WO2021218521A1 - 一种土壤中六价铬呼吸途径的生物可给性测试方法 - Google Patents

一种土壤中六价铬呼吸途径的生物可给性测试方法 Download PDF

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WO2021218521A1
WO2021218521A1 PCT/CN2021/083491 CN2021083491W WO2021218521A1 WO 2021218521 A1 WO2021218521 A1 WO 2021218521A1 CN 2021083491 W CN2021083491 W CN 2021083491W WO 2021218521 A1 WO2021218521 A1 WO 2021218521A1
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soil
hexavalent chromium
bioavailability
testing
experiment
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French (fr)
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夏天翔
梁竞
李宁
贾晓洋
姜昱聪
钟名誉
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北京市环境保护科学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
    • G01N21/73—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited using plasma burners or torches
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/24—Earth materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder

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  • the invention relates to the field of ecological toxicology research, in particular to a method for testing the bioavailability of hexavalent chromium respiratory pathways in soil.
  • Chromium is one of the essential trace elements for the human body. Excessive intake of chromium can cause severe acute or chronic toxicity. In welding, papermaking, printing and dyeing, leather manufacturing, and thermonuclear weapons manufacturing industries, large amounts of Cr-containing by-products will be produced. If chromium is not properly managed, it will inevitably cause it to enter the environment and be enriched in the surrounding soil; chromium in the soil can enter the human body through a variety of channels and endanger human health.
  • Respiratory inhalation is an important way for particulate matter to be exposed to the human body. Due to the complex environment in the human body, the pollutants in the particulate matter will not be completely absorbed after entering the human body. Therefore, the determination of the amount of pollutants in the fine particulate components of the soil can be given. Sex is an important method to objectively evaluate its toxicity. In addition, the total amount of heavy metals (hexavalent chromium) in the soil is generally included in the health risk assessment calculations, including the part of the heavy metals that cannot be absorbed by the human body and endanger human health. This will overestimate the soil. The health risks in the future will affect the decision-making of future soil remediation goals, resulting in waste of resources.
  • the purpose of the present invention is to explore the bioavailable content of hexavalent chromium in soil, to determine the health risk of the soil and to study its influence on the content of hexavalent chromium in the soil according to the relevant physical and chemical properties of the soil.
  • the bioavailability test method of chromium-valent respiratory pathway is to explore the bioavailable content of hexavalent chromium in soil, to determine the health risk of the soil and to study its influence on the content of hexavalent chromium in the soil according to the relevant physical and chemical properties of the soil.
  • a method for testing the bioavailability of hexavalent chromium respiratory pathways in soil including the following steps;
  • the specific method for the determination experiment of soil physical and chemical properties in S3 is as follows:
  • the soil organic matter content analysis experiment in the S4 is as follows: Weigh a certain amount of the tested soil through a 100-mesh sieve, place it in an oven at 105 degrees Celsius for 12 hours, then transfer it to the muffle furnace Bake at 550 degrees Celsius for at least 3 hours, and the weight difference before and after drying in the muffle furnace is the soil organic matter content.
  • the soil heavy metal content analysis experiment in the S4 is as follows: accurately weigh 0.2000g of the test soil in a polytetrafluoroethylene crucible, moisten the soil with a few drops of deionized water, add 10mL HCl, and place it in electric heating After heating the plate at medium temperature to near dryness, add 5 mL HNO 3 , 5 mL HF and 3 mL HClO 4.
  • the SBET method in vitro digestion and extraction experiment in S5 the specific method is as follows: take 0.4mol/L glycine solution, adjust the pH value to 1.5 with 1:1 HCl solution to obtain simulated gastric juice, and then combine the test soil with The simulated gastric juice was mixed in a 250mL Erlenmeyer flask at a ratio of 1:100, and then placed in a constant temperature water bath shaking box. The temperature of the water bath was adjusted to 37 degrees Celsius, the rotation speed was 100r/min, and the mixture was shaken for 1 hour. To be tested.
  • the PBET method in vitro digestion and extraction experiment in the S5 is as follows: 125g pepsin, 0.50g sodium malate, 0.50g sodium citrate, 420 ⁇ L lactic acid and 500 ⁇ L acetic acid are mixed and dilute to 1L, and then use 1: Adjust the pH value of 1 HCl solution to 2.5 to obtain simulated gastric juice; mix sodium bicarbonate, 52.5mg bile salt and 15mg pancreatin to prepare simulated intestinal juice, and then mix the test soil and the simulated gastric juice at a ratio of 1:100 in 250mL In the beaker, place it on a thermostatic magnetic stirrer, adjust the temperature of the outer 1L beaker water bath to 37 degrees Celsius, rotate speed 100r/min, and stir for 1 hour. After adjusting the pH to 7.0 with the simulated intestinal juice, add bile salt and pancreatin and continue stirring Take it out after 2h, filter it with a 045 ⁇ m filter membrane and wait for testing.
