Public Health Risk of Arsenic Species in Chicken Tissues from Live
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Supporting Information for Public Health Risk of Arsenic Species in Chicken Tissues from Live Poultry Markets of Guangdong Province, China Yuanan Hu1, Wenfeng Zhang2, Hefa Cheng3*, Shu Tao3 1 MOE Laboratory of Groundwater Circulation and Evolution School of Water Resources and Environment China University of Geosciences (Beijing) Beijing 100083, China 2 State Key Laboratory of Organic Geochemistry Guangzhou Institute of Geochemistry, Chinese Academy of Sciences Guangzhou 510640, China 3 MOE Key Laboratory for Earth Surface Processes College of Urban and Environmental Sciences Peking University Beijing 100871, China Environmental Science & Technology Date: Feb. 19, 2017 Number of Pages: 27 Number of Tables: 8 Number of Figure: 1 S1 1. Determination of Total Arsenic Contents The contents of total arsenic in the commercial feed and chicken tissue samples were determined after total digestion. The samples (0.20 g) were first pre-digested in digestion vessels with TFM liners using a mixture of 5 mL of HNO3, 1 mL of H2O2, and 1 mL of triple distilled water for 6 h at room temperature. The vessels were then sealed and placed inside a MARS microwave digestion system (CEM, Matthews, NC, U.S.). The mixtures were digested by closed-vessel microwave-assisted digestion with the following program: heating to 120 °C in 10 min and holding at 120 °C for 5 min; heating to 180 °C in 6 min and holding at 180 °C for 6 min; and heating to 190 °C in 5 min and holding at 190 °C for 10 min. After cooling to room temperature, the vessels were opened and the solutions were transferred to PTFE tubes and concentrated to 0.5 mL on an electric heating plate. The residual solutions were then transferred quantitatively into polypropylene vials and diluted to 10 mL by 2% HNO3. The diluted solutions were passed through 0.22 µm cellulose acetate membrane filters, then the contents of arsenic were determined on a 7700 X inductively coupled plasma-mass spectrometer (ICP-MS, Agilent, Santa Clara, CA, U.S.). The chicken feed samples were also digested using similar procedures. Three certified reference materials (CRMs), including a lobster hepatopancreas (TORT-2) obtained from the National Research Council of Canada, a pig liver (GBW10051) and a chicken muscle (GBW10018) supplied by the Chinese Academy of Geological Sciences, were also used in this study. Procedural blanks, at least one of the three CRMs, and duplicates of the samples (10% of the load) were digested in each batch of samples for quality assurance and quality control (QA/QC) purposes. Comparison with the certified values of the CRMs indicates that the recovery rate for total arsenic was 101.0 ± 17.9%. S2 2. Determination of Major Arsenic Species The speciation of arsenic, or the chemical forms in which it occurs, in selected samples of chicken tissues and commercial feeds was determined using the method developed previously in our group.1 In brief, the major inorganic and organic arsenic species, including arsenobetaine (AsB), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenic (i-As), p-arsanilic acid (p-ASA), and roxarsone (ROX), were extracted using microwave-assisted extraction (MAE). MAE was conducted with a MARS microwave system under the following conditions: 100 mg of tissue or feed sample, extracted by 5 mL of 22% v/v methanol, 90 mmol/L (NH4)2HPO4, and 0.07% v/v trifluoroacetic acid (TFA) at pH 10.0 (adjusted by ammonium hydroxide solution), ramping for 10 min to 71 oC, and holding for 11 min. After cooling, the extracts were transferred to 5 mL polypropylene tubes and centrifuged at 15,000 rpm for 10 min. The supernatants were filtered by 0.22 µm PTFE membrane filters, stored at 4 oC and analyzed within 48 h. The inorganic and organic arsenic species in solution samples were detected using a 7700X ICP-MS hyphenated with a 1200 High Performance Liquid Chromatography (HPLC, Agilent, Santa Clara, CA, U.S.). Arsenic species in the solution samples (injection volume: 40 µL) were separated on a PRP-X100 anion exchange chromatographic column (4.6×250 mm, 5 µm; Hamilton, Reno, NV, USA) with gradient elution. Two mobile phases with a combined flow rate of 1.0 mL/min were used for separation of the arsenic species: (A) triple-distilled water, and (B) a buffer solution of 50 mmol/L (NH4)2HPO4 with 0.1% v/v TFA and 0.2% v/v HNO3 at pH 6.0 (adjusted by dilute ammonium hydroxide solution). TFA was added in the mobile phase to improve chromatographic resolution and avoid salting-out of proteins in the column. For efficient separation of the various arsenic species, the following gradient program was run: 0–1 min, 100% phase A linearly decreased S3 to 80%; 