RU2440418C2 - Method to determine toxicity of wastes and soils - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: method includes measurement of microorganisms test function level in presence and in absence of an analysed sample and calculation of toxicity based on the produced results. Dense samples are exposed to testing (wastes, soils). At the same time testing is carried out without preliminary procedure of producing an aqueous extract of the sample. A test item is a culture of a soil bacillus with dehydrogenase activity, and the test function is its dehydrogenase activity, which is identified using resazurin and recorded using a conventional measurement device for spectrophotometry.

EFFECT: increased efficiency of wastes and soils toxicity identification.

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Description

The invention relates to methods for determining the toxicity of solid wastes and soils by bringing them into direct contact with bacteria and recording changes in the dehydrogenase activity of bacteria, as measured using resazurin. The invention can be used in environmental control laboratories, in the development of biotechnologies for waste processing, in the development of technologies for the remediation of contaminated soils, in determining the hazard class of waste, for determining the impact on environmental objects of new materials, industrial formations and waste.

A biosensor is known for determining the toxicity of chemical pollutants and liquid wastes using natural or genetically modified bacteria (Vibrio fischeri, Escherichia coli pUCD615 pRB28, Pseudomonas putida 556) immobilized in a thin film of polyvinyl alcohol GB 19990025788 19991101 (1).

Known microplate method for determining the toxicity of compounds in liquid samples obtained from water, soil and air objects, which uses recombinant luminescent microorganisms Escherichia coli DPD2511, DPD2540, DPD2794 and TV 1061 included in agar, sodium alginate or polyacrylamide, and is based on the increase in luminescence an increase in the concentration of toxicants in liquid samples KR 20010054588 20010905 (2).

The disadvantages of these methods is that they allow you to directly determine the toxicity of only liquid samples, and when testing soil objects they require the preparation of an aqueous extract, which lengthens and complicates the biotesting procedure.

A known method for determining the total toxicity of soils by the intensity of the luminescence of bacteria (3). The method is based on determining changes in the intensity of bioluminescence of a genetically engineered bacterial strain (Ekolyum biosensor) when exposed to toxic substances present in the analyzed sample, compared with the control. The intensity of bioluminescence is determined using a specialized luminometer "Biotox-10". When determining the soil toxicity index, a soil extract prepared by 24-hour soil extraction with 5-fold volume of distilled water is analyzed. The main disadvantages of this method are the inability to determine the toxicity of directly soil samples and the high cost of the measuring device and biosensor.

A known method for the determination of aerobic dehydrogenase activity of soils is based on the acceptance of the hydrogen that is cleaved during dehydrogenation by soil microorganisms by the colorless salts of tetrazolium, in particular 2,3,5-triphenyltetrazolium chloride (TTX), and its conversion to 2,3,5-triphenylformazan having a red color followed by colorimetric measurement of the amount of formazan (4).

The disadvantage of this method is the inability to determine the dehydrogenase activity of samples in which copper is present, associated with the negative effect of copper on the absorption of the product of the reduction of the TTX of 2,3,5-triphenylformazane (5).

The closest analogue, selected as a prototype by the set of matching features and the technical result achieved, is the invention "Vibrio fischeri VKPM B-9580 bacterial strain used as a test culture for determining the toxicity of environmental objects" RU 2346035 C1 (6). Implementation of the use of this strain is as follows. Water extracts from bottom sediments are preliminarily obtained or aqueous solutions of the tested toxicants are prepared. The strain is grown on nutrient agar with 1.7% NaCl, a night culture is prepared, diluted, and solutions of substances or aqueous extracts of bottom sediments are added to aliquots of this suspension. After incubation, luminescence is measured for mixtures containing the analyzed compounds and for the control using an LT-01 luminometer; a 1.7% NaCl solution is used as a control. The method is recommended for monitoring water bodies and bottom sediments.

