RU2110067C1 - Biological method for determining degree of total toxicity and principal toxicants in aqueous media (versions) - Google Patents

Abstract

FIELD: environmental monitoring. SUBSTANCE: method can be utilized in environmental protection service and when monitoring aqueous media with biotesting technique, in particular, when estimating toxicity of water, soils, and bottom sediments polluted by various substances, their contents exceeding maximum permissible concentrations with wide range of exceeding ratios. As test organisms, laboratory culture species are single-time utilized grown by way of closely-related crossing of aquatic organisms under constant maintenance conditions on artificial pure standard water and feed. Detected and compared to each other are behavior reaction spectra of test organisms and their locomotion activity occurring in a series of model toxicant solutions in pure standard water and test aqueous medium. In the other embodiment of method, additionally determined is percentage of test organisms able to find feed within specified period of time. From the comparison data, principal toxicants and their concentrations in test medium are established, and toxicity degree of medium is determined by way of comparing specified concentrations of toxicants with maximum permissible concentration rates for test medium. EFFECT: extended toxicant testing possibilities. 2 cl, 14 tbl

Description

 The invention relates to the field of environmental protection, and in particular to methods of environmental monitoring of aquatic environments for various purposes using biotesting. It is intended to assess the quality of waters contaminated with industrial, agricultural and domestic toxicants and their mixtures, including water extracts of soils and bottom sediments, as well as to establish a non-toxic degree of dilution of the test water environment and for environmental mapping and to identify adverse sections of water bodies and places suitable for water intakes, monitoring of sewage, ground and surface waters, soils and bottom sediments, as well as to establish the degree of toxicity of unknown toxicants and their mixtures.

 The current level of technology is characterized by the following analogues of the proposed method. A large number of methods for biotesting aquatic environments have been developed, using various hydrobionotes as test organisms belonging to animal groups such as protozoa (ciliates, flagella), intestinal (hydra), worms (planaria, leeches), crustaceans (daphnia, gammarus), mollusks (plate-gill, gastropods), fish. According to the literature, the most common methods of biotesting waters, where daphnia is used as test organisms. Apparently, this is due to the fact that, compared with biotesting methods using leeches and fish, methods using daphnia are more sensitive to the main classes of toxicants.

 A biological method for determining the toxicity of water is known, according to which increasing volumes of the test solution are placed in a series of test vessels and water is added for dilution in such a way as to obtain a test solution of a given concentration in each vessel [1]. A predetermined number of Daphnia magna is placed in each vessel. A control vessel is prepared for each test series in which the volume of water for dilution is equal to the volume of the test solutions, the same number of Daphnia magna as in the test solutions. The tests are carried out at a given ambient temperature free of vapors and dust, toxic to Daphnia magna. For testing, animals of at least the third generation obtained during acyclic parthenogenesis under specific breeding conditions of a given size and age are used. The tests are carried out in two stages. At the end of a given test period, the number of mobile Daphnia magna in each vessel is determined. Those animals that cannot swim for 15 seconds after weak mixing of the liquid are considered immobile even if they can still move their antennas. The limit of permissible concentrations, giving immobilization from 0 to 100%, is determined, and any anomalies in the behavior of Daphnia magna are recorded.

 The advantage of the known method lies in the fact that it is an express method, i.e. Assessment of water toxicity is carried out within 24 or 48 hours. The advantages of the method include the fact that for testing they take specially derived third and further generations of animals, in contrast to methods when animals are taken directly from the natural environment. The reliability and accuracy of testing a water sample on specially bred "environmentally friendly" animals is higher than on animals taken from different natural reservoirs and already adapted to the various toxicants available in them. Individuals of Daphnia magna themselves are hypersensitive to a number of major classes of toxicants, which is also an advantage of the method.

 The disadvantage of this method is the fact that individuals of Daphnia magna do not have sufficient resistance to the action of toxicants, which leads to the fact that they essentially assess the toxicity of water indirectly, diluting the test water with previously known toxic-free water, setting different concentrations of the test water in different vessels , and judge the toxicity of water by the behavior of Daphnia magna in dilute water. This reduces the accuracy of the method, and also complicates the entire biotesting method as a whole. In addition, although Daphnia magna was specifically introduced for biotesting, each time it was used for breeding Daphnia magna taken from different water bodies, which reduces the reproducibility of the test results, i.e. also reduces the accuracy of the method. In addition, using this method, the toxicity of water samples is determined only with respect to the species Daphnia magna and only by the degree of animal mobility in these samples, without tying the results of the assessment to the standards applicable to the tested water. This method is not unified in the sense that it does not make it possible to use it to assess the toxicity of waters in accordance with current standards for toxicological indicators for waters for different purposes.

 The closest in essence is a method for determining the toxicity of aqueous media [2]. According to this method, in particular, the locomotor activity of the test organisms of the aquatic organisms Lymnaea lagotis, Lymnaea auricularia, Coretus corneus and Viviparus viviparus in the test aqueous medium is evaluated, physiological parameters characterizing the functional state of the test organisms are determined, these parameters are compared with similar parameters of their functional state obtained by testing aqueous solutions with given concentrations of a model toxicant, the results of the comparison show the toxicity of the tested aqueous medium.

