CN113533685A - Test method for determining acute toxicity of volatile organic compounds in soil to soil organisms-earthworms - Google Patents
Test method for determining acute toxicity of volatile organic compounds in soil to soil organisms-earthworms Download PDFInfo
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Abstract
The invention discloses a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, which comprises the following steps: clearing intestines by using earthworms: selecting earthworms domesticated from uncontaminated soil for clearing intestines; soil contamination: taking soil to carry out contamination pretreatment in a sealed tube; and (3) formal test: placing the tested earthworms into a sealing tube, and recording the toxic effect parameters of the tested earthworms; and (3) toxicity evaluation: calculating the effect concentration LC of the tested soil to the earthworms according to the toxicity effect parametersxAnd evaluating the toxicity of the soil. Aiming at the defect that the existing standard earthworm acute toxicity test method is not suitable for volatile organic compounds, the invention provides the test method for determining the acute toxicity of the volatile organic compounds in the soil to the earthworms in the soil, which is easy to operate, good in test result stability and high in dose-effect correlation.
Description
Technical Field
The invention belongs to the technical field of toxicity detection of pollutants in soil, and particularly relates to a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms.
Background
Soil acts as an active open system, and most of the pollutants released into the environment enter the soil through various routes (atmosphere, water bodies, organisms, etc.). When the content of the pollutants exceeds the soil environment accommodating amount, the soil structure and the physicochemical property are deteriorated, the soil quality and the fertility level are reduced, and even the survival, the propagation and the life safety of human bodies and other organisms are threatened. Therefore, toxicity detection methods and risk assessment for soil pollution are gradually receiving wide attention from the environmental protection field of countries all over the world.
The earthworms are the original organisms in soil, have strong adaptability, are sensitive to pollutants, and are easy to culture and operate in body types, so the earthworms are widely applied to biotoxicity test and soil pollution risk assessment of the pollutants, have important application values in aspects of polluted soil environment risk classification, establishment of pollutant soil quality standards and standards, environment risk assessment of specific polluted sites, remediation effect assessment of the polluted sites and the like, and are known as the most ideal indicator organisms for monitoring soil pollution. A series of standard earthworm acute toxicity experimental methods are published by standardization organizations such as the international union of standardization and the organization of economic cooperation and development. However, none of these standard methods are suitable for the determination of Volatile Organic Compounds (VOCs).
According to the definition of the World Health Organization (WHO), volatile organic compounds refer to various organic compounds with the boiling point of 50-260 ℃ at normal temperature. In China, volatile organic compounds refer to organic compounds with saturated vapor pressure of more than 70Pa at normal temperature and boiling point of 260 ℃ below under normal pressure, or all organic compounds with vapor pressure of more than or equal to 10Pa and volatility at 20 ℃.
When the toxicity of the soil contaminated by volatile organic compounds is tested by using the existing earthworm acute toxicity test method, the earthworm escapes when the concentration of pollutants is low, but the earthworm does not die; when the concentration of contaminants is high, earthworms stay on the soil surface and die quickly. Meanwhile, the existing earthworm acute toxicity experimental method also has the defects of more material and waste materials, long experimental period, easiness in escaping of earthworms and the like, so that the earthworm acute toxicity experimental method which has less material consumption and short period and is suitable for measuring volatile organic compounds is very necessary.
Disclosure of Invention
Aiming at the characteristics of more material and waste, long experimental period, easiness in escaping of earthworms and inapplicability to volatile organic compounds in the conventional earthworm acute toxicity experimental method, the invention provides a method for testing the acute toxicity of the volatile organic compounds in soil to soil organisms, namely earthworms.
The technical scheme of the invention is realized as follows:
a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, comprises the following steps:
s1: earthworm gut purge
Selecting earthworms domesticated by uncontaminated soil, and clearing intestines under test conditions to obtain tested earthworms for later use;
the specific steps of clearing the intestines by the earthworms comprise: a small amount of ultrapure water was added to a 1L flat-bottomed beaker having a layer of filter paper laid on the bottom, preferably just after the filter paper was immersed. Putting earthworms on filter paper, sealing with plastic film, pricking holes, putting the beaker into a climatic chamber with the temperature of 20 +/-2 ℃ and the humidity of 70-90%, culturing in dark and clearing intestines for 24 hours.
