RU2370540C2 - Method of biotoxicity determination for drinking mineral water - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves sampling with further sample degassing. Further, if luminescent sea bacterium Photobacterium phosphoreum B-17 677f is added to test and reference samples, then sample mineralisation level is defined and NaCl is added to the test sample to reach total mineralisation level of 30 g/l. Reference samples are prepared. Further, 0.01 solution of NaCl is added to test and reference samples to reach pH 7.5. Then luminescent sea bacteria Photobacterium phosphoreum B - 17 677f or recombinant luminescent bacteria Esherichia coli K12 TGI with luminescent system genes of Photobacterium leognathi are added to test and reference samples. Further the luminescence level is measure, and toxicity index is calculated by the formula T=((I_r-I_t)/I_r)*100%, where I_r is luminescence intensity in reference sample, I_t is luminescence intensity in test sample. With T<20% water is assessed as non-toxic, with T within 20 to 50% water is considered toxic, and with T>50% it is considered highly toxic.

EFFECT: improved accuracy of method.

2 tbl, 1 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of toxicology and sanitary-hygienic measuring technologies, and in particular to methods of measurement and testing using viable microorganisms.

In particular, the invention is intended for the determination of acute integral toxicity (biotoxicity) of drinking mineral bottled water using luminescent (luminous) bacteria.

The task of testing the biotoxicity of such waters is determined by the need to assess the level of their biological hazard, which determines the possibility of human use for drinking purposes as canteen, medicinal-table or medicinal waters. In this regard, the determination of the biotoxicity of mineral waters is included in the list of studies provided for assessing the quality of bottled, including mineral waters [1].

Thus, the inventive method is intended for use in the laboratories of the centers of hygiene, sanitation and epidemiology and special services of the federal executive authorities, carrying out departmental sanitary and epidemiological surveillance, as well as in production laboratories that carry out internal quality control of drinking mineral waters.

State of the art

Among the various methods of analysis of the toxicity of drinking water, aimed at identifying its danger to human health, biotesting, based on the study of the effect of water on living organisms, is taking an increasingly important place [2]. In contrast to chemical analysis methods focused on the quantitative detection of individual chemical toxicants in water with subsequent comparison of the detected concentrations with standard values - maximum permissible concentrations (MPC), biotesting methods do not allow to judge the chemical nature of water pollution, but make it possible to form an integral idea of total toxicity of the totality of chemicals and compounds present in it, most often characterized by 20% sludge 50% lethality (LD20, LD50) or inhibit any of the vital sides (EC20, EC50) organisms used. At the same time, the important advantages of biotesting are the possibility of an integrated assessment of the toxicity of the entire set of chemicals and compounds present, including those for which methods for the quantitative detection and MPC values are not developed.

Small crustaceans — daphnia [3, 4], protozoa — paramecia, and bacteria [5, 6] were most widely used as objects for biotesting water. In turn, the simplicity of organization, the high rate of metabolism and energy, as well as some other physiological features make the bacteria the most convenient and common tool for biotesting.

Among the bacteria, one of the most popular options is luminescent (spontaneously glowing) bacteria, which is explained by the following main points: 1) the enzyme system for generating luminescence of such bacteria is closely integrated with the main energy flows of the bacterial cell, and therefore it quickly and sensitively changes its activity when the appearance of chemical pollutants in the medium [7], 2) the luminescence intensity can be quantified with high accuracy in real time, which allows quick about quality and to determine the parameters biotoksichnosti a large number of samples analyzed.

One of the first test systems for bioluminescent biotesting of water toxicity based on these principles is the Microtox manufactured by AZUR Environmental (USA) and incorporating lyophilized marine bacteria of the species Photobacterium phosphoreum NRRL B-111 77 (Vibrio fischeri NRRL B B -11177) [8]. In Russia, a similar test system is produced by the Institute of Biophysics of the SB RAS under the name “Microbiosensor B-17677f '' [9]. The biotesting procedure using them involves the introduction of NaCl water into the analyzed sample to a final concentration of 3%, as well as the creation of a control sample based on purified (distilled) water with a similar NaCl content. The necessity of introducing this salt is determined by the environmental characteristics of the microorganism Photobacterium phosphoreum, which naturally lives in sea water. At the next stage, in the experimental and control one sample made by the same amount of rehydrated lyophilized state biosensor was stirred and after 30 minutes (in the embodiment express 5 minutes) incubation at 22-24 ° C measured intensity of luminescence in the test (I _o) and control (I _k) cuvettes on one from the available bioluminometers .. According to the measurement results, the toxicity coefficients of the analyzed sample (7) are calculated, in percent, according to the formula T = ((I _k -I _o ) / I _k ) * 100%, where I _k is the luminous intensity in the control cell, I _o - the intensity of the glow in the experimental cell. At values T <20%, water is assessed as non-toxic, at T from 20 to 50% - as toxic, and at T> 50% - as highly toxic.