 The disadvantages of this method include the fact that, firstly, test organisms are taken from different natural reservoirs each time, which reduces the accuracy and reproducibility of the results of water testing, because animals turn out to be adapted to different living conditions and pollutants, including the model toxicant used. The latter leads to the fact that their resistance and sensitivity to toxicants vary greatly. This creates significant difficulties in interpreting the results of biotesting, thus reducing the accuracy of the method itself, which does not allow us to consider it unified. Secondly, as test organisms, species of aquatic organisms that are obviously different due to their genetic and phylogenetic characteristics in terms of sensitivity and resistance to toxicants are used, which also reduces the accuracy of the method and complicates the interpretation of the results.

 Another disadvantage of the known method is that they do not conduct a direct assessment of the toxicity of the tested aqueous medium, but in its various dilutions, especially since there are no characteristics of the water used for dilution, i.e. it alone may have varying degrees of toxicity. Thus, this method does not take into account the fact that when diluting the test aqueous medium, non-accounting interaction of the test aqueous medium and diluent may occur. This can change the toxicity of the actually tested aqueous medium compared to the aqueous medium taken for testing, which can also lead to a decrease in the accuracy of the method. To reduce the accuracy of the method and the illegality of using it as a unified one, it also leads to the fact that the determination of the general toxicity of the tested aqueous medium is carried out only by comparing with the effect on the test organisms of only one model toxicant (copper sulfate), and is used as physiological parameters for comparison the functional state of test organisms is only their locomotor activity and mortality.

 The problem to which the invention is directed, is that the biotesting method has increased sensitivity and accuracy, allowing the method to be used as a unified and universal biotest for general toxicity and major toxicants of aqueous media. By the term "unified biotest" is meant here that it must possess a combination of such properties that its use would be sufficient to determine the main toxicants and the general toxicity of the tested aqueous medium, taking into account the synergism and antagonism of all the substances that cause this toxicity. The term "universal biotest" here means the possibility of a fairly widespread use of the method for the toxicological assessment of various cultural, domestic and drinking water contaminated with various (domestic, industrial, agricultural and other, possibly unknown) toxicants in a wide range of concentrations, and also water extracts of soils and bottom sediments.

 The technical result achieved by using the invention is that, firstly, the determination of the main toxicants and general toxicity of aqueous media for various purposes by this method is quite accurate, the method is highly reproducible, and secondly, the method has increased sensitivity and a wide measuring range that allows you to determine the various major toxicants present in the tested aqueous medium, in a wide range of excess MPCt (up to Ustimov concentrations of toxicants). In addition, the comparison of the obtained data with the standards applicable to the test environment for toxicological indicators allows the "transfer" of the obtained data from the test organisms used in the method to those organisms, including humans, for which the standards used for comparison have been developed. In other words, the proposed method allows to determine the main toxicants and the general toxicity of aqueous media, regardless of the test organisms used in the proposed method, for higher animals and humans, provided that these animals and humans have standards for defined toxicants or MPCT for the tested aqueous media.

 The task is achieved in the first embodiment of the method in that, as in the known method, a comparison is made of the physiological parameters of the functional state of the test organisms of aquatic organisms in the test sample of the aquatic environment and solutions of a model toxicant and the degree of toxicity of water is determined from the comparison results, but in contrast to the known method, grown on artificial pure reference water and feed with a constant regime of maintenance laboratory culture of test organisms by closely related cross Bani individuals of the same species of aquatic organisms, whose natural habitat is the interval corresponds to the interval salinity water salinity test medium. In the case of testing sea water, instead of pre-growing the laboratory culture of test organisms, it is allowed to obtain a laboratory culture of test organisms by preliminary long-term maintenance of marine hydrobionts of the same species, taken from one ecologically clean area of the sea, on artificial clean reference water and feed at a constant mode laboratory conditions. Then, locomotor activity and the spectrum of behavioral reactions of test organisms as a constant set of values, each of which corresponds to a degree, are initially determined in arbitrary units in the same time interval in vessels smaller in volume than the vessels for holding the laboratory culture of test organisms manifestations of a specific behavioral reaction of the test organism in arbitrary units, for each test organism of the first part of the grown laboratory culture of test organisms in a set differing in concentration series of solutions of each of the model toxicants corresponding to possible toxicants of the test aqueous medium and in fresh artificial pure reference water as in non-toxic control water of the same temperature at which the laboratory culture of test organisms is contained, and all solutions of model toxicants are prepared on a fresh artificial pure reference water, each test organism is placed only once in a model toxicant solution, and the determination is carried out after the protective organisms have disappeared from the test organisms Second reaction process at transferring from one vessel to another vessel. After that, the test organisms of the first part of the laboratory culture of the test organisms are kept in a set of different concentration series of solutions of each of the model toxicants and fresh artificial pure reference water in vessels of a smaller volume than the vessels for maintaining the laboratory culture of the test organisms for the same enough time for physiological adaptation to new aquatic environments and the vessel, with the regime of the content of the laboratory culture of test organisms, and each test organism can withstand exactly that a solution of a model toxicant and fresh artificial pure reference water, in which its locomotor activity and the spectrum of behavioral reactions were primarily determined.