S2: contamination of soil
Taking soil, pretreating in a sealed tube, and adjusting the water-soil weight ratio to 1: 4, rapidly sealing and carrying out balance treatment under test conditions;
s3: official test
Randomly putting the tested earthworms obtained in the step S1 into a sealed tube in the step S2, and recording the toxic effect parameters of the tested earthworms under the test conditions;
the sealing tube is a special sealing tube which is a bottom glass tube (the inner diameter is 2.6cm multiplied by the height is 8 cm).
S4: toxicity evaluation
Calculating the effect concentration LC of the tested soil on the earthworms according to the toxicity effect parameters of the tested earthworms obtained in the step S3xAnd evaluating the toxicity of the soil.
Further, the earthworms in step S1 are Eisenia foetida (Eisenia fetida) or Eisenia andrei (Eisenia andrei).
Furthermore, the two earthworms are healthy earthworms with wet weight of 300-600mg and normal posture and appear in saddle-shaped or zonal genital zones at the front end of the body above 3 months old.
Further, in the step S1, the step S2 and the step S3, the test conditions are that the temperature is 20 +/-2 ℃, the humidity is 70% -90%, and the culture is carried out in a dark place.
Further, in the step S2, selecting actual contaminated soil or standard soil from the soil;
wherein the soil is actual polluted soil, and the actual polluted soil is mixed with reference soil or standard soil before the weight ratio of water to soil is adjusted;
the soil is standard soil, the standard soil is artificial soil, and the artificial soil is subjected to contamination pretreatment before the water-soil weight ratio is adjusted; the artificial soil formula comprises the following components in parts by weight: 10 parts of sphagnum, 20 parts of kaolin clay, 69 parts of industrial quartz sand and 0.3-1.0 part of calcium carbonate.
The reference soil is soil which does not contain pollutants and is collected at a place close to a polluted point. No obvious plant residue in sphagnum, air drying, grinding, pH: 5.5-6.0; the content of kaolinite in the kaolinite clay is not less than 30 percent, the content of fine sand with the particle size of 50-200 mu m in the industrial quartz sand is more than 50 percent, and the calcium carbonate is analytically pure and is ground into powder to play a role in adjusting the pH value of the soil, and the pH value of the soil can be adjusted to 6.0 +/-0.5.
When the standard soil is selected from the soil in the step S2, different contamination pretreatments need to be performed according to the physicochemical and toxicity characteristics of the tested chemical substances.
Furthermore, the soil contamination pretreatment mode is carried out in different modes according to the physicochemical and toxicity characteristics of the tested chemical substances, and the soil contamination pretreatment mode comprises the following steps:
the test substance is a solid test chemical: when the solid chemical substance to be tested is a water-soluble substance, dissolving the solid chemical substance to be tested into ultrapure water, and then uniformly mixing the ultrapure water with soil; or when the tested substance is a hydrophobic substance, fully mixing the solid tested chemical substance with the soil, and then supplementing ultrapure water;
or the test substance is a liquid test chemical: when the liquid chemical substance to be tested is a water-soluble substance, dissolving the substance to be tested in ultrapure water, and then uniformly mixing the substance to be tested with soil; or when the liquid tested chemical substance is a high-toxicity insoluble substance, dissolving the tested substance in a low-toxicity or non-toxic organic solvent, and then uniformly mixing with the soil; or when the liquid chemical substance to be tested is a low-toxicity insoluble substance, directly mixing the liquid chemical substance to be tested with the soil.
Among them, in the liquid chemical substances to be tested, examples of highly toxic poorly soluble substances include, but are not limited to, xylene, examples of less toxic poorly soluble substances include, but are not limited to, trichloroethylene, and examples of less toxic or non-toxic organic solvents include, but are not limited to, dimethyl sulfoxide. Further, in step S3, the toxic effect parameters are death number and toxic symptom of the earthworms, and death is determined by the fact that the front and tail parts of the earthworms do not respond to the mechanical stimulation.
Further, in step S3, the earthworms to be tested are placed in a sealed tube and cultured for 48 hours under the test conditions, and the toxicity effect parameters of the earthworms to be tested are recorded at 24 hours and 48 hours, respectively, and each concentration treatment is repeated 10 times.
