RU2460071C2 - Method of determining bioactivity of soil, water, wastes and preparations based on humic substances - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of determining bioactivity of soil, water, wastes and preparations based on humic substances. To this end, seeds are pre-soaked for a day in a solution of the analysed substance. The seeds are then let to germinate at 25°C for 3 days on a substrate mounted on a vertical transparent plastic container. The container is a base and a rectangular cover of equal size, which are placed in parallel and joined to each other on the entire perimeter by "button" type fasteners. The container allows for installation of a removable central horizontal partition wall for holding the substrate. Before germination, the substrate is moistened, a filter is placed on the substrate and seeds are placed on said filter in a single row along the line of the central horizontal partition wall at a distance of 1 cm from it. After germination, the root length of the sprouts is determined relative control. If the root length in the test samples is shorter than that of control samples by 20% or more, the analysed substance has a toxic effect, if the root length exceeds the control length, the analysed substance has a stimulating effect, in the rest of the cases, the analysed substance has no bioactivity or is weakly toxic.

EFFECT: method enables to conduct serial investigations, is characterised by simplicity and rapidness, seeds are exposed in a short period of time and there is no mechanical damage when determining root length.

3 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of biotechnology, in particular to methods of bioassay and assessment of toxicity or bioactivity of various objects and materials using bioobjects.

In the context of an increase in the technogenic load on the environment, questions of assessing its environmental well-being are becoming increasingly relevant. The emergence of new pollutants, as well as the synthesis under heterogeneous environmental conditions of specific compounds capable of possessing significantly greater toxic potential, leads to the fact that quantitative pollution indicators, such as MPC, PDU, cannot cover the entire variety of pollutants, to give a correct assessment of the ecological well-being of the studied objects . In addition, even non-toxic compounds, when combined, can cause significant toxic effects. In this regard, there is currently growing interest in biotest systems that are able to integrally and quickly give a toxicological characterization of natural and technogenic environments.

Biotesting is considered an effective method of assessing the potential dangers of chemical, physical or biological effects on natural environments, including soil. The principle of the bioassay method in a broad sense is based on the sensitivity of living organisms to exogenous effects. The essence of the method is to determine the effect of the tested substances on specially selected organisms under standard conditions with the registration of various behavioral, physiological or biochemical test reactions. A test reaction (or test function) is defined as one of the naturally occurring responses of a test system to the effects of a complex of external factors. A quantitative expression of a test reaction is a test parameter. The toxicity criterion is the value of the test parameter or the rule on the basis of which a conclusion is drawn about the toxicity of the test sample. The concept of a “test system” includes a spatially limited set of sensitive biological elements (sensors) and the studied environment in which they are located. The terms “test object” and “test culture” (or “test organism”) are widely used to denote the main elements that make up a test system. In this case, the test object is correctly interpreted as a sample or a sample that is being studied and exerts an effect, causing a test reaction.

Various approaches are known to assess the biological effects of toxicants and other environmental objects on living organisms. Various animals, plants, microorganisms (e.g. daphnia, aquatic organisms, algae, microorganisms) are used as test objects (test organisms).

For the same purpose, phytotesting methods are used as the most express and economical.

Phytotesting is widely used not only as a method of toxicological assessment of media, such as soil and water, but also as a very common method for assessing the toxicity or bioactivity of various materials, chemicals, industrial wastes. The phytotests, along with other biotests, have become especially popular in a new field - the rapidly developing technology for assessing the biosafety of nanomaterials (Lin D. Phytotoxicity of nanoparticles: inhibition of seed germination and root growth // Environmental Pollutants, 2007, Vol. 150, Iss.2 , p. 243-250).

There is a method of complex biotesting of water, soil, biologically active substances in phytotests, which involves the identification of cytogenetic, genetic structural, metabolic and morphophysiological disorders in phytotests in Allium sulfur and Phaseolus vulgaris (RU 2322669, 2007).

This method is complex and time-consuming and cannot be reproduced as an express technique.