The main disadvantages of this invention are:

- low efficiency associated with the need to obtain an aqueous extract from the sample. This disadvantage is caused by the fact that in the prototype luminescence is used as a test function; determination of the luminescence intensity does not allow a solid sample to be present in the reaction mixture and necessitates the preparation of an aqueous extract from the sample. However, when testing an aqueous extract, the negative effect on the test organism is estimated of only part of the compounds present in the sample, namely, the water-soluble part, which passes into the aqueous extract. At the same time, according to published data, substances and elements that are in a bound state and do not pass into the aqueous extract can have a negative effect on living organisms (7, 8). This leads to the fact that when determining the toxicity of soils or bottom sediments on the basis of testing their water extracts, only water-soluble toxicants are analyzed, and the effectiveness of evaluating the toxicity of the entire sample as a whole is significantly reduced;

- the complexity of the method associated with the use of marine bacteria Vibrio fischeri. This disadvantage is caused by the fact that Vibrio fischeri is a marine bacterium that requires special conditions for cultivation and biotesting; therefore, thorough preparation of the reaction mixture is required so that the final salt content is 1.7%, which makes the biotesting process difficult. In addition, the enzyme luciferase is very sensitive to temperature changes;

- the high cost of the method associated with the need to purchase to determine the luminescence of the measuring device of the luminograph;

- the difficulty in extrapolating the results of soil and waste biotesting due to the fact that Vibrio fischeri bacteria are not representatives of soil biocenosis affected by toxicants present in soil and waste.

The purpose of the claimed technical solution is:

- increasing the efficiency of determining the toxicity of waste and soil by direct contact of the sample with a test object, which uses a culture of soil bacillus with dehydrogenase activity, which allows us to evaluate the effect of toxic components present in the sample, water-soluble and closely related to the matrix of the sample;

- cheaper through the use of a traditional measuring device;

- reducing the complexity due to the use of soil bacillus culture with dehydrogenase activity, not requiring special cultivation conditions and sample preparation;

- the exclusion of the procedure for extrapolating data by replacing a test object that is not a representative of soil microorganisms with a typical representative.

The method is as follows.

The test sample is sterilized in an autoclave to inactivate the microorganisms present in the sample, then a culture of soil bacillus with dehydrogenase activity, and grown to a phase in which it exhibits maximum activity, is brought into direct contact with the mixture for a certain time, the liquid and solid fractions of the reaction are separated mixtures and in the liquid fraction by changing the indicator of the redox process determine the change in the rate of dehydrogenase reaction occurring under The action of the sample.

To calculate the level of toxicity, the proposed method is implemented in parallel in four versions.

Option 1 ("experience"). A sample is made from the mass of the sterilized sample, which is placed in resealable tubes. A bacterial suspension, distilled water and a solution of resazurin (an indicator of the redox reaction) are added to the test tubes with a hinge. The tubes are placed in a thermostat at 28 ° C and periodically shaken. After 24 hours, the reaction mixture was centrifuged at 6000 rpm and the spectrophotometrically unreduced resazurin was determined in the supernatant.

Option 2 ("experience without bacteria"). A sample is made from the mass of the sample, which is placed in lockable tubes. Distilled water and a resazurin solution are added to the test tubes, but they do not add a bacterial suspension, but replace it with the same amount of distilled water. Next, the procedure is carried out as described above.

Option 3 ("control the activity of bacteria"). Bacterial suspension, distilled water, resazurin solution are poured into the closing tubes, but the sample is not added, replacing it with the same amount of distilled water. Next, the procedure is carried out as described above.

Option 4 (“reagent control”). Instead of a sample, distilled water is placed in closure tubes, and distilled water and resazurin are used instead of a suspension of bacteria. A further procedure is carried out as described above.

All variants of the experiment carried out in triplicate. Then the results of each option are averaged. According to the results of four test options, calculations are made to obtain the toxicity index of the studied soils or waste according to the formula:

, где

where

D _samples - the optical density of the supernatant of the sample "experience";

D _samples - the optical density of the supernatant of the sample experience without bacteria.