Further, the effect concentration LC of step S4xValues were calculated using a unit probability regression analysis.
Further, LCxIs LC50，R2≥0.7。
The invention discloses a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, and provides an experimental method for determining the acute toxicity of the volatile organic compounds in the soil to the earthworms in the soil, which is easy to operate, good in experimental result stability and high in dose-effect correlation, aiming at the defect that the existing standard earthworm acute toxicity experimental method is not suitable for the volatile organic compounds.
Drawings
FIG. 1 is a graph showing the results of acute toxicity tests on earthworms by xylene in example 1;
FIG. 2 is a graph showing the results of acute toxicity tests of trichloroethylene on earthworms in example 2.
Detailed Description
To further illustrate the technical solution of the present invention, the following examples are specifically illustrated.
The pH value of the standard soil in the embodiment 1 and the embodiment 2 is 6.0 +/-0.5, and comprises the following raw materials, by weight, 10 parts of sphagnum moss (no obvious plant residues, air drying and grinding, the pH value is 5.5-6.0), 20 parts of kaolin clay (the content of kaolinite is not less than 30%), 69 parts of industrial quartz sand (the content of fine sand with the particle size of 50-200 mu m is more than 50%), and 0.3-1.0 part of calcium carbonate (analytically pure, ground into powder and used for adjusting the pH value of the standard soil to 6.0 +/-0.5)
Example 1: a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, comprises the following steps:
s1, clearing intestines by earthworms
A small amount of ultrapure water was added to a 1L flat-bottomed beaker having a layer of filter paper laid on the bottom, preferably just after the filter paper was immersed. Putting earthworms on filter paper, sealing the filter paper by using a plastic film, pricking holes, and putting the beaker into a climatic chamber with the temperature of 20 +/-2 ℃ and the humidity of 70-90% for clearing intestines for 24 hours.
The Lumbricus is Eisenia fetida (Eisenia fetida), and the Lumbricus is healthy Lumbricus with wet weight of 600mg and normal body state and with saddle or zonal genital zone at front end of body of more than 3 months old.
S2. soil contamination
Dissolving xylene into dimethyl sulfoxide to prepare a standard solution with corresponding concentration (according to the concentration of the earthworm which is not dead and completely dead in a pre-experiment, the concentration of the standard solution is set to be 5 concentrations of 200mg/kg, 250mg/kg, 300mg/kg, 350mg/kg and 400 mg/kg); setting a solvent control group (namely the concentration of the standard solution is 0mg/kg) and a blank control group (no dimethylbenzene and dimethyl sulfoxide, only soil);
weighing 10g of dried standard soil into a special sealed tube (the special sealed tube is a glass tube with a bottom (the inner diameter is 2.6cm multiplied by 8cm), then quickly and uniformly dripping the standard solution into the standard soil, adjusting the weight ratio of water to soil to be 1: 4 by using deionized water, quickly sealing by using aluminum foil paper, putting the sealed tank into an artificial climate box with the temperature of 20 +/-2 ℃ and the humidity of 70-90%, culturing in a dark place, carrying out balanced treatment for 24h, and tightly sticking the aluminum foil paper on the wall of the sealed tube by using a sealing film to prevent volatilization.
S3, formal test
After the tested soil is balanced for 24 hours, the soil is rapidly stirred, 1 earthworm with the intestines cleared for 24 hours is placed into each sealing tube, the sealing is carried out again, the earthworms are placed into an artificial climate box with the temperature of 20 +/-2 ℃ and the humidity of 70% -90%, the earthworms are cultured for 48 hours in a dark place, the toxic effect parameters of the tested earthworms are recorded for 24 hours and 48 hours respectively, and each concentration treatment is repeated for 10 times.
S4. Experimental results
In the whole experiment process, the blank control group and the solvent control group have no phenomena of earthworm death and discomfort, and the experimental biology and experimental conditions are proved to accord with the quality control standard, and the toxicity experimental result is credible.