  • the PBET method in vitro digestion and extraction experiment in the S5 the specific method is as follows: mix 1.25g pepsin, 0.50g sodium malate, 0.50g sodium citrate, 420 ⁇ L lactic acid and 500 ⁇ L acetic acid to a volume of 1L, and then use 1 :1 HCl solution adjusts the pH to 2.5 to obtain simulated gastric juice.
  • the SGET method in vitro digestion and extraction experiment in S5 the specific method is as follows: mix 3.20g pepsin, 7.0mL concentrated hydrochloric acid and 2.0g NaCl to make 1L simulated gastric juice, and use sodium bicarbonate to prepare simulated intestinal juice. Then mix the test soil and the simulated gastric juice in a 50mL centrifuge tube at 1:100, and then place it in a constant temperature water bath shaking box, adjust the temperature of the water bath to 37 degrees Celsius, rotate at 100r/min, shake for 2h, take it out, and use it. After adjusting the pH of the simulated intestinal juice to 7.0, continue shaking for 2 hours and then take it out, filter it with a 045 ⁇ m filter membrane and wait for measurement.
  • the present invention provides a method for testing the bioavailability of the hexavalent chromium respiratory pathway in soil, which has the following beneficial effects:
  • the bioavailability of soil hexavalent chromium is studied through three in vitro digestion and extraction methods of SBET method, PBET method and SGET method, and the average extraction rate of soil hexavalent chromium is obtained through the average extraction rate of soil hexavalent chromium.
  • the available content is positively correlated with soil pH and organic matter content.
  • the content of iron and manganese in the soil will also affect the bioavailability of soil hexavalent chromium to a certain extent; the present invention explores the bioavailability of soil hexavalent chromium
  • the content is used to judge the health risk of the soil and study its influence on the content of hexavalent chromium in the soil according to the relevant physical and chemical properties of the soil.
  • Figure 1 is a schematic diagram of the total content of hexavalent chromium and the content of in vitro digestion and extraction in a low-polluted soil sample experimental group of a method for bioavailability testing of hexavalent chromium respiratory pathways in soil proposed by the present invention
  • FIG. 2 is a schematic diagram of the total content of hexavalent chromium and the in vitro digestion and extraction content of the experimental group of polluted soil samples in a method for testing the bioavailability of hexavalent chromium respiratory pathways in soil proposed by the present invention
  • FIG. 3 is a schematic diagram of the total content of hexavalent chromium and the in vitro digestion and extraction content of the experimental group of highly contaminated soil samples in a method for testing the bioavailability of the hexavalent chromium respiratory pathway in soil proposed by the present invention.
  • a method for testing the bioavailability of hexavalent chromium respiratory pathways in soil including the following steps;
  • the specific method for determining the physical and chemical properties of the soil in S3 is as follows: Weigh the test soil through a 10-mesh sieve, add carbon dioxide-free deionized water with a solid-liquid ratio of 1:2.5, fully stir the glass rod for 2 minutes, and let it stand for 30 minutes. pH glass electrode measurement.
  • the soil organic matter content analysis experiment in S4 the specific method is as follows: Weigh the test soil through a 100-mesh sieve, place it in an oven and bake it at 105 degrees Celsius for 12 hours, then transfer it to a muffle furnace and bake it at 550 degrees Celsius for at least 3 hours. The weight difference before and after drying in the muffle furnace is the soil organic matter content.
  • the analysis experiment of soil heavy metal content in S4 the specific method is as follows: accurately weigh 0.2000g of the test soil in a polytetrafluoroethylene crucible, moisten the soil with a few drops of deionized water, add 10mL HCl, and place it on an electric hot plate to heat it to near After drying, add 5mL HNO 3 , 5mL HF and 3mL HClO 4. After heating at high temperature to dry, if there is residue, repeat adding triacid until the solution in the crucible is clear and transparent, then transfer to a 50mL volumetric flask after constant volume filtration , Use an inductively coupled plasma spectrometer to analyze the total amount of soil constituent elements.
  • the pH of different types of soil is in the range of 4.44 to 5.31, with an average of 4.79, which is acidic soil; the soil cation exchange capacity is in the range of 8.45 to 20.02 cmol/kg, with an average of 12.97 cmol/kg; the soil organic matter is in the range of 3.61 to 9.69%.