There is also a method for determining the sensitivity of plants to adverse environmental factors, in particular soils containing aluminum ions, based on the fact that plant seeds are germinated in solutions with different concentrations of aluminum chloride (up to 10 ^-4 M). The ability of genotypes of plant varieties to activate coleoptile growth in response to the presence of low concentrations of aluminum ions is determined. The sensitivity of plant varieties is evaluated to determine the most adapted of them to growth on soils containing aluminum ions. In fact, the method is as follows: the seeds are placed on a strip of filter paper (10 cm × 30 cm), placing them on the top of the wide side of the paper, curl the paper in the form of a roll and place them in cups with test solutions. Solutions are prepared on the basis of calcium sulfate (10 ^-4 M). The seedlings are grown for 7-10 days, after which the length of the vegetative part (coleoptile) is measured. The determination of sensitivity to aluminum ions is carried out on the basis of the degree of intensification of growth processes, expressed in an increase in the linear dimensions of coleoptile.

This method allows you to assess the sensitivity of plants to soil contamination by aluminum ions, but it is not convenient enough and does not provide stable reproducibility of the results. The method cannot be used in the field as an express technique.

The current methodology (MP 2.1.7.2297-07 “Justification of the hazard class of waste ...”) involves the germination of seeds in Petri dishes. In this case, the roots curl during the growth process, since they lie on the horizontal plane into which they abut. As a result, root growth vertically down is impossible (Fig. 1). As a result, the roots are intertwined and twisted. At the end of the experiment, each root must be carefully deployed manually and measured using a ruler. Such a procedure is very time-consuming and significantly affects the quality of the results.

There is also a method of growing seeds in vessels with soil - in this case, the roots grow vertically, but they need to be washed from the ground, the method requires long-term germination (from 1 month) and labor-intensive setting up of the experiment (Fig. 2).

The objective of the invention is to provide a method for determining the bioactivity of soils, soils, water, waste, preparations of humic substances, which would allow qualitatively, quickly and relatively simply to carry out phytotesting, as well as evaluate its results.

The technical result of the claimed method is to ensure its simplification, expressness, reducing testing time and ensuring high accuracy of the results. A valuable advantage of the proposed method is the expansion of its scope in relation to the test environments of materials and preparations, as well as the possibility of conducting serial studies. A simplification of the method for determining bioactivity is achieved through the use of transparent plastic containers in which seeds develop in a two-dimensional space. In this case, the unfolding of twisted roots is not required, their mechanical damage and tearing does not occur, which affects the quality of the results. Expression is provided by a 3-day period of seed exposure, which is much faster than the currently existing methods. At the end of the experiment, it is possible to use an automatic method of collecting data using computer processing of digital images (photographs) of seedlings, which gives high accuracy of the results. It is also possible to measure the roots and the ruler directly in containers through a transparent cover, and in this case, the roots are not deformed and not damaged, which occurs when they are removed from Petri dishes or other vessels.

A method is proposed for determining the bioactivity of soils, soils, water, waste, preparations based on humic substances, which provides for the germination of plant seeds both in direct contact with the investigated substrates (on soil, soil, etc.) and in the presence of the test substance by preparing an aqueous extract / suspension from the test object and soaking it with a paper base of filter paper, which acts as a substrate for plant growth. Further, to denote all types of tested substances (both solid and aqueous extracts or suspensions) we will use the term “substrate”. In the case of seed germination directly on a solid substrate, the latter is pre-moistened with distilled water in the amount necessary to achieve the lowest field moisture capacity. Exposure (germination) of seeds is carried out for 3 days at a temperature of 25 ° C.

Germination of seeds is carried out in a vertically installed transparent plastic container, which is a rectangular base and a lid of the same size, installed in parallel and interconnected around the entire perimeter using button-type fasteners, while the container provides for the possibility of installing a removable central horizontal partition for fixing loose substrate placed in one part of the container (soil, soil, etc.). The drawing of the container used is shown in Figure 3. The basis of the container is a container (a certain kind of box) for placing the substrate and supporting plant growth. It is tightly closed with a flat lid that prevents evaporation of moisture from the container. The ratio of length to width at the walls of the container is not more than 1.5. After germination, the length of the roots is determined in the seedlings and, with a decrease in their length in the test samples compared to the control by 20% or more, they make a conclusion about the toxic effect of the test substance, with an increase in the length of the roots compared to the control, they give a stimulating effect, and in other cases they do conclusion about the absence of bioactivity or low toxicity of the test substance. In general terms, the sequence of the main stages of the design of an experiment to evaluate the bioactivity of samples is described below (Scheme 1).