Then, the locomotor activity and the spectrum of behavioral reactions are secondly determined for each test organism of the first part of the laboratory culture of the test organisms in fresh artificial pure reference water at a temperature and in vessels identical to the temperature and vessels used for the initial determination of locomotor activity and the spectrum of behavioral reactions for each test organism of the first part of the laboratory culture of test organisms, during the time interval and in arbitrary units that were used for the initial determination of locomotor activity and the spectrum of behavioral reactions for each test organism of the first part of the laboratory culture of the test organism, and the determination is made after the protective organisms react to the process of transfer from one vessel to another vessel after the test organisms disappear. After that, for each test organism of the first part of the laboratory culture of test organisms, normalized primary and secondary defined spectra of behavioral reactions are calculated by dividing the values of each behavioral response both of the primary and secondary spectra of behavioral responses for each test organism by the value of the following expression:
k ₁ • (k ₂ + LA)
where k ₁ and k ₂ are experimentally determined coefficients, the value of which depends on the type of aquatic organisms used to grow the laboratory culture of test organisms, on a set of behavioral reactions of the determined spectra of behavioral reactions of test organisms, and on arbitrary units in which values of behavioral reactions are measured , and LA is the previously determined locomotor activity of the corresponding test organism.

 Then, average values and standard errors of average values are calculated separately for normalized primary and secondary defined spectra of behavioral reactions, characterizing the functional state of test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of each of the toxic toxicants and a fresh, artificial, pure reference water, by calculating the mean values and their standard errors for each behavioral reaction of the indicated normalized spectra n vedencheskih reactions.

 Then, locomotor activity and the spectrum of behavioral reactions for each test organism of the second part of the laboratory culture of test organisms in the test sample of the aqueous medium are initially determined at a temperature and in vessels identical to the temperature and vessels used to determine the locomotor activity and the spectrum of behavioral reactions for each test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of solution series differing in concentration of model toxicants during the time interval and in arbitrary units that were used for locomotor activity and the behavioral spectrum of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of different series of solutions of model toxicants differing in concentration, each test organism used for testing only once and the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the transfer process from one suck and in another vessel. After that, the test organism of the second part of the laboratory culture of the test organisms is maintained in the test aqueous medium for a time interval and in vessels identical to the time interval and vessels used to withstand the test organisms of the first part of the first part of the laboratory culture of test organisms in fresh artificial clean reference water and a set of different series of solutions of model toxicants differing in concentration in the mode of laboratory culture of test organisms.

 Then, the locomotor activity and the spectrum of behavioral reactions of each test organism of the second part of the laboratory culture of the test organisms in fresh artificial pure reference water are determined for the time interval and in the vessels identical to the time interval and the vessels used to determine the locomotor activity and the spectrum of behavioral reactions of each test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of series differing in concentration solutions of model toxicants, during the time interval and in arbitrary units that were used to determine the locomotor activity and the spectrum of behavioral reactions of each test organism of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of different concentration of series of model toxicant solutions moreover, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the transfer process from one vessel to another vessel.

 After that, for each test organism of the second part of the laboratory culture of the test organisms, the normalized primary and secondarily determined spectra of behavioral reactions are calculated similarly to the calculation of the normalized primary and secondarily determined spectra of behavioral reactions of the test organisms of the first part of the laboratory culture of the test organism, and average values and standard values are calculated errors of mean values separately for normalized primary and secondary defined spectra of behavioral reactions characterizing functionally state test organisms second part of the laboratory culture of test organisms in the tested aquatic environment. After that, the corresponding average values of the normalized primary and secondarily determined spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms are compared with the calculated average values of the spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms, and choose from a set of model toxicants and fresh artificial pure water, like non-toxic control water, those toxicants for which the average values of all behavioral reactions are ized spectra behavioral test organisms, the first part of the laboratory culture of test organisms was not significantly different from the average values of the corresponding behavioral responses normalized spectra behavioral test organisms second part of the laboratory culture of the test organisms. Then, for each selected toxicant, its average concentration is found by averaging all its concentrations selected as a result of the comparison, and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant, and the degree of total toxicity of the tested aqueous medium is determined by comparing certain the main toxicants of the tested aquatic environment with toxicological standards applicable to the tested aquatic environment and the degree of exceeding the maximum permissible concentrations.

 The task is achieved in the second variant of the method in that, as in the known method, a comparison is made of the physiological parameters of the functional state of the test organisms of aquatic organisms in the test sample of the aquatic environment and solutions of the model toxicant and the degree of toxicity of water is determined from the comparison results, but in contrast to the known method, grown on artificial pure reference water and feed with a constant mode of maintenance, the laboratory culture of test organisms by closely related to reschivaniya same species of aquatic organisms, whose natural habitat is the interval corresponds to the interval salinity water salinity test medium. The average time interval required for the grown test organisms of the laboratory culture to determine the same dose of the same type of feed at a constant distance is determined, the type of feed being identical to one of the feeds of the laboratory culture of the test organisms. In the case of testing sea water, instead of pre-growing the laboratory culture of test organisms, it is allowed to obtain a laboratory culture of test organisms by pre-long-term maintenance of marine aquatic organisms of the same species, taken from one ecologically clean area of the sea, on artificial clean reference water and feed with a constant regime of maintenance in laboratory conditions.