  • the average is 7.26%; the results of soil particle classification show that the five soils account for the largest proportion of silt; the total arsenic of the soil is in the range of 0.67 ⁇ 6.3mg/kg, with an average of 1.83mg/kg; the total chromium content of the soil is 28.71 ⁇ 42.03mg /kg range, the average is 97.39mg/kg; the soil total copper content is within the range of 12.64 ⁇ 142.50mg/kg, the average is 50.64mg/kg; the soil total nickel is within the range of 17.87 ⁇ 54.00mg/kg, the average is 31.76mg/ kg; total soil lead content is within the range of 14.5 ⁇ 96.57mg/kg, with an average of 54.15mg/kg; total soil zinc content is within the range of 102.74 ⁇ 265.74mg/kg, with an average of 190.09mg/kg; total soil iron content is 16354.06 Within the range of ⁇ 39444.06mg/kg, the
  • the distinguishing feature is that the SBET method in S5 in vitro digestion and extraction experiment, the specific method is as follows: Take 0.4mol/L glycine solution, adjust the pH with 1:1 HCl solution The value is 1.5 to obtain a simulated gastric juice, and then the test soil and the simulated gastric juice are mixed in a 250mL Erlenmeyer flask at 1:100, and then placed in a constant temperature water bath shaking box, the temperature of the water bath is adjusted to 37 degrees Celsius, and the rotation speed is 100r/ Min, shake for 1h, take it out, filter with 0.45 ⁇ m filter membrane and wait for measurement.
  • the PBET method in vitro digestion and extraction experiment in S5 the specific method is as follows: mix 1.25g pepsin, 0.50g sodium malate, 0.50g sodium citrate, 420 ⁇ L lactic acid and 500 ⁇ L acetic acid to a volume of 1L, and then use a 1:1 HCl solution Adjust the pH to 2.5 to obtain simulated gastric juice, mix sodium bicarbonate, 52.5mg bile salts and 15mg pancreatin to prepare simulated intestinal juice, then mix the test soil and the simulated gastric juice at a ratio of 1:100 in a 250mL beaker, and place On a constant-temperature magnetic stirrer, adjust the outer 1L beaker water bath temperature to 37 degrees Celsius, rotate speed 100r/min, and stir for 1 hour. After adjusting the pH to 7.0 with the simulated intestinal juice, add bile salt and pancreatin and continue stirring for 2 hours before taking it out. After filtering with 045 ⁇ m filter membrane, it is to be tested.
  • the SGET method in vitro digestion and extraction experiment in S5 the specific method is as follows: mix 3.20g pepsin, 7.0mL concentrated hydrochloric acid and 2.0g NaCl to make 1L simulated gastric juice, mix sodium bicarbonate to make simulated intestinal juice, and then test
  • the soil and the simulated gastric juice were mixed at a ratio of 1:100 in a 50mL centrifuge tube, and then placed in a constant temperature water bath shaking box, the temperature of the water bath was adjusted to 37 degrees Celsius, the rotation speed was 100r/min, and the simulated gastric juice was taken out after shaking for 2 hours, and adjusted with the simulated intestinal juice After the pH reaches 7.0, continue to shake for 2h and then take it out, filter it with a 045 ⁇ m filter membrane and wait for measurement.
  • the extraction rate of soil hexavalent chromium by SBET method is in the range of 0-32.49%, and the average extraction rate is 10.25%; the extraction rate of PBET method is 0-32.49%. Within the range of 43.12%, the average extraction rate was 21.71%; the extraction rate of SGET method was within the range of 0-39.74%, and the average extraction rate was 16.83%.
  • the extraction rate of soil hexavalent chromium by SBET method is in the range of 0-4.78%, and the average extraction rate is 1.50%; the extraction rate of PBET method is in the range of 7.29-22.44%, and the average extraction rate is 16.02%;
  • the extraction rate of SGET method is in the range of 0 ⁇ 5.51%, and the average extraction rate is 2.27%.
  • the extraction rate of soil hexavalent chromium by SBET method is in the range of 1.20-90.27%, and the average extraction rate is 59.70%; the extraction rate of PBET method is in the range of 2.20-97.1%, and the average extraction rate is It is 48.17%; the extraction rate of SGET method is in the range of 1.12 ⁇ 92.14%, and the average extraction rate is 59.39%. It can be seen comprehensively that the PBET method has a higher extraction efficiency for soil hexavalent chromium.
  • the extraction rate of soil arsenic by SBET method is in the range of 1.72 to 77.89%, with an average of 36.10%; the extraction rate of cadmium is in the range of 11.93 to 94.43%, with an average of 56.53%; the extraction rate of chromium is 8.86 In the range of ⁇ 94.68%, the average is 46.04%; the extraction rate of copper is in the range of 16.87 to 73.18%, and the average is 46.30%; the extraction rate of nickel is in the range of 3.24 to 82.27%, and the average is 30.00%; for lead removal
  • the extraction rate of Zinc is within the range of 7.22 ⁇ 93.73%, with an average of 50.70%; the extraction rate of zinc is within the range of 08 ⁇ 7.66%, with an average of 41.80%.
