RU2375714C2 - Method for definition of soil toxicity - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention is related to the field of agriculture and soil science. In method chlorella algae cells are radiated with exciting light in presence of analysed toxic substances, and intensity of moderated fluorescence is measured. Radiation and measurement of moderated fluorescence is carried out in aqueous soil suspension after biotesting and exposure in darkness for 30 minutes. At the same time coefficient of surveyed soil toxicity is defined and compared to coefficient of toxicity in conventionally pure soil. Coefficient of relative toxicity that characterises level of soil effect at chlorella is used to identify soil phytotoxicity.

EFFECT: method makes it possible to increase method sensitivity, validity of assessment and comparison of soils phytotoxicity.

2 dwg, 4 tbl

Description

The invention relates to environmental protection, in particular to methods for the study and determination of phytotoxicity of soils, and can be used in environmental activities, agriculture, etc.

A known method for determining the acute toxicity of water extracts from the soil, including recording a decrease in the level of chlorophyll fluorescence of green protococcal algae Scenedesmus quadricauda (Tiger) Breb under the influence of toxic substances present in the aqueous extract from the soil compared to the control culture in samples containing no toxic substances. The criterion of acute toxicity is the suppression of the level of algal chlorophyll fluorescence by 50% or more compared with the control during a 96-hour exposure (Methodology for determining the toxicity of water, water extracts from soils, sewage sludge and waste by changing the level of chlorophyll fluorescence and the number of algae cells. FR. 1.39.2001.00284).

The disadvantage of this technique is the high complexity and duration of its implementation.

A known method for determining the content of phytotoxic substances in aqueous and other solutions, including irradiating the cells of chlorella algae with exciting light in the presence of an analyte of toxic substance, and measuring the intensity of photoinduced delayed fluorescence of algae in a millisecond decay interval under the influence of a toxic substance. In this case, the intensity of delayed fluorescence is measured at induction maxima after switching on the exciting light, first high (50-100 W / m ² ), and then low (5-10 W / m ² ) intensity. As an indicator of phytotoxicity, the ratio of recorded values of delayed fluorescence under the indicated conditions, normalized to a control sample of algae that does not contain a toxicant (patent RU 2069851 C1, publ. 27.11.1996) is used - the prototype.

The disadvantage of this method is the low sensitivity and low level of adequacy of the results under real conditions, since the information obtained characterizes the toxicity of water extract, which can significantly differ from the toxicity of the soil due to the action of the entire complex of pollutants present in it, and not just water-soluble compounds.

The technical task of the claimed invention is to increase the sensitivity of the method and the reliability of the assessment and comparison of phytotoxicity of soils.

The technical problem is achieved by the fact that in the method for determining the phytotoxicity of soils, the cells of chlorella algae are irradiated with exciting light in the presence of the analyzed toxic substances and the delayed fluorescence intensity is measured. Irradiation and measurement of delayed fluorescence is carried out in an aqueous soil suspension after biotesting and exposure in the dark for 30 minutes, the toxicity coefficient of the test soil is determined, it is compared with the toxicity coefficient of relatively clean soil and the value of the relative toxicity coefficient characterizing the level of soil exposure to chlorella, determine the phytotoxicity of the soil.

To determine phytotoxicity, an aqueous soil suspension with soil particle size> 0.315 mm and aged for 2 hours at a temperature of 90 ° C is used. After the introduction of algae cells into the soil suspension, a biotesting is carried out for 2 hours at a temperature of 28-30 ° C and illumination of 75-80 W / m ² followed by an exposure of 20-30 minutes. The level of soil exposure to chlorella is quantitatively expressed by the value of the coefficient of relative toxicity (K _from ). For this, the obtained values of the toxicity coefficients K _{t of the} studied soil are normalized with respect to the conditionally clean soil. (see tables 1 and 2)

Table 1

Influence of illumination and bioassay time on CT (temperature 28-30 ° С) Illumination, W / m ² Bioassay time 1 hour 2 hours 3 hours 65 -0.097 -0.137 -0.331 80 -0,231 -0.525 -0.768

table 2

The effect of biotesting temperature on CT (illumination 80 W / m ² ) sample Temperature ° C 26 29th 36 Soil 1 -0.470 -0.469 -0.213 Soil 2 -0.541 -0.616 -0.306 Soil 3 -0.653 -0.734 -0,401

Water extracts of soils do not have a noticeable effect on the parameters of chlorella PF, the recorded toxicity coefficients range from 0-0.05. The toxic effect begins to appear when working with an aqueous soil suspension. With direct contact of chlorella with soil particles, the action of insoluble forms of pollutants, in particular heavy metals, most of which is associated with soil components, is manifested.

Thermal treatment of soil suspensions before adding a test object enhances the effect. This is due both to an increase in the degree of extraction of toxic elements into the aqueous phase, and to the destruction of the structure of soil particles, which increases the surface of contact with chlorella. A decrease in the size of soil particles contributes to an increase in the sensitivity of chlorella to the action of a toxicant, since this factor, like heat treatment, increases the contact area of the algae with the soil, as well as the completeness and speed of extraction. Soil fractions with a particle size of less than 1 mm were investigated; 0.385-0.315 mm and less than 0.315 mm. It was established that the greatest influence on the parameters of the chlorella ZF is exerted by aqueous suspensions of a fraction of less than 0.315 mm. The values of the toxicity coefficient of aqueous suspensions of a fraction of 0.385-0.315 mm and less than 0.315 mm were respectively -0.520 and -0.917.