 Then the first part of the test organisms of the laboratory culture of the test organisms is maintained in a set of different concentration series of solutions of each of the model toxicants corresponding to the possible toxicants of the test aqueous medium and in fresh artificial pure reference water, as non-toxic control water, in vessels of a smaller volume than vessels for the maintenance of laboratory culture of test organisms, for the same time interval, sufficient for physiological adaptation to new environments and vessels, under the regime the content of the laboratory culture of test organisms, each test organism being placed only once in a model toxicant solution, then locomotor activity and the spectrum of behavioral reactions of test organisms, which is a constant set of values, are determined in arbitrary units for the same time, each of which expresses the degree of manifestation of a specific behavioral reaction of the test organism in arbitrary units, for each test organism of the first part of the culture of test organisms in fresh artificial true reference water of the same temperature as the water used to contain the laboratory culture of the test organisms in the same vessels, smaller in volume than the vessels for the maintenance of the laboratory culture of the test organisms, the determination being made after the protective organisms have disappeared from the test organisms reactions to the procedure of transferring from one vessel to another vessel.

Next, the test organisms of the first part of the laboratory culture of test organisms are divided into groups, each of which contains the same number of test organisms, previously conditioned in the same solution from a set of series of solutions of model toxicants and fresh artificial reference water. Then, each group of test organisms of the first part of the laboratory culture of test organisms is placed in other vessels smaller in volume than the vessels for holding the laboratory culture of test organisms, with fresh artificial pure reference water at the same temperature as the water for containing the laboratory culture test organisms, at the same distance from the same dose in the vessel of the same feed, identical to the dose and feed used to determine the time interval the feed was spent by the test organisms in the laboratory cultures in the vessels for their maintenance, and after the protective organisms respond to the test organisms to the transfer procedure from one vessel to another vessel, the percentage of test organisms that find the same dose of food after the same time interval equal to the previously determined average interval is determined in each group the time required for the test organisms of the laboratory culture of the test organisms to find an identical dose of food at an identical distance in the vessels for maintaining the laboratory culture of the test organisms. After that, for each test organism of the first part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated by dividing the value of each behavioral reaction previously determined for each test organism of the spectrum of behavioral reactions by the value of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the value of which depends on the type of aquatic organisms used to grow the laboratory culture of test organisms, on a set of behavioral reactions of the determined spectra of behavioral reactions of test organisms, and on arbitrary units in which values of behavioral reactions are measured , and LA is the previously determined locomotor activity of the corresponding test organism.

 Then, average values and standard errors of the average values for the normalized spectra of behavioral reactions characterizing the functional state of the test organisms of the first part of the laboratory culture of test organisms in a set of different concentration series of solutions of each model toxicant and fresh artificial pure reference water are calculated by calculating the values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. After that, the average values for each population of all groups of test organisms of the first part of the laboratory culture of test organisms aged in fresh artificial pure reference water and in each of the solutions of a set of series of solutions of model toxicants are calculated, percent values and standard errors of the average percent values of test organisms are calculated. found the same dose of food through the same time interval equal to the previously determined time interval required by the test organisms of the laboratory culture of the test organisms to find identical dose of food at an identical distance in the vessels for the maintenance of laboratory culture of test organisms.

 Then, the second part of the test organisms of the laboratory culture of the test organisms is maintained in the test aqueous medium under the regime of the contents of the laboratory culture of the test organisms during the time interval and in the vessels identical to the time interval and the vessels used to withstand the test organisms of the first part of the laboratory culture test organisms in fresh artificial pure reference water and solutions from a set of series of solutions of model toxicants, and each test organism is used only once and the number test organisms of the second part of the laboratory culture should be a multiple of the number of test organisms of one group of the first part of the laboratory culture of the test organisms. After that, the locomotor activity and the behavioral spectrum of the test organisms of the second part of the laboratory culture in fresh artificial pure reference water at the same temperature are determined in the same arbitrary units and during the same time interval as for the test organisms of the first part of the laboratory culture and in the same vessels that are used to determine the locomotor activity and the behavioral spectrum of the test organisms of the first part of the laboratory culture of the test organisms, and the determination is made after the defensive reaction of the test organisms to the transfer process from one vessel to another vessel disappears.

 After that, the test organisms of the second part of the laboratory culture of the test organisms are divided into groups equal in number of test organisms to the groups of the first part of the laboratory culture of the test organisms, and each group of test organisms of the second part of the laboratory culture is placed in a separate vessel with a fresh, artificial, clean reference water of the same temperature as the temperature of the water for the maintenance of the laboratory culture of the test organisms, at the same distance from the same dose of the same food, and the vessels used, the distance and food They are identical, respectively, to the vessels, distance, and food used to determine in each group of test organisms the first part of the laboratory culture of the test organisms the percentage of test organisms that found the same dose of food for the same time interval, and after the protective organisms didn’t have a defensive reaction to the transfer procedure from one vessel to another vessel is determined in each group by the percentage of test organisms that find the same dose of food after the same time interval equal to the time interval used A definition of each group of test organisms, the first part of the laboratory culture of test organisms per cent of the test organisms have found the same feed dose for the same time period. After that, for each test organism of the second part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated similarly to the calculation of normalized spectra of behavioral reactions of the test organisms of the first part of the laboratory culture of test organisms, and average values and standard errors of the mean values for the calculated normalized spectra of behavioral are calculated reactions characterizing the functional state of test organisms of the second part of the laboratory culture of test organisms in the test one environment.

