RU2125261C1 - Method of biologically monitoring ecological systems and objects - Google Patents

Abstract

FIELD: environmental monitoring. SUBSTANCE: method is designed to determine toxicity of ecological systems and objects: soil, water, foods, drugs, domestic objects, and unknown objects. As test organism, infusoria Paramecium Caudatum are used, and monitoring is conducted consecutively in three steps. First, biological activity of substance of object being monitored is evaluated: effect of this substance on test organism after 0.5-24 h incubation is compared to that of a reference standard medium. In the second step, mixture of standard medium containing test organisms with substance of object being monitored and reference standard medium with test organisms are used. The two media are exposed to functional loads until complete destruction of test organisms is attained, and their life duration since the beginning of functional loads is registered, from which stimulation and inhibitory activities of substance of object being monitored are evaluated in comparison with those of standard medium. In the third step, mixture of standard medium containing 3-4 days- aged test organisms with substance of object revealing biological activity in the first step is used. This mixture is incubated for 3-4 days and then number of test organisms in fixed volume of object is calculated to estimate effect of substance on the rate of test organisms' reproduction. In the first and second steps, 2-3 weeks- aged infusoria Paramecium Caudatum are used as test organisms. Substance of object to be tested is added to medium in concentrations from 1•10^-2 to 1•10^-6 per 1 ml of medium. This mixture and reference standard medium with test organisms are incubated at 18-27 C. EFFECT: potentially dangerous objects rapidly revealed, biological monitoring data banks for ecological systems created, their conditions predicted, and scientifically-founded ways to control them developed. 2 cl, 1 tbl, 6 ex

Description

 The invention relates to the field of ecology, biology, medicine, veterinary medicine, sanitary examination, standardization and certification.

 The proposed method for the biological monitoring of ecological systems and objects (hereinafter the rapid biotest) allows you to quickly, at minimal cost, unify the toxicity, usefulness and specific activity of soil, water, air, food for humans, animal feed, medicines, unknown substances, objects life, unknown items, etc. This is especially important for the sanitary-epidemiological, veterinary-laboratory, customs services, standardization and certification bodies, monitoring centers for land resources, water and air pools, forests.

 A known method for assessing the toxicity of liquids using two-day Daphnia [1].

 A known method for determining the biological activity of drugs using peramecium [2].

 A known method for determining the biological activity of biological products using the ciliates tetrachimene pyrimiformis [3].

 A known method for determining the biological assessment of animal products and feed using the ciliates tetrachimene pyrimiformis [4].

 Known methods for determining the biological activity of chemical compounds using green unicellular algae [5, 6].

 A method for monitoring objects by selecting adaptogen substances using the ciliates-paramecium caudatum, which involves the selection of substances on test organisms and their subsequent evaluation on white mice (7).

 A common drawback of the above analogues and prototype is that each of them is designed to evaluate the substances of the tested object only for any one biological characteristic (e.g. toxicity or bactericidal, fungicidal, stimulating activity), it takes a long time, it is used as test organisms for each operation, different cultures of microorganisms.

 The closest analogue of the claimed invention is the use of ciliates, including paramecium caudatum, for indicating food contamination with mycotoxins (ZA Kurbatskaya. Biological tests for indicating mycotoxins).

 Authors use similar environments for testing. But the method is less informative and testing passively, because when it is carried out, there are no functional loads that more quickly and accurately identify the direction of the biological action of the test objects on the ciliates and it is used exclusively to indicate contamination by fungi and the manifestation of toxicity of producing fungi, while the claimed method allows testing any objects of the external environment, as well as those used by humans to their needs.

 The aim of the proposed invention is the creation of a universal and informative method of biological monitoring of ecological systems and objects.

In the collection of educational materials under the editorship of Tutelyana V.A. "Assessment of food contamination by mycotoxins", t. III, m., 1983, p. 279-280
The difference between the proposed method and the prototype is that it gradually, quickly and accurately assesses the condition and safety of any ecological systems and objects using the same type of test organisms.

This goal is achieved by the fact that the biological effect of the substance of the tested object on ecological systems and objects is determined using the ciliates-Paramecium caudatum (Paramecium caudatum) in three stages:
- at the first, the substance of the test object is introduced into the standard medium with test organisms, and after 0.5 - 24.0 hours, the presence or absence of biological activity of the substance is evaluated in comparison with the action of the control standard medium on test organisms;
- at the second stage, a mixture of a test organisms solution with the substance of the test object incubated for 24 hours and a control solution of test organisms in a standard medium are subjected to functional loads until the test organisms die completely, their life expectancy is recorded from the moment the functional loads begin to act and according to the data obtained evaluate the stimulating or inhibitory activity of the substance of the tested object in comparison with a standard medium;
- on the third mixture of a solution of test organisms with the substance of the test object that showed biological activity, incubate for 3-4 days, after which the number of test organisms in a fixed volume of the mixture is calculated and the effect of the substance of the test object on the reproduction rate of the test is evaluated organisms (determine the index of biological activity of the substance of the tested object).