1–4 min, 80% phase A; 4–5 min, 80% phase A linearly decreased to 0%; 5–20 min, 0% phase A; 20–21 min, 0% phase A linearly increased to 100%; 21–23 min, 100% A. Unidentified arsenic compounds were also extracted from the chicken tissue samples, and their chromatogram peaks were quantitated using the calibration curves of the arsenic species with the closest retention times. More details on instrumentation and elimination of analytical interferences can be found in our previous work.1,2 Due to lack of CRM for chicken tissue with the targeted arsenic species, five spiked chicken tissue samples were prepared by spiking with solution containing DMA, MMA, As(III), As(V), p-ASA, and ROX and used in-house as reference samples.1 The recovery rate for the sum of DMA, MMA, i-As, p-ASA, and ROX determined from extraction of the five spiked chicken tissue samples (total arsenic contents 4.1-6.9 mg/kg) was 104.0±13.8%. At least one spiked chicken tissue samples was extracted with each batch of the chicken tissue and feed samples (10% of them were duplicates). A mixed standard containing DMA, MMA, i-As, p-ASA, and ROX (~20 µg-As/L each) was also analyzed during each batch of analysis, and good reproducibility was observed (relative standard deviations for i-As, DMA, MMA, p-ASA, and ROX were 1.4, 1.1, 2.1, 6.5, and 6.1%, respectively). The limits of detection (LODs) of the speciation analysis method were 3.6 µg/kg for AsB (inferred from As(III)), 18 µg/kg for i-As, 12 µg/kg for DMA, 18 µg/kg for MMA, 31 µg/kg for p-ASA, and 72 µg/kg for ROX in tissue and feed samples (all on the basis of dry weight in term of As mass), respectively. 1 The moisture loss of fresh chicken tissue samples after freeze-drying was determined to be 74±4%. Accordingly, the LODs for arsenic species in the chicken tissues were 0.936 µg/kg for AsB, 4.68 µg/kg for i-As, 3.12 µg/kg for DMA, 4.68 µg/kg for MMA, 8.06 µg/kg for p-ASA, and 18.7 µg/kg for ROX in chicken tissues (wet weight, as As), respectively. S4 3. Tables Table S1. Details on chicken tissue samples collected on live poultry markets in Guangzhou and Lianzhou of Guangdong province, China. Sample Sampling Production Area Sampling location Tissue Chicken breed Note ID time location/producer 1 Guangzhou Live poultry market #1 2013.01.12 Liver Yellow-feathered broiler Unknown 2 Guangzhou Live poultry market #1 2013.01.24 Liver Yellow-feathered broiler Unknown 3 Guangzhou Live poultry market #1 2013.01.25 Liver Yellow-feathered broiler Unknown 4 Guangzhou Live poultry market #1 2013.01.26 Liver Yellow-feathered broiler Unknown 5 Guangzhou Live poultry market #1 2013.01.30 Liver Black-bone chicken Unknown S01 6 Guangzhou Live poultry market #1 2013.01.30 Heart Black-bone chicken Unknown S01 7 Guangzhou Live poultry market #1 2013.02.01 Liver Yellow-feathered broiler Unknown 8 Guangzhou Live poultry market #1 2013.02.01 Gizzard Yellow-feathered broiler Unknown 9 Guangzhou Live poultry market #1 2013.02.20 Liver Yellow-feathered broiler Unknown 10 Guangzhou Live poultry market #1 2013.02.20 Liver Yellow-feathered broiler Unknown 11 Guangzhou Live poultry market #1 2013.02.20 Liver Yellow-feathered broiler Unknown 12 Guangzhou Live poultry market #1 2013.02.20 Gizzard Yellow-feathered broiler Unknown 13 Guangzhou Live poultry market #1 2013.02.20 Gizzard Yellow-feathered broiler Unknown 14 Guangzhou Live poultry market #1 2013.02.21 Liver Yellow-feathered broiler Unknown 15 Guangzhou Live poultry market #1 2013.02.21 Heart Yellow-feathered broiler Unknown 16 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown S02 17 Guangzhou Live poultry market #1 2013.02.28 Heart Yellow-feathered broiler Unknown S02 18 Guangzhou Live poultry market #1 2013.02.28 Liver Black-bone chicken Unknown 19 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown 20 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown 21 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown 22 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown 23 Guangzhou Live poultry market #1 2013.02.28 Liver Yellow-feathered broiler Unknown 24 Guangzhou Live poultry market #1 2013.03.07 Liver Yellow-feathered broiler Unknown S5 25 Guangzhou Live poultry market #1 2013.03.07 Liver Yellow-feathered broiler Unknown S03 26 Guangzhou Live poultry market #1 2013.03.07 Heart Yellow-feathered broiler Unknown S03 27 Guangzhou Live poultry market #1 2013.03.07 Liver Yellow-feathered broiler Unknown S04 28 Guangzhou Live poultry market #1 2013.03.07 Heart Yellow-feathered broiler Unknown S04 29 Guangzhou Live poultry market #1 2013.03.07 Liver Black-bone chicken Unknown 30 Guangzhou Live poultry market #1 2013.03.07 Liver Yellow-feathered broiler Unknown 31 Guangzhou Live poultry market #1 2013.03.07 Liver Yellow-feathered broiler Unknown