Another tool for studying biotoxicity can be recombinant luminescent microorganisms, in particular a recombinant strain of Escherichia coli K 12 TG1 with the cloned luxCDABE genes Photobacterium leiognathi, developed at Moscow State University MV Lomonosov under the commercial name "Ekolyum-9" [10]. Moreover, the biotesting procedure does not differ significantly from those described above, except that it does not provide for the addition of additional amounts of NaCl to the analyzed samples with the appropriate use of distilled water as a control [11].

By the totality of these features, the methods described above should be recognized as closest to the claimed. However, they are intended to determine the chemical toxicity of fresh drinking, surface, ground, sewage and treated wastewater, as well as precipitation, while attempts to include mineral (non-fresh) drinking water in this spectrum run up against significant obstacles. In this case, the main reason preventing the achievement of the desired result when using known methods is the pronounced effect on the bacterial bioluminescence of not only toxic, but also normal components of mineral waters — dissolved gases, cations [12, 13] and anions [14], which can imitate the effects of chemical toxicants and thereby distort biotest results.

Alternative approaches leading to the elimination of this drawback are not described in the literature available to us.

Thus, the claimed method is not known from the prior art, i.e. is new.

SUMMARY OF THE INVENTION

The main task to be solved by the claimed method is aimed at eliminating the influence of the normal component (gas and salt) composition of mineral waters on the results of their bioluminescent biotesting and the resulting increase in the accuracy of determining biotoxicity.

The essence of the proposed method, formulated at the level of functional generalization and underlying it, are the following provisions:

- The main factors present in the composition of mineral waters and capable of exerting influence on the luminescence level of bacterial luminescent biosensors that distort the results of biotoxicity studies are the presence of dissolved gases, mineral salts and high (alkaline) pH values determined by some of them.

- To eliminate the undesirable effect of these factors on the results of biotesting, it is proposed to conduct preliminary sample preparation of the test water by degassing it and adding NaCl and HCl in quantities depending on the initial mineralization and pH values determined in it.

- The use of the proposed sample preparation should be adequate to the environmental features of bacterial luminescent biosensors: the marine luminescent bacteria Photobacterium phosphoreum B-17677f or the recombinant luminescent bacteria Escherichia coli K12 TG1 with the genes of the luminescent system Photobacterium leiognathi.

Accordingly, when implementing the proposed method for determining the biotoxicity of drinking mineral waters using luminescent bacteria, the characteristics of the actions, the order of their implementation and the conditions for implementation in comparison with the prototype method are presented as follows (table 1).

After sampling the mineral water at the first stage of determining its biotoxicity, three sequential actions are carried out: degassing, differentiated mineralization and normalization of pH, which together excludes the influence of the normal component composition of the studied water on the result of its subsequent analysis using luminescent bacterial biosensors.

Moreover, according to claim 1, in the case of the use of the marine luminescent bacterium Photobacterium phosphoreum B-17677f, the following is carried out: 1) degassing of the test water by centrifugation at 6000 rpm for 5 min or settling in flat-bottomed vessels for 3-4 hours at vigorous stirring 300 rpm on a shaker; 2) determination of the level of total mineralization with subsequent addition of NaCl salt in quantities to the test sample, so that the total total mineralization after adding the values of the initial salt content and the introduced amounts of NaCl is equal to 30 g / l; 3) the introduction of 0.01 N. HCl solution until a final pH of 7.5 is reached.

In turn, according to claim 2, in the case of using the recombinant luminescent bacterium Escherichia coli K12 TG1 with the genes of the luminescent Photobacterium leiognathi system, the following is carried out: 1) degassing of the test water by centrifugation at 6000 rpm for 5 min or settling in flat-bottomed vessels in for 3-4 hours with vigorous stirring 300 rpm on a shaker; 2) the introduction of 0.01 N. HCl solution until a final pH of 7.5 is reached.

