RU2751795C1 - Method for determining the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in fecal waste from vehicles - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. A method is proposed for determining the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in fecal waste. The method includes preparing from a sample of fecal runoff test samples with various degrees of dilution, a test liquid with a given concentration of the specified substances, a surfactant and propanol-2, as well as test samples by successive dilutions of the test liquid. Next, the test and test samples are brought into contact with the test culture and the degree of suppression of the growth of the test culture is measured, where the test and test samples with the same degree of suppression of growth are considered to contain equal concentrations of the indicated substances. From the known concentration of these substances in the test sample and the known degree of dilution of the test sample, the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in the sample is calculated.EFFECT: provides an increase in the accuracy of determining the content of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in fecal waste produced in vehicles toilets.2 cl

Description

The invention relates to the field of biochemistry, more precisely, to measurement methods using microorganisms for the quantitative determination of disinfectants (hereinafter referred to as DS) in wastewater of undetermined composition, primarily in fecal wastewater from vehicles: airplanes, passenger cars, buses and ships.

The flushing fluid of the toilets of vehicles includes didecyldimethylammonium chloride (hereinafter - D4 - a common abbreviation) and polyhexamethylenebiguanidine hydrochloride (hereinafter - PHMG-GC - a common abbreviation) as DV. In addition, it contains a number of other chemically active substances such as fragrances and dyes, surfactants.

Faecal effluent from vehicle toilets accumulates at airports, stations and ports. They are subject to deactivation by biological treatment methods. A feature of this type of effluent is the quantitative excess of the flushing liquid over the actual feces by only 2 ... 10 times, while in the sewage drains of urban settlements, the degree of dilution of feces is many tens of times higher, and the content of chemical, including disinfecting, substances is hundreds of times lower ... The content of DV and surfactants in undiluted fecal wastewater from vehicles is so high that none of the bacterial cultures commonly used in wastewater treatment can survive and, moreover, actively multiply in them. Therefore, the neutralization of such effluents is a serious problem.

The solution to this problem is to dilute fecal effluents with water to reduce the concentration of DW to a level at which their biological neutralization becomes possible. However, this greatly increases the volume of wastewater and, accordingly, the payment to the services of treatment facilities that receive them. To resolve the arising contradiction, dilution should be carried out exactly to an acceptable concentration of DWs for the treatment facilities, but no more. Hence, a new problem arises: determination of the concentration of DW in dilute waste streams.

The solution to this problem is complicated not only by the chemically complex composition of fecal wastewater from vehicles, but also by the fact that the D4 and PHMG-GC used in the flushing liquid are very effective as bactericidal agents and therefore their concentration in the wastewater is very low, practically close to the sensitivity limits of chemical analytical methods.

There are a number of methods for the determination of D4 and PHMG-GC by analytical chemistry methods [CN 101943690, JP 2002243719, JP 2002243719, RU 2252413, RU 2336522, RU 2479839, UA 83673]. However, the known methods are unsuitable for the quantitative determination of the content of these substances in the fecal effluents of vehicles due to the presence in the effluents of a number of other chemically active substances that distort the measurement results or make them simply impossible.

There is also known a method for the quantitative determination of polyhexamethylene guanidine by stripping voltammetry, which, as indicated in the patent description, can also be used to control wastewater [RU 2381501]. The disadvantage of this method is the same as that of the above-described analogs.

There are also known methods for the determination of guanidine derivatives based on photoelectrocolorimetry [RU 2487346] or high-speed liquid chromatography [CN 105021721, JP S578447] of a solution of the analyzed sample. Both methods are very sensitive to the presence of other chemicals in the solution and therefore are not suitable for the quantitative determination of the content of guanidine derivatives in fecal effluents.

Most suitable for solving the above problem are methods using microorganisms.

There is a known method for determining the toxicity of the environment, in which the growth indicators of test cultures (micromycetes) are determined in control and experiment, the degree of inhibition of the growth of test cultures in the experiment in relation to the control is calculated and the data obtained are compared with five conditionally established environmental hazard classes [RU 2570637] ... Close to this method is a method for determining the total toxicity of waste, which also compares the growth indicators of test cultures (genus Bacillus), known for their sensitivity to toxicants presumably contained in the test medium, in control and experiment, calculate the integral criterion and compare it with the toxicity criteria [ RU 2202619].

The common disadvantage of these two methods is that they are good for determining only the general toxicity of the test medium, regardless of what kind of substances and in what concentration this toxicity is caused.