 Based on the results of the above steps, a conclusion is drawn about the possibility of using the inspected objects in everyday life, medicine, veterinary medicine, about the condition and safety of the checked ecological system or object (soil in a selected area, water in a selected source, atmosphere in a selected surrounding space, vegetation in a selected area) .

 Example 1. The cultivation and storage of test organisms (ciliates-paramecium-caudatum).

Hay is cut into a measuring cup (without flowers, poured with Lozino-Lozinsky medium (in g: sodium chloride - 0.1; potassium chloride - 0.01; magnesium chloride - 0.01; sodium bicarbonate - 0.02; calcium chloride - 0 , 01; distilled water (pH = 7) - 1 liter) in a ratio of 1: 2 by volume, cover with aluminum foil and boil for 20 minutes.After a little settling, the hay infusion is placed in a thermostat for 72 hours at a temperature of 37 ^o C (for storage in medium of hay bacillus.) After three days, 10 ml of the uterine culture of the test organisms are introduced into the hay infusion, mix well, close gauze, date the sowing, test organisms are cultivated at a temperature of 22 - 25 ^o C. To obtain test organisms in the exponential growth phase (3 days), the culture is re-planted in a new medium every 3-4 days, and in the stationary growth phase (2 weekly) - 2 times a month.

Example 2. Preparation of the substance of the inspected object for research
2.1. Solid objects.

 Solid objects can have a very different shape: dense, soft, loose, structured. Regardless of this, the preparation of the substance of solid objects is carried out according to the following (of the same type) scheme.

The substance is dried at a temperature of 30 ^o C and a portion of 10 g is taken from it, which is ground (if necessary) and sieved through a sieve to obtain particles no larger than 225 microns. 3 weighed samples of 0.1 g each were taken from the obtained particles, placed in test tubes and filled with 10 ml of distilled water. The mixture is kept for 24 hours, shaking 2-3 times, then centrifuged for 15 minutes at 3-5 thousand rpm. For further work using a centrifuge, which is a solution of the test substance 1: 100.

 Soft forms of solid objects are homogenized and the operations described above are performed with them.

 With solid objects that are not amenable to grinding, flushing is done with a minimum amount of distilled water, which is used as a solution of the substance of the object being tested.

 2.2. Liquid objects.

 3 samples of 0.1 g are taken from the substance of the test object, each of them is stirred in 10 ml of distilled water (1: 100) and the solution is kept for 24 hours, periodically shaking. If water does not mix with the substance, before work, they are thoroughly shaken until a uniform suspension, which is used in further work.

 2.3. Gaseous objects.

 A 10 ml bubbler is poured with distilled water and at least 10 liters of a gaseous test object is passed (sparged) through it, i.e., the test gaseous object is dissolved in water until the gas is saturated with water. Thus, three samples are prepared, which are the starting material for further work.

 Example 3. The first stage. Assessment of the presence of biological activity or toxicity of the substance of the tested object. 3.1. Working process.

In 5 test tubes, 9 ml of a solution of test organisms (ciliates-paramecium caudatum) are poured in the stationary phase of growth. In the first test tube add 1 ml of distilled water, mix. In the second test tube add 1 ml prepared in example 2 solution of the substance of the tested object, mix. A concentration of 1: 1000 is obtained. Transferring 1 ml of liquid from the second test tube to the third, from the third to the fourth, from the fourth to the fifth in test tubes, respectively, the concentration of the substance of the tested object is 1:10 000; 1: 100,000; 1: 1 000 000. After this, the tubes are placed in a thermostat with a temperature of 25 ^o C.

After 0.5, 1.0, 3.0, 6.0 and 24 hours, 0.1 ml of solution with test organisms (at least 400 cells) is taken from each tube and five micro-aquariums are filled with it. Under a binocular magnifier, the state of the test organisms in each micro-aquarium is evaluated according to the following criteria:
- IN - indifference (test organisms make uniform Brownian movements);
- BA - bioactivity (test organisms make uneven movements with accelerations);
- BTs-50 - biocidality-50 (50 ± 10% of test organisms died);
- BC-100 - biocide-100 (90 ± 10% of test organisms died).

 In the control tube, at each observation during the day, as well as in micro aquariums, there should be at least 400 test organisms (cells) making uniform Brownian movements.

 3.2. Evaluation of the results.

 IN - the object is biologically inactive.

 BA - an object biologically: (1: 1000) - weakly active; (1:10 000 - medium active; (1: 100 000) - active; (1: 1 000 000) - highly active.