The content of the step is fundamentally different from the known biotesting method, which does not provide for any sample preparation of the studied waters by the parameters of the dissolved gases in them and the establishment of the normative pH level, as well as the fact that when implementing the known method, identical amounts of NaCl are introduced into all the analyzed samples, regardless of initial mineralization of the studied waters. As a result of the proposed sample preparation, conditions are created for detecting the presence of true chemical pollutants in the samples, the effect of which, when implementing the known method, can be masked or imitated by the action of normal components of mineral waters.

Table 1
Comparative characteristics of the actions, the order of their implementation and the conditions for implementation in determining the biotoxicity of mineral water using known and proposed methods Compare Features Known method The inventive method Stage I - sample preparation of the studied mineral waters in order to eliminate factors that could have a negative impact on the results of biotesting - + Degassing no Centrifugation at 6000 rpm for 5 min or settling in flat-bottomed vessels for 3-4 hours with vigorous stirring 300 rpm on a shaker Making additional
amounts of NaCl (in the case of using marine luminescent bacteria Photobacterium phosphoreum B-17677f) At a concentration of 30 g / l Differentiated
adding in quantities depending on the preliminary value of total mineralization, so that a final salt concentration of 30 g / l is composed of their initial presence and exogenously introduced NaCl Bringing the pH to
single regulatory value no The introduction of 0.01 N. Hcl solution to a final pH of 7.5 units. Stage II - the formation of the toxic effect of pollutants present in the sample in relation to the bacterial biosensor + + Used luminescent microorganism Photobacterium phosphoreum B-17677f or Escherichia coli K12 TG1 with genes
Photobacterium leiognathi fluorescent system Also Incubation time 5-30 min Also Incubation temperature 22-24 ° C Also Stage III - registration of the reaction result, calculation and assessment of the level of biotoxicity of the test sample + + Registration method Bioluminometry Also Calculation formula T = ((I _k -I _o ) / I _k ) * 100% Also Biotoxicity Criteria T <20% - non-toxic water; T = 20-50% - toxic water; T> 50% - water is very toxic Also

At the second stage of determining the biotoxicity of mineral waters, a series of three experimental and three control samples is formed in a volume of 900-950 μl, the last of which is a 3% solution of NaCl in distilled water (in the case of biotesting using marine luminescent bacteria Photobacterium phosphoreum B-17677f ) or distilled water itself (in the case of biotesting using the recombinant luminescent bacteria Escherichia coli K12 TG1 with the genes of the luminescent Photoobacterium leiognathi system). Identical amounts (50-100 μl) of one of the named bacterial luminescent biosensors are added to these samples. Samples are incubated for 30 minutes (in express form - 5 minutes) at 22-24 ° C.

The content of the stage — the formation of the toxic effect of the pollutants present in the sample with respect to the bacterial biosensor, which manifests itself in a decrease in its bioluminescence level — does not fundamentally differ from that in the known method.

At the third stage, the cuvettes are placed in the measuring cell of the luminometer and the level of luminescence of the bacterial suspension is measured in the visible blue-green region of the spectrum 420-600 nm. BLM 8802 M (SKTB Nauka, Krasnoyarsk), Biotoks-10 (Nera LLC, Moscow), as well as other domestic and foreign luminometers operating in the indicated spectral range can be used as a measuring device when implementing the proposed method and providing appropriate measurement discreteness. According to the measurement results, the average values of the glow intensity in the experimental (I _о ) and control (I _k ) samples are calculated, and then the relative toxicity coefficient (T) of the analyzed sample is calculated by the formula T = ((I _k -I _o ) / I _k ) * one hundred%. At values T <20%, water is assessed as non-toxic, at T from 20 to 50% - as toxic, and at T> 50% - as highly toxic.

The content of the stage — recording the result of the reaction and calculating the level of biotoxicity of the test sample — also has no significant differences from that in the known method.

The ability to obtain a technical result when performing the above actions in the indicated ranges of values is determined by the following complex of cause-effect relationships:

1) Testing of 22 drinking mineral bottled waters represented by the world (Aqua Minerale (USA), Apollinaris (Germany), Bon Aqua (USA), Evian (France), Donat (Slovenia), Perrier (France), San Pellegrino (Italy), Vichy Celestins (France) and Russian brands (Arkhyz, Essentuki-2, Essentuki-4, Essentuki-17, Narzan, etc.) using the prototype method showed that 100% of them are characterized by toxicity indices T> 50% (table 2 ), which, in accordance with existing standards, made them assess as highly toxic. At the same time, the study of these waters for the presence of the most significant of chemical pollutants (in accordance with SanPiN 2.1.4.1116-02) testified to their complete harmlessness to human health.Thus, the obtained data showed the inapplicability of the prototype methods for the stated purposes, due to the effects of normal components of the studied mineral waters.