  • the extraction rate of soil arsenic by PBET method is in the range of 0.50-66.57%, with an average of 12.44%; the extraction rate of cadmium is in the range of 1.11-66.00%, with an average of 14.03%; the extraction rate of chromium is in the range of 6.68-84.08% , The average is 29.60%; the extraction rate of copper is in the range of 4.47 to 84.68%, with an average of 28.58%; the extraction rate of nickel is in the range of 5.44 to 47.25%, with an average of 15.65%; the extraction rate of lead is in the range of 0.06 to In the range of 3.69%, the average is 0.86%; the extraction rate of zinc is in the range of 0-7.93%, and the average is 1.55%.
  • the extraction rate of soil arsenic by SGET method is in the range of 0-31.36%, with an average of 7.79%; the extraction rate of cadmium is in the range of 0-42.00%, with an average of 5.21%; the extraction rate of chromium is in the range of 0-93.91% , The average is 20.62%; the extraction rate of copper is in the range of 0 ⁇ 87.21%, the average is 30.80%; the extraction rate of nickel is in the range of 1.34 ⁇ 26.32%, the average is 7.64%; the extraction rate of lead is 0 ⁇ In the range of 1.14%, the average is 0.10%; the extraction rate of zinc is in the range of 0-0.01%, and the average is 0.001%.

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Abstract

一种土壤中六价铬呼吸途径的生物可给性测试方法，属于生态毒理研究领域。通过SBET法、PBET法和SGET法三种体外消化提取方法对土壤六价铬的生物可三种体外消化提取方法对土壤六价铬的生物可给性进行研究，通过对六价铬污染土壤的平均提取率，得出土壤六价铬的生物可给态含量与土壤pH、有机质含量呈正相关关系，土壤中铁、锰的含量同样会在一定程度上影响土壤六价铬的生物可给性。通过探索土壤六价铬的生物可给态含量，用以判断土壤的健康风险并根据土壤相关理化性质研究其对土壤六价铬含量的影响。

Description

一种土壤中六价铬呼吸途径的生物可给性测试方法
本申请要求于2020年04月29日提交中国专利局、申请号为202010353005.8、发明名称为“一种土壤中六价铬呼吸途径的生物可给性测试方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生态毒理研究领域，尤其涉及一种土壤中六价铬呼吸途径的生物可给性测试方法。
背景技术
铬是人体必需的微量元素之一，摄入过量的铬会引起严重的急性或慢性毒性，在焊接、造纸、印染、皮革制造、热核武器制造行业会产生出大量的含Cr副产物，这些含铬物质若不经妥善管理，必定导致其进入环境，并在周边土壤中富集；土壤中的铬可以通过多种渠道进入到人体内，危害人体健康。
呼吸吸入是颗粒物暴露于人体的一种重要途径，由于人体内的复杂环境，颗粒物中的污染物在进入人体后并不会被完全吸收，因此，测定土壤细颗粒组分中污染物的可给性是客观评价其毒性的一种重要方法。除此之外，一般进行健康风险评估计算时都带入重金属(六价铬)在土壤中的总量，包括其中不能被人体吸收利用进而危害人类健康的那一部分形态重金属，这样会高估土壤的健康风险，影响未来土壤修复目标决策，造成资源浪费。故探究土壤细颗粒组分中六价铬呼吸途径的生物可给性对人体健康风险评价及污染场地的风险管控具有重要意义。鉴于上述原因，设计一种土壤中六价铬呼吸途径的生物可给性测试方法就显得非常有必要了。