1. An empty container (“base”) is placed on a horizontal surface (for example, on a desktop).

2. Filter paper (substrate) is placed in one of the compartments of the main part of the container with tweezers, impregnated with an aqueous solution of the preparation (or with an aqueous extract from solid samples, which adheres tightly to the container base and is fixed between the container walls.

3. Seeds of plants pre-soaked for 24 hours in aqueous solutions or aqueous extracts from the tested samples are laid out at a distance of 0.5-1 cm from the septum. Departing from the partition by more than 1 cm is not recommended, since with further exposure of the container in an upright position, the roots growing down can reach the lower boundary of the container. When placing seeds at a distance of more than 1 cm from the septum, the lower part of the root can begin to bend, which will lead to the need for manual straightening and complicate the experiment. As a test culture, white mustard seeds (Sinapis alba L.) are usually used.

4. The container with seeds on the substrate is closed with a “lid”, which is fixed with locks and buttons, and is installed vertically. Seeds germinate at 25 ° C for 3 days.

5. After three days at the seedlings, the length of the roots is measured (using a ruler or automatically by transferring digital images of containers with seedlings to a computer). Measurements are taken from the point where the root begins to grow to its lower boundary.

The root growth start point is usually clearly visible - it is slightly thicker than the root and stem itself and differs in color from them.

Scheme 1. The main stages of determining bioactivity

Example

The example discusses the features of determining the bioactivity of various forms of the tested substances - both in solid form and in the form of aqueous extracts / suspensions.

A) Solid substrates (for example, soil)

As a control, a reference substrate (reference unpolluted soil) is used, the choice of which is determined by the research objectives, or a substrate saturated with distilled water. The experiment is carried out in 3 replicates (3 containers with control and 3 containers with the test object are prepared).

1. Preparation of the substrate

Testing is carried out on objects saturated with moisture to the level of the lowest field moisture capacity. Take about 1 dm ^{3 of the} test soil and sift it through a sieve with a diameter of 2 mm to avoid the ingress of coarse material. 90 cm ^{3 of} soil are placed in a graduated laboratory beaker and 50 ml of distilled water is added. The soil is stirred with water until it is saturated with liquid, and then it is defended, after which excess liquid emerges on the surface of the soil. Depending on the particle size distribution, the moisture saturation process can take from several minutes (for sandy soils) to several hours (for clay soils). The volume of water required for water saturation is calculated. This volume is equal to the volume of water added to the soil, minus the volume of water formed on its surface after settling.

With a beaker, measure 90 ml of sifted soil and transfer it to the lower compartment of the container. The soil is moistened by adding distilled water in the amount necessary to achieve the lowest field moisture capacity. Use a spatula to level the moistened soil in the lower compartment of the container.

2. Seed placement

10 seeds of one plant species, previously soaked for 24 hours in a solution containing the test substance (extract), are placed on the soil surface in one row at an equal distance from each other. Seeds are laid out approximately 1 cm from the central septum in the container. Next, the dividing wall is removed, the container is covered with a lid and the buttons snap.

A sticker is stuck on the upper left corner of the base of the container indicating the type of experiment (soil type, seed type, repetition number),

3. Seed incubation

6 filled containers with seeds (3 with test soil and 3 with control) are placed vertically. The containers are placed in an incubator and exposed at 25 ° C for 3 days.

4. Analysis of the results

Counting the number of sprouted seeds

Count the number of sprouted seeds in the control experiments. The percentage of seed germination should be at least 80%, otherwise the seeds are characterized by poor quality and their use is not reliable.

Measuring the root lengths of germinated seeds.