 After that, the values of the normalized spectra of behavioral reactions of test organisms of the second part of the culture of test organisms are compared with the calculated average values of the spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms and are selected from a set of model toxicants and fresh artificial pure water as non-toxic control water, those toxicants and their concentrations for which the average values of all behavioral reactions of normalized spectra of behavioral reactions are test organ zmov first part of the laboratory culture of test organisms was not significantly different from the average values of the corresponding behavioral responses normalized spectra behavioral test organisms second part of the laboratory culture of the test organisms. Then, for each selected toxicant, its average concentration is found by averaging all the concentrations selected as a result of the comparison and the presence of the main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant. After that, the average values for the percentage for each population of all groups of test organisms of the second part of the laboratory culture of test organisms are calculated and the standard errors of the average percent values for test organisms that find the same dose of food after the same time interval. Then compare the average values of the percentage of test organisms for all groups of the second part of the laboratory culture of the test organisms that found the same dose of feed for the same time interval, with the average values of the percentage of test organisms for each set of all groups of test organisms of the first part of the laboratory culture of the test organisms who have found the same dose of feed at the same time interval and are selected from all model toxicants, including fresh artificial pure reference water as non-toxic control water, those oxicants and their concentrations, for which the average values of the percentage of test organisms in the first part of the laboratory culture of test organisms that find the same dose of food after the same time interval do not statistically significantly differ from the corresponding average values of the percentage of test organisms in the second part of the laboratory culture of test organisms .

 Then, for each selected toxicant, its average concentration is found by averaging all the concentrations selected as a result of the comparison and judge the presence of each main toxicant in the test aqueous medium based on the found average concentrations of the corresponding toxicant. Next, determine the preliminary value of the toxicity of the test aqueous medium, first by comparing the toxicological parameters and the degree of exceeding the maximum permissible concentrations determined as a result of the first comparison of the main toxicants of the test aqueous medium with the toxicants and then by comparing the main toxicants tested as a result of the second comparison aquatic environment with toxicological standards applicable to the tested aquatic environment azatelyam powers and maximum allowable concentrations, after which finally determine the degree of general toxicity test aqueous environment as the highest value of the two predefined degrees of overall toxicity.

 The inventive step of the proposed method is proved by the fact that the combination of essential features leads to the fact that using the proposed method it is possible to determine with sufficient accuracy the main toxicants in a wide range of degrees of exceeding their maximum permissible concentrations (MPC) and the degree of general toxicity of the tested aqueous medium. Indeed, the fact that for testing according to this method, individuals from a pre-grown by closely related crossing individuals of one species of hydrobionts on artificial pure reference water and feeds are used as test organisms for a constant mode of laboratory culture of test organisms whose natural habitat is the salinity range corresponds to the salinity range of the tested aqueous medium, significantly increases the accuracy of determination of toxicants. In addition, the determination of the main toxicants and the general toxicity of the test aqueous medium is carried out by comparing the effects on the test organisms of the test aqueous medium and a whole set of different series of solutions of model toxicants corresponding to possible toxicants of the test aqueous medium and artificial pure reference water, which differ in concentration use for comparison, as physiological parameters of the functional state of test organisms, a whole range of their behavioral reactions, representing is a constant set of values, each of which corresponds to the degree of manifestation of a certain behavioral reaction of the test organism in arbitrary units, and when comparing the spectra of behavioral reactions of test organisms, the locomotor activity of test organisms determined during the process is taken into account, which leads to a significant increase in sensitivity and accuracy of the method due to the fact that the determination of the main toxicants and the general toxicity of the test aqueous medium is based on such physiological pairs meters that most fully reflect the most significant changes in the functional state of test organisms that occur in them under the influence of toxicants contained in the test aqueous medium. The increase in the accuracy and sensitivity of the method also occurs to a large extent from the fact that the determination of the physiological parameters of the functional state of test organisms in the test aqueous medium is performed twice in both versions of the method.

 The method allows it to be used widely for biotesting of aquatic environments contaminated with various toxicants in a wide range of concentrations, as well as water extracts of soils and bottom sediments, firstly, because the test organisms taken from previously grown by closely related crosses of individuals of one species of hydrobionts on artificial pure reference water and feed with a constant mode of maintenance of laboratory culture of aquatic organisms, the natural habitat of which according to the salinity interval corresponds to the salinity range of the tested aqueous medium, they are highly sensitive in combination with sufficient resistance to exposure to a wide range of toxicants, and, secondly, the main toxicants and the degree of general toxicity of the tested aqueous medium are determined by comparing the effects on the test organisms of the tested aqueous medium and a whole set of different concentration of series of solutions of model toxicants corresponding to possible toxicants of the tested aqueous medium, and artificial pure reference water, and not solutions of one model toxicant. In addition, this contributes to the possibility with the proposed method of "transfer" of the obtained results from the used test organisms to those organisms, including humans, for which the standards for the tested aqueous medium used for comparison in the methods have been developed, i.e. the possibility of the proposed method for determining the main toxicants and the general toxicity of aquatic environments, regardless of the used test organisms for higher animals and humans, provided that for these animals and humans there are standards for the determined toxicants or MPCT for the tested aquatic environments. All this together leads to the universality of the method.