发明内容
本发明的目的是为了探索土壤六价铬的生物可给态含量，用以判断土壤的健康风险并根据土壤相关理化性质研究其对土壤六价铬含量的影响，而提出的一种土壤中六价铬呼吸途径的生物可给性测试方法。
为了实现上述目的，本发明采用了如下技术方案：
一种土壤中六价铬呼吸途径的生物可给性测试方法，包括以下步骤；
S1、取用受到Cr 6+污染的土壤，并依据污染程度的高低将其分为低浓度六价铬污染土壤样品实验组、中浓度六价铬污染土壤样品实验组和高浓度六价铬污染土壤样品实验组；
S2、将土壤样品置于阴暗处室温风干，待土壤干燥后对土壤进行研磨；
S3、将研磨后的土壤过10目筛，然后进行土壤理化性质的测定实验；
S4、将研磨后的土壤过100目筛，然后进行土壤有机质和重金属含量分析实验；
S5、将研磨后的土壤过60目筛，分别利用SBET法、PBET法和SGET法进行体外消化提取实验；
S6、将体外消化提取得到的待测液分别利用电感耦合等离子体光谱仪和紫外线分光光度计测量金属和Cr 6+的含量；
S7、对实验数据进行整理和分析，得出土壤理化性质与六价铬生物可给态含量的关系。
优选的，所述S3中土壤理化性质的测定实验，具体方法如下：
称取过10目筛供试土壤，以固液比1:2.5加入无二氧化碳去离子水，玻璃棒充分搅拌2min，静置30min后用pH玻璃电极测定。
优选的，所述S4中土壤有机质含量分析实验，具体方法如下：称取一定量过100目筛供试土壤，置于烘箱中于105摄氏度环境烘12h之后，将其转移至马弗炉中于550摄氏度环境烘至少3h，马弗炉烘干前后重量差即为土壤有机质含量。
优选的，所述S4中土壤重金属含量分析实验，具体方法如下：准确称取0.2000g供试土壤于聚四氟乙烯坩埚中，以若干滴去离子水润湿土壤后加入10mL HCl，置于电热板上中温加热至近干后，加入5mL HNO 3、5mL HF和3mL HClO 4，高温加热近干后，若还有残渣剩余，重复加入三酸，直至坩埚中溶液澄清透明，之后转移至50mL容量瓶中定容过滤后，用电感耦合等离子体光谱仪进行土壤组成元素全量分析。
优选的，所述S5中SBET法体外消化提取实验，具体方法如下：取 0.4mol/L的甘氨酸溶液，用1：1的HCl溶液调节pH值为1.5，得到模拟胃液，然后将供试土壤与所述模拟胃液以1：100混合于250mL的锥形瓶中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡1h后取出，用0.45μm滤膜过滤后待测。
优选的，所述S5中PBET法体外消化提取实验，具体方法如下：将125g胃蛋白酶、0.50g苹果酸钠、0.50g柠檬酸钠、420μL乳酸及500μL醋酸混合定容至1L，然后用1：1的HCl溶液调节pH值为2.5，得到模拟胃液；将碳酸氢钠，52.5mg胆汁盐和15mg胰液素混合调配成模拟肠液，接着将供试土壤与所述模拟胃液以1：100混合于250mL烧杯中，置于恒温磁力搅拌器上，调节外层1L烧杯水浴温度为37摄氏度、转速100r/min，搅拌1h，用所述模拟肠液调节pH至7.0后，加入胆汁盐和胰液素后继续搅拌2h后取出，用045μm滤膜过滤后待测。
优选的，所述S5中PBET法体外消化提取实验，具体方法如下：将1.25g胃蛋白酶、0.50g苹果酸钠、0.50g柠檬酸钠、420μL乳酸及500μL醋酸混合定容至1L，然后用1：1的HCl溶液调节pH值为2.5，得到模拟胃液，将52.5mg胆汁盐和15mg胰液素混合调配成模拟肠液；接着将供试土壤与所述模拟胃液以1：100混合于250mL烧杯中，置于恒温磁力搅拌器上，调节外层1L烧杯水浴温度为37摄氏度、转速100r/min，搅拌1h；用碳酸氢钠调节pH至7.0后，加入胆汁盐和胰液素混合调配而成的模拟肠液后继续搅拌2h后取出，用045μm滤膜过滤后待测。
优选的，所述S5中SGET法体外消化提取实验，具体方法如下：将3.20g胃蛋白酶、7.0mL浓盐酸和2.0gNaCl混合后定容成1L的模拟胃液，用碳酸氢钠调配成模拟肠液，接着将供试土壤与所述模拟胃液以1:100混合于50mL的离心管中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡2h后取出，用所述模拟肠液调节pH至7.0后，继续振荡2h后取出，用045μm滤膜过滤后待测。
与现有技术相比，本发明提供了一种土壤中六价铬呼吸途径的生物可给性测试方法，具备以下有益效果：
本发明通过SBET法、PBET法和SGET法三种体外消化提取方法对土壤六价铬的生物可给性进行研究，通过对六价铬污染土壤的平均提取 率，得出土壤六价铬的生物可给态含量与土壤pH、有机质含量呈正相关关系，土壤中铁、锰的含量同样会在一定程度上影响土壤六价铬的生物可给性；本发明通过探索土壤六价铬的生物可给态含量，用以判断土壤的健康风险并根据土壤相关理化性质研究其对土壤六价铬含量的影响。