D _Ka - optical density of the supernatant of the sample "control the activity of the culture."

D _SKA - the optical density of the supernatant of the sample "control reagents".

The conclusion about the toxicity or non-toxicity of the sample is made in a well-known manner based on the results of determinations of the dehydrogenase activity of bacteria in control and experiment. A sample is considered toxic if the T value is 50% or more. In this case, to quantify the toxicity of the sample, the effective concentration of the sample, EC _{50, is established} .

The method is implemented as follows and is illustrated in tables 1, 2, 3.

Example 1

The culture of soil bacillus was carried out in test tubes on meat and peptone agar. Cultivation was carried out at 28 ° C in 250 ml flasks with 50 ml of L-broth (g / l): peptone - 10, yeast extract - 5, NaCl - 10. For biotesting, the culture was incubated in L-broth medium (g / l) : peptone - 10, yeast extract - 5, NaCl - 10 at a temperature of 28 ° C until reaching an optical density of 1.5-1.7 opt. (λ = 600 nm, cuvette 10 mm).

Aliquots of this culture (3 ml) were transferred to screw cap tubes, 3 g of sample or water was added, and analysis was performed in accordance with the four process options described above. Toxicity was assessed by the relative difference in the dehydrogenase reaction of bacteria, an indicator of which was the restoration of resazurin, in the presence and absence of the analyzed sample (in experiment and control).

For comparison, the samples were tested according to the method used in the investigated prior art, including in the prototype. For this, water extracts were obtained from the samples. To prepare the aqueous extract, a 1 g soil sample was diluted with dechlorinated water in a ratio of 1:10, the mixture was shaken on a rotator (1 h, 60 rpm), settled for 24 hours, then filtered. During testing, the obtained extracts (3 ml) were used instead of 3 g of the analyzed samples.

To quantify the toxicity of the sample, the characteristic EC ₅₀ (effective concentration) was used.

In the case of determining the toxicity of the sample by the proposed method, the EU ₅₀ is the effective concentration of the sample, causing 50% inhibition of the dehydrogenase activity of the culture compared to the activity of the culture in the absence of the sample. In the case of determining the toxicity of the sample on the basis of determining the toxicity of the water, the proposed method for a sample with a chromium content of 992 mg / kg 54 mg, which is 3.2 times higher than the value established in the analysis by the method of obtaining an aqueous extract (as indicated in prototype). Also, for example, when analyzing the toxicity of a sample containing cadmium 58 mg / kg, the differences in EC ₅₀ values were 4 times, and the EC ₅₀ value established by the proposed method was lower. The lower the value of the EU ₅₀ , the more effective the determination method. From the above data it follows that the values of the EU ₅₀ established when testing the aqueous extract from the samples (as is customary in the prototype) is higher, i.e. less dilution is required to achieve a 50% inhibitory effect, therefore, the method proposed in this technical solution is more effective.

Example 2

Industrial waste was tested: Emergency Situations - Black Solar from a plant for the production of road bitumen, ШГ - Sludge from a thermovoltaic workshop, ШВП - Sludge from degreasing baths, ШГ2 - Galvanic sludge, filter press station, ШФ - Phosphating sludge from a phosphating bath.

For a comparative assessment, the characteristics of the EC ₅₀ and EC _{10 were used} . In the case of determining the toxicity of the sample by the proposed method, the EU ₅₀ and EU ₁₀ is the effective mass of the sample, causing 50% and 10% inhibition of the dehydrogenase activity of the culture compared with the activity of the culture in the absence of the sample.

The results of the biotesting are shown in table 2.