2) The degassing of the studied mineral waters allowed reducing the determined values of their toxicity to values T <20% in 6-8 and to values T = 20-50% in 10 out of 22 studied mineral waters (table 2). The obtained results indicate the importance of removing CO ₂ from the analyzed water, which is in the dissolved state in the form of the compound H ₂ CO ₃ and is capable of exerting a direct bactericidal effect on the bacterial luminescent biosensors used, as well as acting as a competitor to molecular oxygen in the biochemical reaction leading to the appearance of a glow .

3) A study of the reasons for the suppression of the bioluminescence of bacterial biosensors used by degassed mineral waters made it possible to ascertain the development of a similar effect mainly in the group of medium mineralized (1-10 g / l) and highly mineralized (10-15 g / l) mineral waters. At the same time, the study of the effect of the main cations present in these waters in the form of chlorides and sulfates allowed us to form the series of increased biotoxicity of Ca ²⁺ > Na ⁺ > Mg ²⁺ > K ⁺ . Accordingly, the presence of significant named cations in the composition of the studied mineral waters, especially against the background of undifferentiated (independent of the initial level of total mineralization) addition of additional amounts of NaCl, can lead to suppression of bioluminescence imitating the effect of chemical pollutants.

The elimination of this factor is possible during biotesting using the marine luminescent bacterium Photobacterium phosphoreum B-17677f, which involves adding additional NaCl to the test sample at a rate of 30 g / l. In this case, a preliminary determination of the level of initial mineralization of the studied waters and subsequent differentiated introduction of NaCl into the analyzed samples in amounts equal to the difference between the final value of 30 g / l and a certain value of the total mineralization allowed reducing the determined toxicity values to T <20% in 15 of 22 of the studied mineral waters and up to the values of T = 20-50% in 7 of the 22 studied mineral waters (table 2). However, the positive effects of such a procedure were formed mainly in the group of low-saline and medium-saline water, not significantly affecting the results of bio-testing of highly saline water.

4) The final analysis of the reasons for suppressing the bioluminescence of bacterial biosensors with highly mineralized waters after their degassing and differentiated (depending on the initial level of "mineralization) introduction of additional amounts of NaCl allowed us to state that the preservation of such effects is determined by the presence of a significant amount of dissolved salts in the form of hydrocarbons (НСО ₃ ^- ), shifting the pH level of the analyzed water in the zone of high (alkaline) values.

In order to eliminate such an obstacle, the pH values of the studied mineral waters were normalized by the controlled addition of 0.01 n to them. Hcl solution with a stepwise increase in the recorded pH by 0.5 units.

The implementation of this approach, applicable both when using the marine luminescent bacterium Photobacterium phosphoreum B-17677f, and the recombinant luminescent bacterium Escherichia coli K12 TG1 with the genes of the luminescent system Photobacterium leiognathi, allowed us to state that the optimum pH value, upon reaching which the level is negative, the level of bioluminescence of bacterial biosensors, is a pH value of 7.5. Moreover, the values of toxicity indices T <20% were achieved in 20 of the 22 studied waters (table 2). Conducting the complete sample preparation procedure “degassing → differentiated mineralization → normalization of pH” available during biotesting using the marine luminescent bacteria Photobacterium phosphoreum B-17677f, all 22 of the 22 studied mineral waters were evaluated as non-toxic, which led the bioluminescent biotesting data to be consistent with the results of their chemical analysis (in accordance with SanPiN 2.1.4.1116-02).

5) The claimed sequence of actions "degassing → differentiated mineralization → normalization of pH" is determined by the following considerations. Firstly, the removal of dissolved gases, the leading one of which is CO ₂ , allows you to transfer liquids to the normal (non-foamed) state necessary for all of the following actions and measurements. Secondly, the removal of CO ₂ , which is in the dissolved state in the form of the compound H ₂ CO ₃ , is a necessary condition for studying the pH, in the determination of the value of which this weak acid may introduce an error. Thirdly, the introduction of additional amounts of NaCl may follow degassing, but should precede the normalization of pH, since the introduction of even neutral compounds can have the so-called “Salt effect”, which consists in changing the degree of dissociation of individual cations and anions.