There is a known method for determining the bactericidal and bacteriostatic concentration of DW, including the creation of a number of test samples by repeated serial dilutions of DW in a sterile liquid, bringing the test samples into contact with the test culture, cultivation during the test time, and assessing the concentration of DW by the dilution parameters in which bacterial growth [UA 89484]. The method is designed to assess the toxicity of DV, uses dilutions of a known amount of DV in a sterile liquid and therefore is not suitable for determining the unknown concentration of DV in the fecal wastewater of vehicles containing a variety of chemical and organic substances.

A known method for the detection of antimicrobial substances in a test sample, in which a test culture is introduced, which is a strain of Bacillus or Streptococcus, and at least two redox indicators in an agar medium, after which the test sample is brought into contact with an agar medium and the content is assessed antimicrobial substances by comparing the growth of the test culture with control samples [RU 94045954 A1]. The disadvantage of the known method is that it only determines whether the test sample contains antimicrobial substances or not, without quantitative assessment.

There is a known method for determining the toxicity of samples, according to which test cultures of Pseudomonas yamanorum VKM B-3033D are incubated under certain conditions in the presence of test samples and in their absence, after a certain time, the indicators characterizing the degree of activation or inhibition of test samples (light intensity, optical density, etc.) are measured. intensity of photofluorescence) and calculate this degree according to a specially developed formula [RU 2688745]. The disadvantage of the known method is that it only determines the degree of toxicity of the tested samples, regardless of what substances and in what concentration this toxicity is caused.

The closest to the proposed in technical essence and the achieved result is a method for determining DW in a liquid sample, in which this sample is brought into contact with a test culture selected from thermophilic strains of Bacillus and Streptococcus, control samples of the test culture are exposed to a known amount of DW, compare , after a certain period of time, the growth of the test and control samples of test microorganisms [RU 96119987 A].

The disadvantage of this method is the low accuracy of determining the quantitative content of the antibacterial compound, since it does not take into account the presence in the test sample of other, non-antibacterial, chemical compounds that can affect the growth / inhibition of bacterial colonies. In addition, thermophilic strains of Bacillus and Streptococcus are very diverse and include strains with both increased and decreased sensitivity to didecyldimethylammonium chloride and polyhexamethylene biguanidine hydrochloride.

The objective of the present invention is to improve the accuracy of the method for the quantitative determination of the content of D4 and PHMG-GC in fecal effluents of a complex composition produced in toilets of vehicles.

The technical result from the use of the proposed method consists in increasing the accuracy of determining the content of D4 and PHMG-GC in fecal effluents of a complex composition, produced in the toilets of vehicles.

The specified technical result is achieved by the fact that a number of test samples are prepared from a sample of fecal runoff with varying degrees of dilution of the sample, a test liquid is prepared that is closest in composition to the test streams and contains a given concentration of D4 and PHMG-GC, and which is human feces in the mixture with water in a ratio of 1: (2-10) with the addition of a surfactant, which is oxyethylated monoalkylphenols, and propanol-2, prepare a number of test samples from serial dilutions of the test liquid, bring the test and test samples into contact with the test culture, then the samples are incubated, the degree of suppression of the growth of the test culture is measured, where the test and test samples with the same degree of suppression of growth are considered to contain equal concentrations of didecyldimethylammonium chloride and polyhexamethylene biguanidine hydrochloride, after which, according to the known concentration of didecyldimethylammonium chloride and polyhexamethium Lenbiguanidine hydrochloride in the test sample and a known dilution of the test sample, calculate the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in the fecal runoff sample and, if necessary, refine the result by fractional dilution of the test liquid near the initially established dilution with the same degree of suppression of growth, as well as this point.

In addition, neonol selected from the group: Neonol AF 9-9, Neonol AF 9-10, Neonol AFB-10, Neonol AF 9-12, Neonol AFB-12 in an amount of (0.02. ..0,1) of the total mass of the zero dilution test liquid.

In addition, bacterial strains selected from the series: Paenalcaligenes sp., Staphylococcus pasteuri, Enterococcus faecium, Micrococcus endophyticus, Enterococcus casseliflavus, Bacillus subtilis, Alcaligenes aquatilis are used as a test culture.

Due to the creation of a number of test subjects and a number of test samples with different degrees of dilution of the starting material, the accuracy of determining the content of D4 and PHMG-GC is significantly increased, since dilutions can be carried out both multiple and fractional.