When biotesting according to the proposed method, the values of the EU ₅₀ were significantly lower compared to the EU ₅₀ established by the method of obtaining an aqueous extract. So, in the analysis of the emergency ES ₅₀ waste, determined by the proposed method, amounted to 60 mg, while the method of obtaining an aqueous extract (as in the prototype) - 2300 ml, etc. This indicates that not only water-soluble toxicants affect the test function (dehydrogenase activity) inhibitory. In addition, the table shows that the differences between the established values of the EU ₅₀ significantly differ for each of the tested waste. So, for emergency situations, the differences were 38 times, for emergency events - 3.5 times, for ballscrews - 1.9 times, for emergency balloons - 3 times, for emergency ships - 6.4 times. A similar picture is observed when analyzing differences in the EU ₁₀ . So, the differences were 43, 2.4, 1.3, 2.3 and 3.9 times for wastes of emergency situations, SHG, ball screws, SHG2 and ShF, respectively. These differences indicate that the composition of the waste contains substances with different solubilities in water, this explains the different values of the EU ₅₀ and EU ₁₀ . The different solubility of substances determines the different negative effect of toxicants in waste on living organisms and confirms the need to evaluate not only water-soluble compounds in waste, but also in a bound state. Thus, the data presented demonstrate the advantage of the claimed technical solution.

Example 3

Soil samples contaminated with metals and an organic toxicant, fungicide alto-super, were tested by the proposed method and method to obtain an aqueous extract. In the same samples, before they were sterilized in an autoclave, the dehydrogenase activity of indigenous microflora was determined in order to find out how adequately the results of biotesting reflect the negative effect of toxicants on the real community of microorganisms in the soil and, therefore, how easy it will be subsequently interpreted. For comparison, the toxicity of the samples was determined. The test results are presented in table 3.

Firstly, the table shows that when testing by the proposed method, the level of inhibitory effect is much higher. For example, when soil is contaminated with fungicide alto super at a concentration of 33.5 mg / kg, the level of inhibition differed by 1.9 times, when contaminated with Cu (70 mg / kg), by 1.5 times, Cr (15.6 mg / kg ) - 1.6 times, etc.

Secondly, in order to establish the results of which biotesting method most adequately reflect the real situation, a correlation analysis was carried out. The established correlation coefficient (R) between the results of biotesting by the proposed method and the results of determining the dehydrogenase activity of indigenous microflora turned out to be 0.96. In a similar analysis of the results of biotesting by the method with obtaining an aqueous extract and the results of determining the dehydrogenase activity of indigenous microflora, the correlation coefficient (R) was 0.62. Thus, the results obtained by the proposed method more adequately reflect the real situation and therefore more easily interpretable.

The claimed technical solution meets the criterion of "novelty" presented to the invention, because from the studied prior art has not been identified a method that has a combination of the characteristics claimed in the claims, which allows to simultaneously obtain the claimed group of technical results (goals), namely, high efficiency of determining the toxicity of waste, man-made structures and soils due to direct contact of the sample with the test object and low laboriousness during the analysis.

The claimed technical solution meets the criterion of "inventive step" for inventions, because The claimed technical solution does not follow explicitly for a person skilled in the art and is not obvious to a person skilled in the art and allows to resolve conflicting tasks, namely: on the one hand, the method should be simple and cheap and at the same time have high efficiency in determining toxicity of wastes, technogenic formations and soils.

The claimed technical solution meets the criterion of "industrial applicability" to the invention, because it was realized on real industrial waste (black solarium from a plant for the production of road bitumen, sludge from a thermogalvanic workshop, sludge from degreasing baths, galvanic sludge from a filter press station, phosphate sludge from a phosphating bath), and the stated results were obtained, namely, more high efficiency of the claimed technical solution, as evidenced by tabular data. At the same time, standard laboratory equipment is required to implement the claimed industrial solution, traditional reagents and available microorganisms, for example, bacilli, are used.

Information sources:

(1) GB 19990025788 19991101 Immobilized bacteria.

(2) KR 20010054588 20010905 Kit for analysis of toxicity of toxic materials in sample usig fixed recombinant luminescent microorganisms.

(3) MP No. 11-1 / 11-09. Determination of general soil toxicity by the intensity of bacterial bioluminescence.