The root length is counted from the point where the root begins to grow until it ends, and is measured using a ruler. Digital image processing is also possible. According to the obtained data, the average root length for the control and test soil is calculated.

The percentage of inhibition or stimulation of root growth is calculated by the formula:

Where

A - the average length of the roots in the control soil

B is the average root length in the tested soil

An example calculation is shown in Table 1.

B) Water extracts / suspensions

As a control, a substrate soaked in distilled water is used. The experiment is carried out in 3 replicates (3 containers with control and 3 containers with the test object are prepared).

1. Preparation of the substrate

Water extracts / suspensions are prepared from the test objects under study (in accordance with GOST). Next, filter paper measuring 15 × 9 cm (half the area of the container) is lowered with tweezers into the prepared solution / suspension, removed, wait for the gravity water to drain off for several seconds and placed in one of the container compartments.

2. Seed placement

Seeds are placed at a distance of 1 cm from the top edge of the paper base (the top edge corresponds to the middle of the container)

Subsequent operations (2-4) are carried out similarly to the described example for solid substrates (A).

TABLE 1 An example of processing results in determining bioactivity Sample Control, unpolluted soil
Root length mm Root number one 2 3 four 5 6 7 8 9 10 The average inside the repetition, mm The final average, mm Standard deviation between replicates, mm Bioactivity,% Toxicity Repeatability 1 70 72 43 48 52 35 64 60 66 57 56 3 Repeat 2 78 75 52 63 45 60 44 45 58 56 58 Repeat 3 32 65 71 58 47 65 fifty 36 62 37 52 Sample 1.1% phosphogypsum contaminated soil Root length mm Root number one 2 3 four 5 6 7 8 9 10 The average inside re-sti, mm The final average, mm Standard deviation between replicates, mm Bioactivity,% Toxicity Repeatability 1 70 52 45 35 54 48 49 65 52 54 four -3.2 non toxic Repeat 2 47 55 69 60 70 60 65 37 58 Repeat 3 40 38 42 39 39 80 73 29th 74 57 51 Sample Soil contaminated with phosphogypsum at a concentration of 3.3% Root length mm Root number one 2 3 four 5 6 7 8 9 10 The average inside re-sti, mm The final average, mm Standard deviation between replicates, mm Bioactivity,% Toxicity Repeat 1 75 57 42 35 43 40 37 35 48 47 46 45 one -18.7 non toxic Repeat 2 thirty 54 55 33 44 35 45 44 61 45 Repeat 3 60 51 41 37 36 42 52 17 68 45 Sample Soil contaminated with phosphogypsum at a concentration of 7.5% Root length mm Root number one 2 3 four 5 6 7 8 9 10 The average inside the repetition, mm The final average, mm Standard deviation between replicates, mm Bioactivity,% Toxicity Repeatability 1 35 32 2 thirty 27 27 35 27 27 2 -50.7 toxic Repeat 2 43 27 31 28 thirty 19 23 24 38 32 thirty Repeatability 3 45 eighteen 31 thirty fourteen 28 twenty twenty 26

Claims (1)

A method for determining the bioactivity of soil, soil, water, waste, preparations based on humic substances, providing for the germination of plant seeds on test substrates or in the presence of the test substance, determining a test reaction of germinated seeds and evaluating the results of germination, providing that the seeds are pre-soaked for days in a solution of the test substance, after which the seeds are germinated at 25 ° C for 3 days on a substrate placed in a vertically installed transparent plastic an onteiner, which is a base and a lid of rectangular shape, the same size, installed in parallel and connected together around the entire perimeter using button-type fasteners, while the container provides for the possibility of installing a removable central horizontal partition to accommodate the substrate, moisten the substrate before germinating , a filter is laid on it, on which the seeds are placed in one row along the line of the central horizontal partition at a distance of 1 cm from it, then after germination In seedlings, the root length is determined in relation to the control; if the root length in the test samples is reduced by 20% or more compared to the control, a conclusion is made about the toxic effect of the test substance, and if the root length is increased compared to the control, the stimulating effect is In other cases, they conclude that there is no bioactivity or low toxicity of the test substance.

Images