Quality control criteria and test method for the gastrointestinal in-vitro bioavailability test using arsenic-contaminated soils

• Main Authors: Chia-Yu Lin, 林家玉
• Other Authors: Chow-Feng Chiang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/79466744598160168834

碩士 === 中國醫藥大學 === 環境醫學研究所 === 96 === In order to preserve underground water resource for sustainable use, most countries in the world have promulgated heavy metal standards for soil. Recently Taiwan and many other countries have proposed a variety of risk assessment meth-ods for site remediation. But most of the methods normally assume 100% bioavail-ability for contaminated soils. The US Environmental Protection Agency (USEPA) allows for a modification of bioavailability in the Superfund Program. The absolute bioavailability factor (ABF) is defined as the dose percentage of contaminant in soil entering the blood circulation system, while relative bioavailability factor (RBF) is defined as the ABF ratio in percentage between soil samples to the standard sample. Various in-vitro bioavailability test methods based on gastrointestinal physiology have been proposed since 1992. The aim of this study is to propose a standard test procedure for bioavailability focusing on establishing quality control (QC) criteria and simplifying test procedure and apparatus. This study refers to the physiologically based extraction test (PBET) developed by USEPA Region VIII and the method developed in the early study by our group. The first phase of this method simulates stomach condition (pH 1.8) and the second phase simulates intestinal condition (pH 7.0). Many test parameters proposed in these methods have widely accepted in the literature for sample pretreatment, tem-perature, enzyme dose, pH, and reaction time. This study used sodium arsenate (Na2HAsO4•7H2O) and NIST 2710 soil as the standards to investigate the effect of mixing intensity, liquid to solid (L/S) ratio, extraction time, and extract pretreatment methods for the determination of bioavailability. Testing at a constant L/S ratio of 1000:1 mL/gm, all the ABFs (n = 5) obtained for the 2 phases from the 3 velocity gradients (G) of 0, 470, and 1000 1/sec had no significant difference (95 % CI) for both standards. The ABFs (n = 5) from both phases were 82-96 % for sodium arsenate and were 26-35% for the NIST soil. When the test was conducted at a constant G of 470 1/sec with 3 L/S ratios of 200:1, 1000:1, and 5000:1 mL/gm, only the ABFs obtained from the intestinal phase for the standard soil resulted in significant difference (p =0.02). The ABFs (n= 5) ob-tained from both phases were 85-101 % for sodium arsenate and were 25-33 % for the standard soil. The As concentrations extracted for both standards from the intes-tinal phase were within 10% of difference for 2-11 hours of extraction time based on the first hour of extraction. Thus 1 hour of extraction appears to be appropriate. Sample pretreatment using centrifugation with or without a follow-up filtration yielded no significant difference. In conclusion, we propose 3 types of QC criteria: (1) a concentration less than the method detection limit (MDL) for the reagent blank; (2) a ABFG and ABFI of 90±8 % and 90±5 % for sodium arsenate, respectively, (3) a RBFG and RBFI of 35±2 % and 30±3 % for NIST 7210 soil, respectively. Based on the test results, this study proposes a standard procedure for the two-phase bioavailability test. The soil sample is first dried by air and sieved for the particle size of 250 μm or less. A 0.5 gram of sample is fed into a serum bottle with a liquid volume of 500 mL at a liquid to solid (L/S) ratio of 1000:1 mL/gm in the stomach phase. A velocity gradient (G) of 500 1/sec is provided using magnetic stir-ring and a temperature of 37±0.5 oC is maintained in a air-circulated incubator. The extraction liquid contains 0.15 M of NaCl and 1% of porcine pepsin and the pH is adjusted to 1.8±0.1 by HCl stock solution. After 1 hour of extraction, a sample of extract is taken and centrifuged to remove particulate matters. The supernatant is determined for total arsenic. In the intestinal phase, pancreatin and bile are added to the extract liquid and is adjusted to pH 7.0±0.1 by NaHCO3 stock solution. After I hour of extraction, a sample extract is taken and centrifuged for the measurement of arsenic. In order to simply the method, we further propose using magnetic stirring with weighted ring in stead of argon sparging; using air-circulated incubator in stead of water bath for better observation and sample collection; using centrifuging in stead of filtration for extract pretreatment; using one phase of simplified bioaccessibility extraction (SBET) method instead of the two-phase method for a better estimate to the in-vivo method.