Due to the use of the test liquid, which is the closest in composition to the tested effluents and contains a given concentration of the indicated substances, the accuracy of determining the D4 content and PHMG-GC significantly increases, since the test cultures find themselves in an environment that is practically identical to that of the investigated effluent.

Due to the fact that human feces are used as a test liquid, in a mixture with water in a ratio of 1: (2 ... 10) with the addition of a surfactant, the accuracy of determining the content of D4 and PHMG-GC increases, since the test cultures are in an environment that is almost identical the environment of the investigated runoff.

Due to the close coincidence of the chemical compositions of the fecal runoff sample and the test liquid, the determination accuracy increases, since the results do not depend on small deviations from the composition and growing conditions of test cultures, which are the same for the tested and test samples.

The use as a surfactant of oxyethylated monoalkylphenols, mainly selected from the group Neonol AF 9-9, Neonol AF 9-10, Neonol AFB-10, Neonol AF 9-12, Neonol AFB-12 in an amount of (0.02 ... 0, 1) from the total mass of the test liquid of zero dilution, increases the accuracy of determining the content of D4 and PHMG-GC, since it brings the composition of the test liquid and, therefore, the growth conditions of the test culture to the composition and conditions of the test runoff to the greatest extent.

The use as a test culture of bacterial strains selected from the series: Paenalcaligenes sp., Staphylococcus pasteuri, Enterococcus faecium, Micrococcus endophyticus, Enterococcus casseliflavus, Bacillus subtilis, Alcaligenes aquatilis contributes to an increase in the accuracy of determining the content of D4 and PGMG-H being sensitive to D4 and PHMG-GC, they provide at the same time good stability of results from one experiment to another.

Due to the fact that the determination result is clarified by fractional dilution of the test liquid near the initially established dilution with the reactions of the test culture close to the test samples, the concentration determination accuracy is sufficient to calculate the required dilution of fecal effluent.

The proposed method is carried out as follows.

A number of identical test culture samples are prepared by inoculating a microbial suspension of a certain density in a Petri dish on nutrient agar.

A test liquid is prepared, which is almost similar in composition to the faecal effluent from vehicles.

Prepare a series of test samples from dilutions of the test liquid (eg, 1:10 to 1: 600) with sterile water.

Prepare a number of test samples from a sample of fecal runoff by diluting it to varying degrees with sterile water.

The test culture samples are brought into contact with the test and test samples.

The plates are incubated in a thermostat at a certain temperature for a certain time, after which the result is taken into account by measuring the degree of suppression of the growth of the test culture.

The test and control samples with the same degree of growth inhibition are considered to contain equal concentrations of AI. The dilutions of the subject and the test specimens may differ in this case.

Based on the known concentration of DV in the test sample and the known degree of dilution of the test sample, the concentration of DV in the sample of fecal runoff is calculated.

If necessary, clarify the result by fractional dilution of the test liquid near the initially established dilution with the same degree of growth suppression, as well as by conducting a series of repeated tests near this point.

Below, as an example, described in more detail one of the possible options for the implementation of the proposed method.

1. Prepare a number of identical samples of the test culture by plating a microbial suspension of a certain density in a Petri dish on nutrient agar.

2. Preliminary studies have shown that the composition of toilet flushing fluids is approximately the same, regardless of the country where they were manufactured.

Moreover, all of them use a disinfectant manufactured by Sweden under the trade designation "Arquad 2.10-50", which has a complete analogue of the Swiss agent "Bardac-22". They are an aqueous solution of D4 and 2-Propanol with a concentration of 50 g / l and 20 g / l, respectively.

In domestic transport equipment for flushing toilets, a liquid is used under the trade designation "Deodorant agent" Latrina ", which is an aqueous solution of" Arquad 2.10-50 "(100 g / l), PGMG-GH (7 g / l), produced by the domestic industry under trade designation "Polisept", PGMG-GC (7 g / l), a surfactant which is a technical mixture of polyethylene glycol ethers of monoalkylphenols, known under the trade designation "Neonol AF 9-10" (50 g / l), as well as fragrances and dye in small concentrations.

Since the compositions of the flushing liquids are approximately the same in all countries, and the sources of raw materials for their manufacture are the same, the results of the described process practically do not depend on whether the source of fecal waste was domestic or foreign vehicles.

Therefore, to prepare a test solution, first prepare a test analogue of a toilet flushing fluid in the form of an aqueous solution of the following composition:

- Arquad 2.10-50 (Arquad) - 100 g / l (D4 content 50 / g / l);

- Polysept (PGMG-GC) - 7 g / l:

- Neonol AF 9-10-50 g / l.