附图说明
图1为本发明提出的一种土壤中六价铬呼吸途径的生物可给性测试方法的低污染土壤样品实验组六价铬总含量与体外消化提取含量示意图；
图2为本发明提出的一种土壤中六价铬呼吸途径的生物可给性测试方法的中污染土壤样品实验组六价铬总含量与体外消化提取含量示意图；
图3为本发明提出的一种土壤中六价铬呼吸途径的生物可给性测试方法的高污染土壤样品实验组六价铬总含量与体外消化提取含量示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
在本发明的描述中，需要理解的是，术语“上”、“下”、“前”、“后”、“左”、“右”、“顶”、“底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
实施例1：
一种土壤中六价铬呼吸途径的生物可给性测试方法，包括以下步骤；
S1、取用受到Cr 6+污染的土壤，并依据污染程度的高低将其分为低浓度六价铬污染土壤样品实验组、中浓度六价铬污染土壤样品实验组和高浓度六价铬污染土壤样品实验组；
S2、将土壤样品置于阴暗处室温风干，待土壤干燥后对土壤进行研磨；
S3、将研磨后的土壤过10目筛，然后进行土壤理化性质的测定实验；
S4、将研磨后的土壤过100目筛，然后进行土壤有机质和重金属含量分析实验；
S5、将研磨后的土壤过60目筛，分别利用SBET法、PBET法和SGET法进行体外消化提取实验；
S6、将体外消化提取实验得到的待测液分别利用电感耦合等离子体光谱仪和紫外线分光光度计分别测量金属和Cr 6+的含量；
S7、对实验数据进行整理和分析，得出土壤理化性质与六价铬生物可给态含量的关系。
所述S3中土壤理化性质的测定实验，具体方法如下：称取过10目筛供试土壤，以固液比1:2.5加入无二氧化碳去离子水，玻璃棒充分搅拌2min，静置30min后用pH玻璃电极测定。
S4中土壤有机质含量分析实验，具体方法如下：称取过100目筛供试土壤，置于烘箱中于105摄氏度环境烘12h之后，将其转移至马弗炉中于550摄氏度环境烘至少3h，马弗炉烘干前后重量差即为土壤有机质含量。
S4中土壤重金属含量分析实验，具体方法如下：准确称取0.2000g供试土壤于聚四氟乙烯坩埚中，以若干滴去离子水润湿土壤后加入10mL HCl，置于电热板上中温加热至近干后，加入5mL HNO 3、5mL HF和3mL HClO 4，高温加热近干后，若还有残渣剩余，重复加入三酸，直至坩埚中溶液澄清透明，之后转移至50mL容量瓶中定容过滤后，用电感耦合等离子体光谱仪进行土壤组成元素全量分析。
不同类型土壤pH在4.44～5.31范围内，平均为4.79，属于酸性土壤；土壤阳离子交换量在8.45～20.02cmol/kg范围内，平均为12.97cmol/kg；土壤有机质在3.61～9.69％范围内，平均为7.26％；土壤颗粒分级结果显示五种土壤占比重最大均为粉粒；土壤总砷在0.67～6.3mg/kg范围内，平均为1.83mg/kg；土壤总铬含量在28.71～42.03mg/kg范围，平均为97.39mg/kg；土壤总铜含量在12.64～142.50mg/kg范围内，平均为50.64mg/kg；土壤总镍在17.87～54.00mg/kg范围内，平均为31.76mg/kg；土壤总铅含量在14.5～96.57mg/kg范围内，平均为54.15mg/kg；土壤总锌含量在102.74～265.74mg/kg范围内，平均为190.09mg/kg；土壤总铁含量在16354.06～39444.06mg/kg范围内，平均为24420.73mg/kg；土壤总铝含量在27394.20～57667.53mg/kg范围内，平均为39083.53mg/kg；土壤总锰 含量在104.93～248.70mg/kg范围内，平均为151.08mg/kg；土壤总磷含量在752.40～1340.07mg/kg范围内，平均为1094.80mg/kg；土壤总硫含量在64.73～338.36mg/kg范围内，平均为207.11mg/kg。
实施例2：
请参阅图1～3，与实施例1相比，其区别特征在于，S5中SBET法体外消化提取实验，具体方法如下：取0.4mol/L的甘氨酸溶液，用1：1的HCl溶液调节pH值为1.5，得到模拟胃液，然后将供试土壤与所述模拟胃液以1:100混合于250mL的锥形瓶中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡1h后取出，用0.45μm滤膜过滤后待测。