 BTs-50 - the object is toxic.

 BTs-100 - toxic effect: (1: 1000) - weak; (1:10 000) - average; (1: 100,000) - strong; (1: 1,000,000) - very strong.

 BA-24 hours - biological activity is very stable.

 BA-3.0-6.0 hours - the biological activity is stable.

 BA-0.5-1.0 hours - biological activity is slightly stable.

 BTs-100 - 0.5 - 1.0 hour - rapid damage to vital mechanisms.

 BU-100 - 3.0 - 6.0 hours - gradual damage to vital mechanisms.

 BTs-100 - 24 hours - slow damage to vital mechanisms.

 Example 4. The second stage. Assessment of the biological activity of the substance of the tested object by the resolving method.

 The essence of the method is to identify, with the help of an additional resolving unfavorable factor, the biological action of the substance of the tested object on the adaptation mechanisms and resistance of test organisms (cells). In the work, test organisms from the first stage are used, which were in contact with various concentrations of the substance of the tested object for 24 hours, and not in contact (control).

 4.1. Working process.

 From the control tube, 1 ml of a solution of test organisms is taken into several test tubes and 8% sodium chloride solution (0.1 to 0.5 ml) or a solution of another electrolyte or alcohol is added to them, so that a 100% test -organisms died within 5 minutes. The death of test organisms is controlled in micro aquariums under a binocular magnifier using a stopwatch. Then take in several test tubes 1 ml of a mixture of a solution of test organisms with the substance of the test object, add the same amount of sodium chloride solution to it and measure the life of the test organisms to 100% of their death. The experiments are repeated as many times as necessary, and the arithmetic mean value is used for further work.

 4.2. Evaluation of the results.

The calculations are carried out according to the formula IBA = T _O / T _K ,
Where
IBA - the index of biological activity of the substance of the tested object;
T _O - life expectancy of test organisms (in minutes) under the influence of a resolving factor, who have lived previously 24 hours in an environment with a selected concentration of the substance of the tested object;
T _To - the life expectancy of the control test organisms (in minutes) under the influence of the resolving factor, who lived previously 24 hours in the control environment.

 Received IBA evaluated as follows.

 IBA = 1,000 ± 0,1000 - the substance of the object is biologically inactive.

 IBA> 1,000 ± 0,1000 - the substance of the object increases the viability of test organisms.

 IBA <1,000 ± 0,1000 - the substance of the object reduces the viability of test organisms.

 The value of the IBA of the substance of the tested object and its concentration in the solution with test organisms characterize the degree of its biological activity.

 Example 5. The third stage. Assessment of the biological activity of the substance of the tested object by the intensity of reproduction of test organisms.

 5.1. Working process.

 Use test organisms in the exponential growth phase.

 To count test organisms in 1 ml of solution, i.e. to determine the density of the inoculum, add 20 μl of a Lugol solution or 5% alcohol solution of iodine to a test tube with 1 ml of solution, mix thoroughly, fill the solution with a Fuchs-Rosenthal chamber, calculate the number of test organisms in 10 squares, dividing them by 10, calculate the average number test organisms in one square (the volume of one square is 0.0001 ml) and multiplied by 10 000. The resulting figure shows the number of test organisms in 1 ml of solution, i.e. inoculum density - PI.

Further, the work is carried out according to the first paragraph of clause 3.1. example 3, after which the tubes are placed in a thermostat with a temperature of 25 ^o C for 3 days. During exposure, the mixture is aerated by periodically shaking the tubes several times a day.

 After 3 days, the density of PI inoculum is determined in each tube as described above. If necessary, put several experiments and average the results for reliability.

 5.2. Evaluation of the results.

где
ИИР - индекс интенсивности размножения тест-организмов;
ПИОК - плотность инокулята в опыте в конце инкубации;
ПИКН - плотность инокулята в контроле в начале инкубации;
ПИКК - плотность инокулята в контроле в конце инкубации;
ПИОН - плотность инокулята в опыте в начале инкубации.Calculations are carried out according to the formula:

Where
IIR - the index of the intensity of reproduction of test organisms;
PIEC - inoculum density in the experiment at the end of incubation;
PIKN - inoculum density in the control at the beginning of incubation;
PICC - inoculum density in the control at the end of incubation;
PION - inoculum density in the experiment at the beginning of incubation.

 Obtained by the calculation of R&D is evaluated as follows.

 IIR = 1,000 ± 0,1000 - the substance of the object is biologically inactive.

 IIR> 1,000 ± 0,1000 - the substance of the object stimulates the reproduction of test organisms.

 IIR <1,000 ± 0,1000 - the substance of the object inhibits the reproduction of test organisms.

 The value of the IIR in combination with the concentration of the substance of the tested object in the mixture characterizes the degree of its influence on the reproduction mechanism of test organisms.