The experimental result shows that the earthworms are exposed in the low-concentration dimethylbenzene (the dimethylbenzene is less than 300mg/kg), and have the phenomena of body stiffness, slow peristalsis, slow reaction and the like; when the earthworms are exposed in high-concentration dimethylbenzene (the dimethylbenzene is more than or equal to 300mg/kg), the earthworms struggle violently on the soil surface, secrete body fluid, break the body and the like. The mortality of earthworms increases with the increase of the concentration of xylene and the increase of the exposure time.
As shown in FIG. 1, xylene has acute toxicity to earthworms and has a significant dose-effect relationship. Obtaining the 24h and 48h semilethal concentrations LC of the xylene to the earthworms by a unit probability regression analysis method50300.23mg/kg and 221.62mg/kg respectively (see Table 1 for specific results).
Example 2:
a test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, comprises the following steps:
s1, clearing intestines by earthworms
A small amount of ultrapure water was added to a 1L flat-bottomed beaker having a layer of filter paper laid on the bottom, preferably just after the filter paper was immersed. Putting earthworms on filter paper, sealing with plastic film, pricking holes, putting the beaker into a climatic chamber with the temperature of 20 +/-2 ℃ and the humidity of 70-90%, culturing in dark and clearing intestines for 24 hours.
The Lumbricus is Eisenia fetida (Eisenia fetida), and the Lumbricus is healthy Lumbricus with wet weight of 600mg and normal body state and with saddle or zonal genital zone at front end of body of more than 3 months old.
S2. Standard soil contamination
Weighing 10g of dried standard soil into a special sealed tube (the special sealed tube is a glass tube with a bottom (the inner diameter is 2.6cm multiplied by 8cm) and then quickly and uniformly dripping trichloroethylene into the standard soil (the adding concentration of the trichloroethylene is set to 5 concentrations of 0mg/kg, 4000mg/kg, 5000mg/kg, 6000mg/kg, 7000mg/kg and 8000mg/kg according to the concentration of the earthworms which are not dead and completely dead in the preliminary experiment), adjusting the weight ratio of water and soil to 1: 4 by using deionized water, quickly sealing by using aluminum foil paper, putting the sealed tank into an artificial climate box with the temperature of 20 +/-2 ℃ and the humidity of 70-90 percent, culturing in a dark place, carrying out balance treatment for 24h, and tightly pasting the aluminum foil paper on the wall of the sealed tube by using a sealing film to prevent volatilization.
S3. formal experiment
After 24h of balance, the soil is quickly stirred, 1 earthworm with the intestines cleared for 24h is placed into each sealing tube, the sealing is carried out again, the earthworms are placed in an artificial climate box with the temperature of 20 +/-2 ℃ and the humidity of 70% -90%, the earthworms are cultured for 48h in a dark place, the toxicity effect parameters of the tested earthworms are recorded for 24h and 48h respectively, and each concentration treatment is repeated for 10 times.
S4. Experimental results
In the whole experiment process, the blank control group (namely in the standard soil) has no phenomena of earthworm death and discomfort, and the experimental biology and experimental conditions are proved to accord with the quality control standard, and the toxicity experimental result is credible.
The experimental result shows that when the earthworm is exposed to low-concentration trichloroethylene (the trichloroethylene is less than 6000mg/kg), the earthworm has the phenomena of body stiffness, slow peristalsis, slow reaction and the like; when the earthworms are exposed to high-concentration trichloroethylene (the trichloroethylene is more than or equal to 6000mg/kg), the earthworms violently struggle on the soil surface, secrete body fluid, break the body and the like. The mortality of earthworms increases with increasing trichloroethylene concentration and with increasing exposure time.
As can be seen from fig. 2, trichloroethylene has an acute toxic effect on earthworms and has a significant dose-effect relationship. Obtaining the 24h and 48h semilethal concentrations LC of the trichloroethylene to the earthworms by a unit probability regression analysis method505332.36mg/kg and 4522.41mg/kg, respectively (see Table 1 for specific results).
TABLE 1 acute toxicity of xylene and trichloroethylene to earthworms
The evaluation criteria for toxicity of volatile organic compounds are the chemical acute toxicity transdermal exposure grading criteria of table 2.
TABLE 2 grading Standard of acute toxicity
According to the acute toxicity of chemical substances, the toxicity of xylene to earthworms is high, and the toxicity of trichloroethylene to earthworms is low, which is known by the skin grading standard (see table 2).