6) It is fundamentally important that the sample preparation described above, aimed at eliminating the non-specific inhibitory effects of the normal component composition of mineral waters on luminescent biosensors, does not simultaneously affect the sensitivity of the latter to the effects of true toxicants.

This statement is supported by the test results of samples of highly mineralized water Essentuki-17, in which one of the model toxicants was preliminarily introduced - the organic compound benzene, which is one of the priority pollutants in the aquatic environment, which belongs to the 2nd hazard class and in this regard is most often used in assessing the sensitivity of various biotesting methods. In a series of experiments, the final concentrations of benzene in the analyzed samples varied from 0.5 to 5 g / L. Subsequently, these samples were investigated in accordance with the prototype method and the claimed method for compliance with the regulatory values of the biotoxicity EC ₂₀ and EC _{50 of the} used toxicant defined in the control, corresponding to its concentrations, which determine 20% and 50% quenching of bacterial bioluminescence.

The EC ₂₀ and EC ₅₀ parameters determined for benzene were 3.10 ± 0.32 and 5.20 ± 0.54 g / L, respectively, which approximately corresponded to the values previously determined for one of the biosensors used [11]. At the same time, when conducting similar studies in the mineral water of Essentuki-17 using the known method, the named calculation parameters turned out to be much lower, which was explained by quenching of the luminescence of bacterial biosensors not only as a result of exposure to pollutant, but also due to the co-directed effect of normal components of this mineral water. In turn, the conduct of sample preparation in accordance with the claimed method resulted in an increase in the values of the determined parameters, ceasing to significantly differ from the standard values.

In general, summarizing the above materials about the essence of the proposed method, the characteristics of the actions, the order of their implementation and the conditions for implementation, we can state that the claimed method does not follow from the prior art and should be evaluated as meeting the criterion of "inventive step".