 The invention can be used for biotesting of various water environments, including water extracts of soils and bottom sediments, i.e. for environmental monitoring of surface, ground, and wastewater, soils and bottom sediments, as well as for establishing a non-toxic degree of dilution of the test aqueous medium. According to the first variant of the method, the laboratory culture of test organisms is preliminarily grown on artificial pure reference water and feed with a constant mode of maintenance by closely interbreeding individuals of one species of hydrobionts, whose natural habitat, according to the salinity interval, corresponds to the salinity interval of the tested aqueous medium. In the case of testing sea water, instead of pre-growing a laboratory culture of test organisms, it is allowed to obtain a laboratory culture of test organisms by pre-long-term maintenance of marine aquatic organisms of the same species, taken from one ecologically clean area of the sea on artificial clean reference water and feed under constant conditions in the laboratory . Then, locomotor activity and the spectrum of behavioral test organisms are determined in arbitrary units during the same time interval in vessels smaller in volume than the vessels for containing laboratory culture of test organisms, each of which corresponds to the degree of manifestation a specific behavioral reaction of the test organism in arbitrary units, for each test organism of the first part of the grown laboratory culture in a set of different series of solutions of each from model toxicants corresponding to possible toxicants of the tested aqueous medium, and in fresh artificial pure reference water, as in non-toxic control water at the same temperature at which the laboratory culture of test organisms is contained, and all solutions of model toxicants are prepared in fresh artificial pure reference water, each test organism is placed only once in a model toxicant solution, and the determination is made after the protective organisms react to the process Renos from one vessel to another vessel.

 After that, the test organisms of the first part of the laboratory culture of the test organisms are kept in a set of different concentration series of solutions of each of the model toxicants and fresh artificial pure reference water in vessels of a smaller volume than the vessels for maintaining the laboratory culture of the test organisms for the same enough time for physiological adaptation to new aquatic environments and the vessel, with the regime of the content of the laboratory culture of test organisms, and each test organism can withstand exactly that a solution of a model toxicant and fresh artificial pure reference water, in which its locomotor activity and the spectrum of behavioral reaction were primarily determined.

Then, the locomotor activity and the spectrum of behavioral reactions are secondly determined for each test organism of the first part of the laboratory culture of the test organisms in a fresh artificial pure reference will at a temperature and in vessels identical to the temperature and vessels, respectively, used for the initial determination of locomotor activity and the spectrum of behavioral reaction for each test organism of the first part of the laboratory culture of test organisms, during the time interval and in arbitrary units that were used for initial determination of locomotor activity and the spectrum of behavioral reactions for each test organism of the first part of the laboratory culture of test organisms, and the determination is made after the protective organisms react to the process of transfer from one vessel to another vessel after the test organisms disappear. After that, for each test organism of the first part of the laboratory culture of test organisms, normalized primary and secondary defined spectra of behavioral reactions are calculated by dividing the values of each behavioral response both of the primary and secondary spectra of behavioral responses for each test organism by the value of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the value of which depends on the type of hydrobionts used to grow the laboratory culture of test organisms, on a set of behavioral reactions of certain spectra of behavioral reactions of test organisms, and on arbitrary units in which values of behavioral reactions are measured , and LA is the previously determined locomotor activity of the corresponding test organism.

 Then, average values and standard errors of average values are calculated separately for normalized primary and secondary defined spectra of behavioral reactions, characterizing the functional state of test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of each of the toxic toxicants and a fresh, artificial, pure reference water, by calculating the mean values and their standard errors for each behavioral reaction of the indicated normalized spectra n vedencheskih reactions. Then, locomotor activity and the spectrum of behavioral reactions are first determined for each test organism of the second part of the laboratory culture of the test organisms in the test sample of the aqueous medium at a temperature in vessels identical to the temperature and vessels used to determine the locomotor activity and the spectrum of behavioral reactions for each test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of solution series differing in concentration in model toxicants, during the time interval and in arbitrary units that were used to determine the locomotor activity and the behavioral spectrum of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of different series of solutions of model toxicants differing in concentration, each test -organism is used for testing only once and the determination is made after the protective organisms react to the process of transfer from one vessel into another vessel.

 After that, the test organisms of the second part of the laboratory culture of the test organisms are maintained in the test aqueous medium for a time interval and in vessels identical to the time interval and the vessels used to withstand the test organisms of the first part of the laboratory culture of the test organisms in a fresh, artificial, clean reference water and a set of different series of solutions of model toxicants differing in concentration in the mode of laboratory culture of test organisms. Then, the locomotor activity and the spectrum of behavioral reactions of each test organism of the second part of the laboratory culture of the test organisms in fresh artificial pure reference water are determined for the time interval and in the vessels identical to the time interval and the vessels used to determine the locomotor activity and the behavioral spectrum of each test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of series differing in concentration solutions of model toxicants, during the time interval and in arbitrary units that were used to determine the locomotor activity and the spectrum of behavioral reactions of each test organism of the first part of the laboratory culture of test organisms in fresh artificial pure reference water and a set of different concentration of series of model toxicant solutions moreover, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the transfer process from one vessel to another vessel.