3. In two ... ten parts of the prepared test analogue of toilet flushing fluid, 1 part of human feces, including feces and urine, is diluted. The resulting test liquid has a concentration of DV approximately the same as that of faecal effluent. The indicated proportions were established experimentally taking into account the content of substances in the flushing fluids and provide a quick convergence of the process for determining the content of D4 and PHMG-GC.

4. Prepare a number of test samples from dilutions of sterile water samples of fecal runoff and a number of test samples from dilutions of the test liquid in proportions from 1:10 to 1: 600. Samples are applied to paper disks in an amount (5-20 μl).

5. In a Petri dish on nutrient agar LB (Broth, Miller), inoculate a microbial suspension of a certain density (equivalent to a turbidity standard of 0.5 (at OD 600 nm)) of one of the above test cultures.

6. Paper disks with applied samples are placed on the agar. Diffusion of disinfectants into agar leads to the formation of a zone of suppression of the growth of microorganisms around the discs. After incubation of the plates in a thermostat at 28 ° C for 24 ... 48 hours, the result is taken into account by measuring the growth inhibition zone around the discs. The diameter of the zone is used to judge the reaction of the test culture and the bactericidal effect of samples with a given dilution. This method is considered the most convenient of all other methods of bringing the samples into contact with the test culture.

7. Identify the test and test samples with the same reaction of the test culture and, knowing the degree of dilution of the samples, calculate the content of DV in the sample of fecal runoff. All tests are carried out in triplicate; standard statistical methods are used to process the results.

8. Knowing the content of DW in the sample of fecal runoff, the required dilution rate of this runoff is calculated, at which the concentration of DW does not exceed a given value. Document the result.

9. If it turns out that in the previous dilution the diameter of the growth inhibition zone of the test samples is larger, and in the subsequent dilution it is less than that of the test samples, apply a fractional dilution of the test liquid in the range between the preceding and subsequent dilutions by the dichotomy method in order to obtain a more accurate result. This is important, because when dilutions are multiples of 2, an error per dilution means a doubling of the volume of effluents delivered to treatment facilities and, accordingly, a doubling of the payment for their reception.

The results of control experiments carried out with a test liquid consisting of an aqueous solution of only D4 and PHMG-GC showed a multiple discrepancy between the results obtained by the proposed method.

The complexity of the proposed method is small, it does not require technically complex and expensive equipment, like chemical or chemical-physical methods, the determination accuracy does not depend on errors in the preparation of test cultures. The main thing is that it allows you to get results with sufficient accuracy for practice on such material with which none of the known methods simply gives any results.

Claims (3)

1. A method for determining the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in the fecal wastewater of vehicles, in which a number of test samples with varying degrees of sample dilution are prepared from a sample of the fecal drainage, a test liquid is prepared that is closest in composition to the test wastewater and contains a given concentration of these substances and which is human feces mixed with water in a ratio of 1: (2-10) with the addition of a surfactant, which is oxyethylated monoalkylphenols, and propanol-2, a number of test samples are prepared from serial dilutions of the test liquid, the subjects are given and test samples in contact with the test culture, then the samples are incubated, the degree of suppression of the growth of the test culture is measured, where the test and test samples with the same degree of suppression of growth are considered to contain equal concentrations of didecyldimethylammonium chloride and polyhexamet ilenbiguanidine hydrochloride, after which, according to the known concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in the test sample and a known dilution of the test sample, calculate the concentration of didecyldimethylammonium chloride and polyhexamethylenebiguanidine hydrochloride in the sample of fecal effluent, and dilute the result of the fractional dilution with the same dilution in the sample the degree of inhibition of growth, as well as conducting a series of repeated tests near this point. 2. The method according to claim 1, characterized in that the surfactant used is neonol selected from the group: Neonol AF 9-9, Neonol AF 9-10, Neonol AFB-10, Neonol AF 9-12, Neonol AFB -12 in the amount of 0.02-0.1 of the total mass of the test liquid of zero dilution. 3. The method according to any one of claims 1, 2, characterized in that the test culture used bacterial strains selected from the series: Paenalcaligenes sp., Staphylococcus pasteuri, Enterococcus faecium, Micrococcus endophyticus, Enterococcus casseliflavus, Bacillus subtilis, Alcaligenes aquatilis ...