table 2
The values of the toxicity indexes during the biotesting of mineral waters using the marine luminescent bacterium Photobacterium phosphoreum B-17677f and the recombinant luminescent bacterium Escherichia coli K12 TG1 with the genes of the luminescent system Photobacterium leiognathi in various sample preparation options Explored Mineral Waters Toxicity Index Values when using P. phosphoreum B-17 677f " when using E. coli K.12 TG1 with genes of the luminescent system of P. leiognathi prototype method after degassing after degassing and rationing of mineralization after degassing and normalizing pH≤7.5 the claimed method according to claim 1 of the claims prototype method after degassing the claimed method according to claim 2 of the claims Aqua minerale 67.1 ± 0.29 ** 5.89 ± 0.07 1.6 ± 0.05 <0 <0 98.3 ± 0.01 ** <0 <0 Apollinaris 79.8 ± 0.07 ** 29.4 ± 0.06 * <0 17.8 ± 0.06 <0 93.0 ± 0.01 ** 83.5 ± 0.03 ** 1.7 ± 0.11 Bonaqua 66.1 ± 0.30 ** <0 0.8 ± 0.04 18.1 ± 0.30 <0 98.4 ± 0.01 ** <0 <0 Donat 72.9 ± 0.15 ** 72.9 ± 0.03 ** 31.0 ± 0.04 * 24.5 ± 0.02 * 13.4 ± 0.07 87.0 ± 0.01 ** 95.7 ± 0.01 ** 14.8 ± 0.12 Evian 73.1 ± 0.03 ** 43.4 ± 0.21 * <0 17.3 ± 0.29 <0 64.3 ± 0.03 ** <0 <0 Perrier 75.1 ± 0.07 ** 17.3 ± 0.08 <0 5.2 ± 0.06 <0 98.3 ± 0.01 ** 38.3 ± 0.11 * 14.9 ± 0.13 San pelgrino 89.6 ± 0.02 ** <0 44.9 ± 0.03 * <0 1.7 ± 0.03 88.7 ± 0.02 ** 22.6 ± 0.05 * <0 Vichy celestina 86.1 ± 0.01 ** 33.3 ± 0.01 * <0 18.1 ± 0.01 <0 81.7 ± 0.02 ** 71.3 ± 0.09 ** 13.3 ± 0.02 Arkhyz 93.6 ± 0.02 ** 26.7 ± 0.11 * 2.0 ± 0.03 18.2 ± 0.01 5.6 ± 0.02 91.3 ± 0.02 ** 44.3 ± 0.14 * 15.4 ± 0.03 Volzhanka 79.8 ± 0.01 ** <0 25.3 ± 0.01 * <0 0.8 ± 0.02 86.1 ± 0.02 ** 17.3 ± 0.02 8.2 ± 0.01 Essentuki-2 83.5 ± 0.14 ** 55.8 ± 0.07 ** 17.9 ± 0.02 <0 0.7 ± 0.04 58.3 ± 0.02 ** 61.3 ± 0.48 ** <0 Essentuki-4 92.2 ± 0.03 ** 62.5 ± 0.06 ** 26.0 ± 0.06 * 17.5 ± 0.01 11.1 ± 0.02 68.7 ± 0.01 ** 68.4 ± 0.07 ** <0 Essentuki-17 83.5 ± 0.07 ** 76.1 ± 0.05 ** 35.6 ± 0.08 * 20.9 ± 0.02 * 11.6 ± 0.01 74.8 ± 0.01 ** 86.9 ± 0.05 ** 13.3 ± 0.10 Living water 76.1 ± 0.21 ** 21.5 ± 0.12 * 19.8 ± 0.11 18.2 ± 0.11 18.6 ± 0.01 96.5 ± 0.01 ** 24.5 ± 0.13 * 8.1 ± 0.13 Krasnoglinskaya 88.7 ± 0.04 ** <0 <0 <0 <0 80.9 ± 0.01 ** 31.3 ± 0.02 * 31.3 ± 0.06 * Menzelinskaya 85.2 ± 0.07 ** 28.7 ± 0.05 * <0 18.1 ± 0.02 <0 86.2 ± 0.02 ** 18.1 ± 0.29 12.1 ± 0.04 Narzan 68.7 ± 0.28 ** 24.3 ± 0.07 * 17.6 ± 0.06 <0 <0 71.3 ± 0.02 ** 18.2 ± 0.16 <0 Rameno 60.9 ± 0.36 ** 18.2 ± 0.09 8.1 ± 0.07 8.1 ± 0.10 13.6 ± 0.01 94.8 ± 0.01 ** 24.2 ± 0.14 * 13.8 ± 0.29 Holy spring 76.1 ± 0.21 ** 20.2 ± 0.07 19.1 ± 0.08 0.8 ± 0.03 <0 86.1 ± 0.02 ** 25.5 ± 0.19 * 13.1 ± 0.06 Slavyanovskaya 91.3 ± 0.02 ** 24.4 ± 0.17 * 20.9 ± 0.02 * 1.2 ± 0.01 1.9 ± 0.02 80.8 ± 0.03 ** 44.9 ± 0.07 * 25.5 ± 0.02 * Sulak 66.1 ± 0.20 ** 21.4 ± 0.07 * 17.3 ± 0.07 0.9 ± 0.08 1.7 ± 0.01 78.9 ± 0.04 ** 31.3 ± 0.08 * <0 Yaik 58.3 ± 0.20 ** 31.7 ± 0.11 * 20.9 ± 0.06 * 0.9 ± 0.02 1.5 ± 0.01 73.9 ± 0.02 ** 24.3 ± 0.06 * 1.7 ± 0.01 Note ** - water is rated as highly toxic; * - water is assessed as toxic, without designations - water is assessed as non-toxic

Information confirming the possibility of carrying out the invention

To determine the biotoxicity of drinking mineral water by bioluminescent biotesting, a sample of 100-200 ml is taken in a sterile glass container.

In the future, the sequence of actions during the implementation of the proposed method, their conditions and modes are as follows:

1) to achieve degassing, the test water is centrifuged at 6000 rpm for 5 minutes or settled in flat-bottomed vessels for 3-4 hours with vigorous stirring at 300 rpm on a shaker;

2) when performing the method according to claim 1 in the part of the sample with a volume of 100 ml, the level of total mineralization (OM) is determined by evaporating it and weighing the dry residue on the balance, allowing to estimate the value of the latter with an accuracy of ± 0.001 g;

3) when performing the method according to claim 1 of the claims, a weighed portion of the sodium chloride category of grade H. in the amount of X g from the calculation of X = 30 - OM and mix thoroughly until it is completely dissolved, so that the final level of mineralization of the sample is 30 g / l;

4) measure the pH in the sample using a pH meter that allows you to evaluate the values of this indicator with an accuracy of 0.1 units;