S5中PBET法体外消化提取实验，具体方法如下：将1.25g胃蛋白酶、0.50g苹果酸钠、0.50g柠檬酸钠、420μL乳酸及500μL醋酸混合定容至1L，然后用1：1的HCl溶液调节pH值为2.5，得到模拟胃液，将碳酸氢钠，52.5mg胆汁盐和15mg胰液素混合调配成模拟肠液，接着将供试土壤与所述模拟胃液以1：100混合于250mL烧杯中，置于恒温磁力搅拌器上，调节外层1L烧杯水浴温度为37摄氏度、转速100r/min，搅拌1h，用所述模拟肠液调节pH至7.0后，加入胆汁盐和胰液素后继续搅拌2h后取出，用045μm滤膜过滤后待测。
S5中SGET法体外消化提取实验，具体方法如下：将3.20g胃蛋白酶、7.0mL浓盐酸和2.0g NaCl混合后定容成1L的模拟胃液，用碳酸氢钠调配成模拟肠液，接着将供试土壤与所述模拟胃液以1：100混合于50mL的离心管中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡2h后取出，用所述模拟肠液调节pH至7.0后，继续振荡2h后取出，用045μm滤膜过滤后待测。
从图1中可以看出：3#土壤样品六价铬含量最低，几种体外消化提取方法都无法将其中六价铬提取出，2#样品土壤样品六价铬无法被SBET方法提取出，低浓度样品提取效果重复性较差。图2中可以看出：8#土壤样品六价铬含量虽然较其余三个土壤样品低，但几种体外消化提取方法提取四个样品六价铬含量比较接近。图3中可以看出10#样品虽然六价铬含量达到1052.56mg/kg，但几种提取方法对其六价铬的提取最高的PBET 方法也仅有23.14mg/kg。纵观以上三图，PBET方法对六价铬提取较高，仅有9#样品PBET方法提取六价铬含量最低，9#样品直观表现为黑色。
三种体外消化试验对三种不同浓度六价铬污染土壤的提取率统计结果如表1所示。
表1体外消化方法对土壤六价铬提取率，％
Figure PCTCN2021083491-appb-000001
从表1中可以看出：对于低浓度六价铬污染土壤样品，SBET方法对土壤六价铬的提取率在0～32.49％范围内，平均提取率为10.25％；PBET方法提取率在0～43.12％范围内，平均提取率为21.71％；SGET方法提取率在0～39.74％范围内，平均提取率为16.83％。对于中浓度六价铬污染土壤样品，SBET方法对土壤六价铬提取率在0～4.78％范围内，平均提取率为1.50％；PBET方法提取率在7.29～22.44％范围内，平均提取率为16.02％；SGET方法提取率在0～5.51％范围内，平均提取率为2.27％。对于高浓度六价铬污染土壤样品，SBET方法对土壤六价铬的提取率在1.20～90.27％范围内，平均提取率为59.70％；PBET方法提取率在2.20～97.18％范围内，平均提取率为48.17％；SGET方法提取率在1.12～92.14％范围内，平均提取率为59.39％。综合可以看出，PBET方法对土壤六价铬的提取效率较高。其中几种体外消化提取试验对中浓度六价铬污染土壤中六价铬的提取较为稳定，波动范围较窄；低浓度污染土壤次之；高浓度污染土壤最为不 稳定，波动范围宽；高浓度污染土壤生物可给态六价铬含量最高，低浓度污染土壤次之，中浓度污染土壤最低。SBET、PBET、SGET三种方法对土壤六价铬的提取率分别为23.82％、28.81％、26.16％。
同理可以得到：SBET法对土壤砷提取率在1.72～77.89％范围内，平均为36.10％；对镉的提取率在11.93～94.43％范围内，平均为56.53％；对铬的提取率在8.86～94.68％范围内，平均为46.04％；对铜的提取率在16.87～73.18％范围内，平均为46.30％；对镍的提取率在3.24～82.27％范围内，平均为30.00％；对去铅的提取率在7.22～93.73％范围内，平均为50.70％；对锌的提取率在08～7.66％范围内，平均为41.80％。PBET法对土壤砷提取率在0.50～66.57％范围内，平均为12.44％；对镉的提取率在1.11～66.00％范围内，平均为14.03％；对铬的提取率在6.68～84.08％范围内，平均为29.60％；对铜的提取率在4.47～84.68％范围内，平均为28.58％；对镍的提取率在5.44～47.25％范围内，平均为15.65％；对铅的提取率在0.06～3.69％范围内，平均为0.86％；对锌的提取率在0～7.93％范围内，平均为1.55％。SGET法对土壤砷提取率在0～31.36％范围内，平均为7.79％；对镉的提取率在0～42.00％范围内，平均为5.21％；对铬的提取率在0～93.91％范围内，平均为20.62％；对铜的提取率在0～87.21％范围内，平均为30.80％；对镍的提取率在1.34～26.32％范围内，平均为7.64％；对铅的提取率在0～1.14％范围内，平均为0.10％；对锌的提取率在0～0.01％范围内，平均为0.001％。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (8)

  1. 一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：包括以下步骤；