The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.
Claims (10)
1. A test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, is characterized by comprising the following steps:
s1: earthworm gut purge
Selecting earthworms domesticated by uncontaminated soil, and clearing intestines under test conditions to obtain tested earthworms for later use;
s2: contamination of soil
Taking soil to be treated in a sealed tube, and adjusting the water-soil weight ratio to be 1: 4, rapidly sealing and carrying out balance treatment under test conditions;
s3: official test
Randomly putting the tested earthworms obtained in the step S1 into a sealed tube in the step S2, and recording the toxic effect parameters of the tested earthworms under the test conditions;
s4: toxicity evaluation
Calculating the effect concentration LC of the tested soil on the earthworms according to the toxicity effect parameters of the tested earthworms obtained in the step S3xAnd evaluating the toxicity of the soil.
2. The method as claimed in claim 1, wherein the earthworms in step S1 are Eisenia foetida (Eisenia foetida)Eisenia fetida) Or Eisenia foetida (Eisenia andrei）。
3. The test method for determining the acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, as claimed in claim 1 or 2, wherein the earthworms in step S1 are healthy earthworms with wet weight of 600mg at 300-600mg and normal posture and saddle-shaped or zonal genital zone at the front end of the body at 3 months of age.
4. The test method for determining the acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, as claimed in claim 1, wherein the test conditions in the steps S1, S2 and S3 are 20 ± 2 ℃ and 70% -90% humidity, and the soil organisms are cultured in the absence of light.
5. The test method for determining acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, as claimed in claim 1, wherein in step S2, actual contaminated soil or standard soil is selected from the soil;
wherein the soil is actual polluted soil, and the actual polluted soil is mixed with reference soil or standard soil before the weight ratio of water to soil is adjusted;
the soil is standard soil, the standard soil is artificial soil, and the artificial soil is subjected to contamination pretreatment before the water-soil weight ratio is adjusted; the artificial soil formula comprises the following components in parts by weight: 10 parts of sphagnum, 20 parts of kaolin clay, 69 parts of industrial quartz sand and 0.3-1.0 part of calcium carbonate.
6. The test method for determining the acute toxicity of volatile organic compounds in soil to soil organisms, namely earthworms, as claimed in claim 5, wherein the artificial soil contamination pretreatment mode is as follows:
the test substance is a solid test chemical: when the solid chemical substance to be tested is a water-soluble substance, dissolving the solid chemical substance to be tested into ultrapure water, and then uniformly mixing the ultrapure water with soil; or when the tested substance is a hydrophobic substance, fully mixing the solid tested chemical substance with the soil, and then supplementing ultrapure water;
or the test substance is a liquid test chemical: when the liquid chemical substance to be tested is a water-soluble substance, dissolving the substance to be tested in ultrapure water, and then uniformly mixing the substance to be tested with soil; or when the liquid tested chemical substance is a high-toxicity insoluble substance, dissolving the tested substance in a low-toxicity or non-toxic organic solvent, and then uniformly mixing with the soil; or when the liquid chemical substance to be tested is a low-toxicity insoluble substance, directly mixing the liquid chemical substance to be tested with the soil.
7. The method as claimed in claim 1, wherein the parameters of toxic effect in step S3 are death number and toxic symptom of earthworms, and the death is determined by the non-response of the front and tail parts of earthworms to mechanical stimulation.
8. The test method for determining the acute toxicity of soil-associated volatile organic compounds on soil organisms, namely earthworms, as claimed in claim 1, wherein the earthworms to be tested in step S3 are placed into a sealed tube and cultured for 48h under test conditions, and the toxicity effect parameters of the earthworms to be tested are recorded for 24h and 48h respectively, and each concentration treatment is repeated for 10 times.
9. The method as claimed in claim 1, wherein the effect concentration LC of step S4 is determined by the method of determining the acute toxicity of volatile organic compounds in soil to soil organism-earthwormxValues were calculated using a unit probability regression analysis.
10. The test method for determining the acute toxicity of soil-associated Volatile Organic Compounds (VOCs) to soil organisms (earthworms) according to claim 9, wherein LCxIs LC50，R2≥0.7。
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