 After that, for each test organism of the second part of the laboratory culture of test organisms, normalized primary and secondary defined spectra of behavioral reactions are calculated similarly to the calculation of primary and secondary determined spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms, and average values and standard errors are calculated separate mean values for normalized primary and secondary defined spectra of behavioral reactions characterizing a functional state test organisms of the second part of the laboratory culture of test organisms in the test aqueous medium. After that, the corresponding average values of the normalized primary and secondarily determined spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms are compared with the calculated average values of the spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms, and choose from a set of model toxicants and fresh artificial pure water, like non-toxic control water, those toxicants for which the average values of all behavioral reactions are ized spectra behavioral test organisms, the first part of the laboratory culture of test organisms was not significantly different from the average values of the corresponding behavioral responses normalized spectra behavioral test organisms second part of the laboratory culture of the test organisms.

 Then, for each selected toxicant, its average concentration is found by averaging all its concentrations selected as a result of the comparison, and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant, and the degree of total toxicity of the tested aqueous medium is determined by comparing certain the main toxicants of the tested aquatic environment with toxicological standards applicable to the tested aquatic environment and the degree of exceeding the maximum permissible concentration.

 According to the second variant of the method, the laboratory culture of test organisms is preliminarily grown on artificial pure reference water and feed with a constant regime of maintenance by closely interbreeding individuals of one species of hydrobionts, whose natural habitat, according to the salinity interval, corresponds to the salinity interval of the tested aqueous medium. The average time interval required for the grown test organisms of the laboratory culture to determine the same dose of the same type of feed at a constant distance is determined, the type of feed being identical to one of the feeds of the laboratory culture of the test organisms. In the case of testing sea water, instead of pre-growing the laboratory culture of test organisms, it is allowed to obtain a laboratory culture of test organisms by pre-long-term maintenance of marine aquatic organisms of the same species taken from one ecologically clean area of the sea on artificial clean reference water and feed under a constant regime of maintenance under laboratory conditions .

 Then the first part of the test organisms of the laboratory culture of the test organisms is maintained in a set of different concentration series of solutions of each of the model toxicants corresponding to the possible toxicants of the test aqueous medium, and in fresh artificial pure reference water, as non-toxic control water, in smaller vessels, than vessels for the maintenance of laboratory cultures of test organisms, during the same time interval, sufficient for physiological adaptation to new environments and vessels, with the content of the laboratory culture of test organisms, each test organism being placed only once in a model toxicant solution, then locomotor activity and the spectrum of behavioral reactions of test organisms, representing a constant set of values, are determined in arbitrary units for the same time, each of which expresses the degree of manifestation of a specific behavioral reaction of the test organism in arbitrary units, for each test organism of the first part of the culture of test organisms in fresh artificial h true reference water of the same temperature as the water used to contain the laboratory culture of the test organisms in the same vessels, smaller in volume than the vessels for the maintenance of the laboratory culture of the test organisms, the determination being made after the protective organisms have disappeared from the test organisms reactions to the procedure of transferring from one vessel to another vessel.

Next, the test organisms of the first part of the laboratory culture of test organisms are divided into groups, each of which contains the same number of test organisms, previously conditioned in the same solution from a set of series of solutions of model toxicants and fresh artificial reference water. Then, each group of test organisms of the first part of the laboratory culture of test organisms is placed in other vessels smaller in volume than the vessels for holding the laboratory culture of test organisms, with fresh artificial pure reference water at the same temperature as the water for maintaining the laboratory culture test -organisms at the same distance from the same dose of the same feed in the vessel, identical to the dose and feed used to determine the time interval the feed was spent by the test organisms of the laboratory the ulcers in the vessels for their maintenance, and after the protective organisms react to the test organisms with the disappearance of the transfer procedure from one vessel to another vessel, the percentage of test organisms that find the same dose of food after the same time interval equal to the previously determined average interval is determined in each group the time required for the test organisms of the laboratory culture of the test organisms to find an identical dose of food at an identical distance in the vessels for maintaining the laboratory culture of the test organisms. After that, for each test organism of the first part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated by dividing the values of each behavioral reaction previously determined for each test organism of the spectrum of behavioral reactions by the values of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the value of which depends on the type of aquatic organisms used to grow the laboratory culture of test organisms, on a set of behavioral reactions of the determined spectra of behavioral reactions of test organisms, and on arbitrary units in which values of behavioral reactions are measured , and LA is the previously determined locomotor activity of the corresponding test organism.

 Then, average values and standard errors of the average values for the normalized spectra of behavioral reactions characterizing the functional state of the test organisms of the first part of the laboratory culture of test organisms in a set of different concentration series of solutions of each of the model toxicants and in fresh artificial pure reference water are calculated by calculating mean values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. After that, the average values for each population of all groups of test organisms of the first part of the laboratory culture of test organisms aged in fresh artificial pure reference water and in each of the solutions of a set of series of solutions of model toxicants are calculated, percent values and standard errors of the average percent values of test organisms are calculated. found the same dose of food after the same time interval equal to the previously determined time interval required by the test organisms of the laboratory culture of the test organisms to find identical dose of food at an identical distance in the vessels for the maintenance of laboratory culture of test organisms.