    S1、取用受到Cr 6+污染的土壤，并依据污染程度的高低将其分为低浓度六价铬污染土壤样品实验组、中浓度六价铬污染土壤样品实验组和高浓度六价铬污染土壤样品实验组；
    S2、将土壤样品置于阴暗处室温风干，待土壤干燥后对土壤进行研磨；
    S3、将研磨后的土壤过10目筛，然后进行土壤理化性质的测定实验；
    S4、将研磨后的土壤过100目筛，然后进行土壤有机质和重金属含量分析实验；
    S5、将研磨后的土壤过60目筛，分别利用SBET法、PBET法和SGET法进行体外消化提取实验；
    S6、将体外消化提取实验得到的待测液分别利用电感耦合等离子体光谱仪和紫外线分光光度计测量金属和Cr 6+的含量；
    S7、对实验数据进行整理和分析，得出土壤理化性质与六价铬生物可给态含量的关系。
  2. 根据权利要求1所述一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S3中土壤理化性质的测定实验，具体方法如下：称取过10目筛供试土壤，以固液比1:2.5加入无二氧化碳去离子水，玻璃棒充分搅拌2min，静置30min后用pH玻璃电极测定。
  3. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S4中土壤有机质含量分析实验，具体方法如下：称取过100目筛供试土壤，置于烘箱中于105摄氏度环境烘12h之后，将其转移至马弗炉中于550摄氏度环境烘至少3h，马弗炉烘干前后重量差即为土壤有机质含量。
  4. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S4中土壤重金属含量分析实验，具体方法如下：准确称取0.2000g供试土壤于聚四氟乙烯坩埚中，以若干滴去离子水润湿土壤后加入10mL HCl，置于电热板上中温加热至近干后，加入5mL  HNO 3、5mL HF和3mL HClO 4，高温加热近干后，若还有残渣剩余，重复加入三酸，直至坩埚中溶液澄清透明，之后转移至50mL容量瓶中定容过滤后，用电感耦合等离子体光谱仪进行土壤组成元素全量分析。
  5. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S5中SBET法体外消化提取实验，具体方法如下：取0.4mol/L的甘氨酸溶液，用1：1的HCl溶液调节pH值为1.5，得到模拟胃液，然后将供试土壤与所述模拟胃液以1:100混合于250mL的锥形瓶中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡1h后取出，用0.45μm滤膜过滤后待测。
  6. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S5中PBET法体外消化提取实验，具体方法如下：将125g胃蛋白酶、0.50g苹果酸钠、0.50g柠檬酸钠、420μL乳酸及500μL醋酸混合定容至1L，然后用1：1的HCl溶液调节pH值为2.5，得到模拟胃液，将碳酸氢钠，52.5mg胆汁盐和15mg胰液素混合调配成模拟肠液；接着将供试土壤与所述模拟胃液以1：100混合于250mL烧杯中，置于恒温磁力搅拌器上，调节外层1L烧杯水浴温度为37摄氏度、转速100r/min，搅拌1h；用所述模拟肠液调节pH至7.0后，加入胆汁盐和胰液素后继续搅拌2h后取出，用045μm滤膜过滤后待测。
  7. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S5中PBET法体外消化提取实验，具体方法如下：将1.25g胃蛋白酶、0.50g苹果酸钠、0.50g柠檬酸钠、420μL乳酸及500μL醋酸混合定容至1L，然后用1：1的HCl溶液调节pH值为2.5，得到模拟胃液，将52.5mg胆汁盐和15mg胰液素混合调配成模拟肠液；接着将供试土壤与所述模拟胃液以1:100混合于250mL烧杯中，置于恒温磁力搅拌器上，调节外层1L烧杯水浴温度为37摄氏度、转速100r/min，搅拌1h；用碳酸氢钠调节pH至7.0后，加入胆汁盐和胰液素混合调配而成的模拟肠液后继续搅拌2h后取出，用045μm滤膜过滤后待测。
  8. 根据权利要求1所述的一种土壤中六价铬呼吸途径的生物可给性测试方法，其特征在于：所述S5中SGET法体外消化提取实验，具体方法如下：将3.20g胃蛋白酶、7.0mL浓盐酸和2.0gNaCl混合后定容成1L的 模拟胃液，用碳酸氢钠调配成模拟肠液，接着将供试土壤与所述模拟胃液以1:100混合于50mL的离心管中，然后置于恒温水浴振荡箱中，调节水浴温度为37摄氏度，转速100r/min，振荡2h后取出，用所述模拟肠液调节pH至7.0后，继续振荡2h后取出，用045μm滤膜过滤后待测。
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