barnstead International (Dubuque, IA). Oasis hydrophilic-lipophilic balance (HLB) SPE 500 mg/6mL cartridges were purchased through Waters (Mildford, MA) and aminopropyl (NH2) 500 mg/6mL cartridges were purchased from Phenomenex (Torrence, CA).
Glass and Plasticware Treatment All glassware was soaked in 10% nitric acid for 8 h, rinsed with NANOpure TM water, and baked at 250°C overnight to reduce adsorption of tetracyclines onto the glassware (Tso et al., 2011). HDPE bottles were soaked in 2% nitric acid for 2 hours, rinsed with NANOpure TM water, and air-dried prior to use. Disposable polypropylene centrifuge tubes were used as received.
Standard Preparation and Stability Primary standard solutions of analytes were prepared at 1 mg/mL concentrations in methanol and stored at -40°C. Due to limited solubility in methanol, primary standards of SPD were prepared in 1% (v/v) ammonium hydroxide in methanol. From primary standard solutions, individual calibration standard solutions of sulfonamides and tetracyclines at 10 μg/mL were prepared. Primary and calibration standards were analyzed against freshly prepared standards for stability. Both sulfonamide and tetracycline standards were stable for at least 3 months. Spiking standards of 500 ng/mL in methanol were prepared daily from calibration standards. Quality control standards of 10 ng/mL were prepared from spiking solutions in water/methanol (95/5, v/v) and 0.1% glacial acetic acid.
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Extraction, Cleanup, and Analysis of Antibiotics Liquid Manure Liquid manure samples were extracted according to a previously described method (Wallace and Aga, 2016). Briefly, 5mL of liquid manure was added to a 15mL polypropylene centrifuge tube and the pH was adjusted to 4.0±0.2 with glacial acetic acid, and spiked with 25µL of surrogate standards (500ng/mL) d4-SMX,
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C6-SMZ and DMC. One mL of 0.1M
EDTA/McIlvaine buffer (pH=4.0) was added to each sample and vortexed for 30s. Five mL of methanol was then added, vortexed vigorously for 60s and centrifuged at approximately 4000g for 10min at 10ºC. The supernatant was decanted into a clean 500mL HDPE bottle and the extraction was repeated a second time. Extracts were pooled and diluted to 250mL with NANOpure water to reduce organic content to