 Example 6. Evaluation of the biological activity of animal feed.

 Evaluation of the biological activity of the feed was carried out on the example of cake and silage. Feed samples were prepared according to paragraph 2.1. example 2, after which the test samples were investigated according to paragraphs. 3.1. and 3.2. example 3, p.p. 4.1. and 4.2. example 4, p.p. 5.1. and 5.2. Example 5. The test results are presented in the table.

 Conclusions about the biological safety and reliability of the tested ecological systems and facilities are made on the basis of the analysis of the results of the above stages.

The proposed method (rapid biotest) allows you to:
1. Quickly, inexpensively, massively determine the toxicity of soil, water, air, feed, food, animal feed, unknown substances with gradation according to the degree of hazard type - Attention! Caution! Biocide! Poison! In these cases, the rapid biotest allows you to determine that the object cannot be used to avoid an emergency and that it requires a detailed study to make a final conclusion about safety and suitability for use.

 2. Quickly, inexpensively, massively determine the given usefulness of food products, feed, nonspecific activity of medicinal mixtures, etc. with a gradation according to the degree of their usefulness type - Defective! Excess stimulation! In these cases, the rapid biotest allows you to determine that the object being tested does not comply with the certificate and to make a final decision it is necessary to evaluate the specific parameters of the non-compliance.

 3. Quickly, inexpensively, massively, constantly conduct biological monitoring of ecological systems of any level with gradation according to the degree of hazard of the type - Attention! The ecological system is in danger! In these cases, the rapid biotest allows you to determine what is close to an emergency that threatens man and nature, and that urgent measures are needed to curb the damaging effects and restore ecosystem stability.

 4. To create databanks of biological monitoring of ecological systems, which allows real opportunities to predict possible situations, develop scientifically sound ways to manage the safety and reliability of ecological systems, given their main quality - biological nature.

 5. Quickly and efficiently identify low-quality products when they are imported into a protected area, and in other cases when it is necessary to quickly determine whether an object has biological activity, whether it poses a threat to the health of people and animals (before conducting a comprehensive study and examination for certificate compliance) to prevent entry through the border and use.

Literature
1. USSR author's certificate N 1234770, G 01 N 33/18.

 2. USSR author's certificate N 1623426, G 01 N 33/15.

 3. Methodical recommendations. N.N. Maksimyuk, L.Ya. Telishevskaya / Ed. A.P. Prostyakova. - M .: 1996. Determination of the biological activity of biological products using a test culture of infusoria-tetrachimen pyrimiformis and test strains of microorganisms).

 4. Guidelines for the biological assessment of animal products and feed using the test organism tetrachimene pyrimiformis. VASKHNIL, - M ,: 1972.

 5. The use of ciliates-tetrachimen pyrimiformis as a test object in biological research in agriculture. Overview information. UPPER. - M :: 1978.

 6. The use of ciliary infusoria for high-quality rapid assessment of food and feed technology in the Far North. - Tyumen: 1979.

 7. Guidelines for determining the biological value of agricultural products. VASKHNIL, Southern Branch. - Kiev: 1981.

 8. French patent N 2141006, G 01 N 33/00.

 9. British patent N 1358760, C 12 K 1/06.

 10. Copyright certificate of the USSR N 990189 A 61 B 10/00.

Claims (2)

 1. The method of biological monitoring of ecological systems and objects, providing for the assessment of the biological effect of the substance of the tested object when introduced into a standard medium with test organisms Paramaecium candatum, characterized in that the monitoring is carried out in three stages, while the biological activity of the substance of the tested substance is evaluated at the first stage of the object by comparing the action of this substance and the control standard medium on test organisms after 0.5 - 24.0 hours of incubation; in the second stage, incubated for For 24 hours, a mixture of a standard medium with test organisms with the substance of the test object obtained in stage I and a control standard medium with test organisms are subjected to functional loads until the test organisms are completely dead, and their lifespan is recorded from the moment the functional loads begin to act and the obtained data evaluate the stimulating or inhibitory activity of the substance of the tested object in comparison with the standard medium, and at the third stage use a mixture of standard medium with test organisms in 3–4 days old, with the substance of the test object that showed biological activity in stage I, they are incubated for 3–4 days, after which the number of test organisms in a fixed volume of the mixture is counted and the effect of the substance of the test object on the propagation speed of the test is evaluated. organisms. 2. The method according to p. 1, characterized in that for the first and second stages, paramecium-caudatum infusoria are used as test organisms at the age of 2 to 3 weeks, the substance of the test object is introduced into the medium in concentrations from 1 • 10 ^-2 to 1 • 10 ^-6 per 1 ml of medium, incubate this mixture and the control standard medium with test organisms at 18 - 27 ^o C.

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