5) 0.01 n is added dropwise into the sample. HCl hydrochloric acid solution, dynamically measuring the dynamics of pH shift; after reaching a pH of 7.5 units the introduction of acid is stopped;

6) form a series of three experimental and three control samples: a 3% solution of NaCl in chemically pure distilled water in a volume of 950 μl (in the case of research according to claim 1) or chemically pure distilled water in a volume of 900 μl (during research according to claim 2);

7) when performing the method in accordance with claim 1 of the invention, the vial of a cooled lyophilized culture of marine luminescent bacteria Photobacterium phosphoreum B-17677f is reactivated by adding 2 ml of a 1.5% NaCl solution. The resulting suspension of bacteria is shaken several times until a homogeneous solution, and then kept for 30 minutes in a refrigerator at 2-4 ° C to stabilize the level of luminescence, bring the temperature of the suspension of bacteria to room 22-24 ° C. When performing the method according to claim 2, reactivation of the lyophilized recombinant strain of Escherichia coli K 12 TG1 with the cloned luxCDABE genes of P. leiognathi is carried out by adding 10 ml of distilled water. The resulting suspension of bacteria is shaken several times until a homogeneous solution, and then incubated for 30 minutes in a refrigerator at 2-4 ° C to stabilize the level of glow, bring the temperature of the suspension of bacteria to room 22-24 ° C;

8) identical amounts of one of the aforementioned luminescent bacteria are introduced into experimental and control samples: in a volume of 50 μl when performing the method in accordance with claim 1 and in a volume of 100 μl when performing the method in accordance with claim 2;

9) cuvettes with experimental and control samples are kept under identical conditions for 30 minutes (in the express version - 5 minutes) at 22-24 ° C;

10) the cuvettes are sequentially placed in the measuring cell of the bioluminometer and the level of luminescence is measured in the visible blue-green region of the spectrum at 420-600 nm for 30-60 seconds, the values of the luminous intensity in three experimental (I _o ) and three control (I _k ) samples average;

11) calculate the biotoxicity T of the studied drinking mineral water according to the formula T = ((I _k -I _o ) / I _k ) * 100%;

12) at values T <20%, water is assessed as non-toxic, at T from 20 to 50% - as toxic, and at T> 50% - as highly toxic.

Example

An enterprise producing bottled drinking water has developed a new mineral water, the Ural Springs, which is bottled in consumer containers and intended for use as treatment and table water. This water meets the requirements of technical conditions and is produced according to the current technological instructions in compliance with the "Sanitary Rules for the Processing and bottling of drinking mineral waters." According to organoleptic, microbiological indicators, permanganate oxidation, the content of toxic elements and radionucleotides, Ural Springs medicinal table water meets the requirements of GOST 13273-88 and SanPiN 2.3.560 "Hygienic requirements for the quality and safety of food raw materials and food products".

To study the medicinal-table water "Ural springs" by the method of bioluminescent analysis by a known method, several bottles of mineral water were selected from different lots. The studies were carried out according to existing methods using the marine luminescent bacterium Photobacterium phosphoreum B-17677f (produced by the IBSO RAS under the commercial name Microbiosensor B-17677F ') and the recombinant luminescent bacterium Escherichia coli K12 TG1 with the genes of the luminescent system Photobacterium M. leiogn. V. Lomonosov under the commercial name "Ekolyum-9"). When performing this study, 950 μl of mineral water was taken, previously salted at the rate of 300 mg of NaCl per 10 ml of mineral water (using Microbioseno B-17677f) or 900 μl of mineral water (when using Ekolyum-9) and added to measuring cuvettes, 50 or 100 μl of a suspension of luminescent bacteria Microbiosensor B-17677f and Ekolyum-9, respectively, were added thereto. the cuvettes were placed 950 μl of a 3% NaCl solution using the Microbiosensor B-17677f or 900 μl of distilled water using Ecolyum-9 and 50 or 100 μl of a suspension of the same luminescent bacteria. After a 30-minute incubation at 22-24 ° C, the sample cuvettes were placed in a bioluminometer measuring the biological glow of the BLM 8802 bioluminometer (SKTB Nauka, Krasnoyarsk) and the luminescence level was measured in the control and experiment. The toxicity index (T) was calculated by the formula T = ((I _k -I _o ) / I _k ) * 100%, where I _k is the intensity of the glow in the control cell, I _about is the intensity of the glow in the experimental cell.