(4) Khaziev F.Kh. Methods of soil enzymology / F.Kh. Khaziev; Institute of Biology Ufim. SC - M .: Nauka, 2005 .-- 252 p.

(5) Obbard J.P. Measurements of dehydrogenase activity using 2-p-iodophenyl-3-p-nitrophenyl-5-pheniltetrazolium chloride in the presence of copper // Biol. Fertil. Soils. - 2001. - V.33. - P.328-330.

(6) RU 2346035 C1 08.13.2007. Application 2007130940/13. Posted 02/10/2009. Bull. Number 4. The strain of bacteria Vibrio fischeri, used as a test culture to determine the toxicity of environmental objects.

(7) Brandt, K.K. Decreased abundunce and diversity of culturable Pseudomonas spp. Populations with increasing copper exposure in tghe suger beet rhizosphere [text] / K.K.Brandt, A.Petersen, P.Holm, O. Nybroe // Microbiol Ecology. - 2006. - No.56. - P.281-291.

(8) Ivask, A. Recombinant luminescent bacterial sensors fotr the measurement of bioavailability of cadmium and lead in soils polluted by metal smelters [text] / A.Ivask, M.Fracois, A.Kahru, H.-C. Dobourguier, M .Virta, F. Douay // Chemosphere. - 2004. - No.55. - P.147-156.

The method for determining the toxicity of waste and soil

Table 1 Sample EC ₅₀ (mg) determined by the proposed method EC ₅₀ (μl), determined by the method of obtaining an aqueous extract Chromium contaminated soil 600 3000 (VI) at a concentration of 7.8 mg / kg Chromium contaminated soil 54 176 (VI) at a concentration of 992 mg / kg Chromium contaminated soil 29th 136 (VI) at a concentration of 3971 mg / kg Cadmium contaminated soil in 1500 3000 concentration of 3 mg / kg Cadmium contaminated soil in 300 3000 concentrations of 11.6 mg / kg Cadmium contaminated soil in 107 429 concentration of 58 mg / kg

table 2 Biological testing of the proposed method Biological testing according to the method of obtaining an aqueous extract EU ₅₀ EU ₁₀ EU ₅₀ EU ₁₀ Emergency 60 24 2300 173 SH 62 22 218 53 Ball screw one hundred 39 190 44 SHG2 112 40 334 90 Bf 34 fifteen 216 58

Table 3 Toxicant
in the soil Concentration, mg / kg Toxicity,% The proposed method A method using an aqueous extract A method based on the assessment of indigenous microflora Cu 5 28 22 16 fifteen fifty 38 47 thirty 55 41 57 60 70 47 78 70 72 49 81 120 80 61 83 180 87 74 89 Cr 0.31 41 0 45 3,1 46 0 57 7.8 79 29th 84 15.6 93 58 93 992 96 63 96 3971 one hundred 66 99 Ni 10 10 0 four twenty 17 eleven eighteen 40 45 eleven 48 60 62 13 64 one hundred 80 41 80 250 92 54 94 Pb fifteen 9 7 5 fifty 61 fourteen 60 one hundred 77 21 75 200 81 59 81 250 86 64 89 500 92 80 one hundred 1000 95 90 one hundred 1500 one hundred 99 one hundred Fungicide 0.3 0 0 0 alto 0.7 2 0 0 super 0.85 6 0 3 1,1 27 0 9 1.3 28 19 13 6.7 57 21 37 33.5 84 44 75 67.8 88 58 87

Claims (1)

A method for determining the toxicity of samples, which consists in measuring the level of the test function of microorganisms in the presence and absence of an analyzed sample and calculating toxicity based on the results, characterized in that dense samples (waste, soil) are tested, and testing is carried out without a preliminary procedure for obtaining an aqueous extract of the sample , as a test object, a culture of soil bacillus with dehydrogenase activity is used; as a test function, it is used dehydrogenase activity as determined using Resazurin and recorded using a conventional measuring apparatus for spectrophotometry.