Table 3

The effect of thermal treatment of soil suspension on the value of CT Sample Extraction conditions 3 hours at 80 ° C 2 hours at 90 ° C Soil 1 -0.246 -0.222 Soil 2 -0.314 -0.302 Soil 3 -0.394 -0.427

To obtain the most objective comparison of soil phytotoxicity, it is necessary that all samples of the test culture be taken from one suspension, which is impossible when analyzing a large number of samples. The introduction of soil into the control sample, against which the toxicity coefficients are calculated, did not give satisfactory results, since the level decreased and the interval of variation of the obtained toxicity coefficients of soils with different degrees of contamination decreased. Therefore, conditionally clean soil is introduced as the “internal standard”, relative to which the level of influence of a particular soil on chlorella is calculated and compared, which is quantitatively expressed by the value of the coefficient of relative toxicity.

The claimed combination of features was not identified as a result of patent information research, therefore, the claimed invention meets the criterion of "inventive step".

Comparison of the proposed solution with the prototype shows that it also meets the criterion of "novelty."

The inventive method is implemented by the inventors and can be used in various fields related to soil research, therefore, it meets the criterion of "industrial applicability".

The method is as follows

A weighed portion of the soil fraction <0.315 mm is placed in 10 ml glass bottles, add 2 ml of distilled water, mix thoroughly with a glass spatula, close with stoppers and incubated for 2 hours in an oven at a temperature of 90 ° C, stirring the contents of the bottles several times with shaking. The volume of soil suspension, and therefore the size of the sample of the studied soil and the volume of the aqueous phase, are determined by the size of the ditches of the recording device. As a result of the studies, it was shown that the amount of soil of 50-100 mg and 2 ml of water provides the optimal ratio of solid and liquid phases during extraction and allows you to identify the difference in soils by their effect on the fluorescence characteristics of chlorella.

Then, 3 ml of an aqueous suspension of chlorella with an optical density of 0.54-0.56 are added to the soil suspensions cooled to room temperature. After keeping the mixture in the phytotester for 2 hours at an illumination of 80 W / m ² and a temperature of 29 ° C, the contents of the vials are thoroughly mixed by shaking and transferred to the phytotoximeter cuvettes. The option of careful decantation is possible. Then give a 30-minute exposure in the dark. Irradiation of algae cells with an analyzed soil suspension and measuring the intensity of photoinduced delayed fluorescence of algae in the millisecond attenuation interval under the influence of toxic substances of the soil suspension is carried out in the Photon 7-1 phytotoximeter (developer and manufacturer of Krasnogorsk State University).

A series of experiments consisted of 11 determinations, two of which were control (suspension of chlorella and water, which underwent the same heat treatment as the soil suspension), three-chlorella in a suspension of 100 mg of the “internal standard”, six - chlorella in a suspension containing two different samples of the investigated soil (three replicates of each). The number of determinations in the series was determined by the design features of the phytotoximeter. For each of the three replicates, the average toxicity coefficient relative to chlorella was calculated. To determine the relative toxicity of the studied soil, a graph of the dependence of CT on the logarithm of the soil mass is constructed (straight line 1, Fig. 1).

The CT value of the “internal standard” is expressed as a straight line parallel to the abscissa axis (straight line 2). From the abscissa, the points of intersection of the straight lines find the mass of the studied soil, which has the same effect as the mass of the "internal standard" (the so-called reduced mass). The ratio of the mass of the "internal standard" to the given mass will express the coefficient of relative toxicity of the soil - Cat. The larger this value, the more significant the influence of the experimental soil on chlorella in comparison with relatively clean soil.

, где m - масса "внутреннего стандарта", мг;

where m is the mass of the "internal standard", mg;

m _x - reduced mass, mg.

The correctness of this approach to determining the coefficient of relative toxicity is based on the linear nature of the dependence of the value of CT on the logarithm of the soil mass (Fig. 2).

The technique was tested on soils with a known content of priority technogenic elements: copper, cadmium, zinc and lead. Background soil is used as the “internal standard”. Analysis of the results of determining the coefficients of relative toxicity of soils, presented in table 4, shows that heavily contaminated soils have a higher phytotoxicity.

Table 4

Metal content and soil toxicity ratios The soil Cu (mg / kg) Cd (mg / kg) Zn (mg / kg) Pb (mg / kg) K _from (m ± Δm) Coef. variations,% Background soil 25.19 0.65 32.91 32,46 - - Sample 1 714.57 10.30 564.29 173.91 2.13 ± 0.07 5.7 Sample 2 89.62 1.31 71.45 36.66 1.33 ± 0.03 4.9 MPC in the soil 66.0 1,0 110.0 65 - -

Thus, the claimed invention through the use of an aqueous soil suspension, a set of conditions for its preparation (soil particle size, temperature, time), biotesting conditions for chlorella cells (time, temperature, light, contact with soil particles) increases the sensitivity of the known method, allows to determine phytotoxicity soils of varying degrees of pollution and obtain results that are adequate to real conditions. The introduction of conditionally pure soil as the “internal standard”, relative to which the level of influence of a particular soil on chlorella is calculated, which is quantitatively expressed by the value of the coefficient of relative toxicity, makes it possible to maximally objectively compare the phytotoxicity of soils when analyzing a large number of samples.

Claims (1)

A method for determining soil phytotoxicity, comprising irradiating with light of cells of chlorella algae in the presence of analyzed toxic substances and measuring the intensity of delayed fluorescence, characterized in that the radiation and measurement of delayed fluorescence is carried out in an aqueous soil suspension after biotesting and exposure in the dark for 30 minutes, determine the toxicity coefficient of the studied soil, compare it with the toxicity coefficient of relatively clean soil and the value of the coefficient of relative toxicity characterizing the level of soil exposure to chlorella, determine the phytotoxicity of the soil.

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