The results obtained using the known method showed that this mineral water is characterized by toxicity indices equal to 89% and 92% (T> 50%), which, in accordance with existing standards, made it evaluate as highly toxic. However, the study of this water for the presence of the most significant chemical pollutants testified to their absence or presence in quantities significantly lower than the established MPC. Thus, when conducting research using the known method, an erroneous result was obtained that contradicts the results of other studies.

When determining the biotoxicity of the studied mineral water in accordance with the claimed method, its preliminary three-stage sample preparation was carried out. The test water was poured into 10 ml tubes, centrifuged at 6000 rpm for 5 min or by settling in flat-bottomed vessels for 3-4 hours with vigorous stirring at 300 rpm on a shaker, after which the degassed samples were combined. In a biotoxicity study using the B-17677f Microbiosensor, 100 ml of the combined degassed sample was evaporated, after which the weight of the dry residue was found to be 732 mg (i.e., the total salinity of the test water was 7.32 g / l). Based on the data of calculations in other 100 ml, taken from the combined degassed sample, contributed 2268 mg of NaCl salt, so the final mineralization level thus established was 30 g / L. Further, in this degassed and differentiated saline (when using Microbiosene ora B-17677f "), as well as only degassed (using" Ekolyum-9 ") samples, we measured pH using an Expert-001 fluid analyzer (Econix-Expert LLC, Moscow), which turned out to be equal to 8.7 units and 8.8 units After that, a 0.01 N hydrochloric acid HCl solution was dropwise added to both samples, dynamically measuring the pH shift dynamics until a pH of 7.5 was reached. Subsequently, samples prepared in this way were introduced into measuring cuvettes in volumes of 950 μl (degassed, differentially salted, and normalized by pH water) or 900 μl (d non-carbonated and pH-normalized water), after which 50 or 100 μl of a suspension of luminescent bacteria Microbiosensor B-17677f and Ekolyum-9, respectively, were added thereto. 950 μl of a 3% NaCl solution was placed in the control measuring cuvettes using the Microbiosensor B-17677f or 900 μl of distilled water using the Ecolum-9 and 50 or 100 μl of the suspension of the same luminescent bacteria. After a 30-minute incubation at 22- At 24 ° C, the sample cuvettes were placed in a device that recorded biological glow — a BLM 8802 bioluminometer (SKTB Nauka, Krasnoyarsk), the luminescence level was measured in the control and experiment, after which the toxicity index (7) was calculated using the above formula.

The claimed sequence of actions "degassing → differentiated mineralization → normalization of pH" (in the case of using "Microbiosensor B-17677f") and "degassing → normalization of pH" (in the case of using "Ekolyum-9") made it possible to exclude the action of non-specific inhibitory effects of the normal component composition of the mineral water to luminescent biosensors, which resulted in a decrease in certain toxicity values to 7.6% and 9.2% (T <20), indicating its non-toxicity and consistent with other research data SanPiN 2.3.560.

Information sources

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Claims (2)

1. A method for determining the biotoxicity of drinking mineral water, which involves sampling, its degassing, determining the level of total mineralization, introducing NaCl into the test sample to a final value of total mineralization of 30 g / l, followed by 0.01 n. Hcl solution until reaching the final pH value of 7.5, preparing a control sample, adding Photobacterium phosphoreum B-17 677f to the test and control samples of the marine luminescent bacterium, measuring the luminescence level, calculating the toxicity index by the formula T = ((I _k -I _o ) / I _k ) · 100%, where I _k is the luminescence intensity in the control, I _o is the luminescence intensity in the experiment, while at values of T <20% water is assessed as non-toxic, at T from 20 to 50% as toxic, at T > 50% as highly toxic. 2. A method for determining the biotoxicity of drinking mineral water, which involves sampling, its degassing, making 0.01 N. Hcl solution until a final pH of 7.5 is reached, preparing a control sample, introducing a recombinant luminescent bacterium Escherichia coli K 12 TG1 into the test and control samples with the genes of the luminescent system Photobacterium leiognathi, measuring the luminescence level, calculating the toxicity index by the formula T = ((I _k -I _o ) / I _k ) · 100%, where I _k is the luminous intensity in the control, I _o is the luminous intensity in the experiment, while at T <20% the water is evaluated as non-toxic, at T from 20 to 50% - as toxic, at T> 50% - as highly toxic.