 Then, the second part of the test organisms of the laboratory culture of the test organisms is maintained in the test aqueous medium under the regime of the contents of the laboratory culture of the test organisms for the time interval and in the vessels identical to the time interval and the vessels used to withstand the test organisms of the first part of the laboratory culture test organisms in fresh artificial pure reference water and in solutions from a set of series of solutions of model toxicants, and each test organism is used only once and of test organisms second part of the laboratory culture should be a multiple of the number of test organisms of one group of the first part of the lab culture of the test organisms.

 After that, the locomotor activity and the behavioral spectrum of the test organisms of the second part of the laboratory culture in fresh artificial pure reference water at the same temperature are determined in the same arbitrary units and during the same time interval as for the test organisms of the first part of the laboratory culture and in the same vessels that were used to determine the locomotor activity and the behavioral spectrum of the test organisms of the first part of the laboratory culture of the test organisms, the determination being made after the defensive reaction of the test organisms to the transfer process from one vessel to another vessel disappears. After that, test organisms of the second part of the laboratory culture of test organisms are divided into groups equal in number of test organisms to the groups of the first part of the laboratory culture of test organisms, and each group of test organisms of the second part of the laboratory culture is placed in a specific vessel with a fresh, artificial, clean reference water of the same temperature as the temperature of the water for the maintenance of the laboratory culture of the test organisms, at the same distance from the same dose of the same feed, and the vessels used, the distance and m are identical, respectively, to the vessels, distance and feed used to determine in each group of test organisms the first part of the laboratory culture of the test organisms the percentage of test organisms that found the same dose of food for the same time interval, and after the defensive reaction of the test organisms disappeared the transfer procedure from one vessel to another vessel is determined in each group of test organisms that find the same dose of food after a time interval equal to the time interval used to determine in each group of test organisms of the first part of the laboratory culture of test organisms, the percentage of test organisms that found the same dose of food for the same time interval.

 After that, for each test organism of the second part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated similarly to the calculation of normalized spectra of behavioral reactions of the test organisms of the first part of the laboratory culture of test organisms, and average values and standard errors of the mean values for the calculated normalized spectra of behavioral are calculated reactions characterizing the functional state of test organisms of the second part of the laboratory culture of test organisms in the test one environment. After that, the average values of the normalized spectra of behavioral reactions of test organisms of the second part of the culture of test organisms are compared with the calculated average values of the spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms, and they are selected from a set of model toxicants and fresh artificial pure water, as non-toxic control water, those toxicants and their concentrations for which the average values of all behavioral reactions of the normalized spectra of behavioral reactions are those st organisms of the first part of the laboratory culture of test organisms do not statistically significantly differ from the average values of the corresponding behavioral reactions of the normalized spectra of behavioral reactions of the second part of the laboratory culture of test organisms.

 Then, for each selected toxicant, its average concentration is found by averaging all its concentrations selected as a result of the comparison, and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant. After that, the average percent for each population of all groups of test organisms of the second part of the laboratory culture of test organisms is calculated and the standard errors of the average percent values of test organisms that find the same dose of food after the same time interval. Then, the average values of the percentage of test organisms for all groups of the second part of the laboratory culture of test organisms that find the same dose of food for the same time interval are compared with the average values of the percentage of test organisms for each set of all groups of test organisms of the first part of the laboratory culture of test organisms who found the same dose of feed after the same period of time, and choose from all model toxicants, including fresh artificial pure reference water, as non-toxic control water, those toxicants and their concentrations, for which the average percentage of test organisms of the first part of the laboratory culture of test organisms that find the same dose of food after the same period of time, do not statistically significantly differ from the corresponding average values of the percentage of test organisms of the second part of the laboratory culture of test organisms. Then, for each selected toxicant, its average concentration is found by averaging all its concentrations selected as a result of the comparison, and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant.

 Next, determine the preliminary value of the toxicity of the test medium, first by comparing the toxicological indicators and the degree of exceeding the maximum permissible concentrations determined as a result of the first comparison of the main toxicants of the test aqueous medium with the toxicological indicators, and then by comparing the basic toxicants determined by the second comparison of the main toxicants of the test aqueous environment with toxicological standards for the tested aquatic environment pits and degrees of exceeding the maximum permissible concentrations, after which the degree of general toxicity of the tested aqueous medium is finally determined as the largest value of the two predefined degrees of general toxicity.

 Specifically, the main toxicants and the degree of general toxicity of the test aqueous medium are determined according to the first embodiment of the method as follows.

 Example No. 1. In August 1995, a sample of water from a lake located in the territory of a comprehensive environmental review of the production activities of a chemical plant and monitoring the state of surface and ground waters in the Altai Territory was taken for biological determination of the main toxicants and the degree of general toxicity. The sample volume was 2 liters. It was known that the water taken belongs to the mineralized fresh and slightly saline waters (the content of the main salt components does not exceed 4 - 5 MPCT for cultural water). In addition, heavy metals can be present in it as in the water of a surface reservoir located not far from a small settlement, railway and highway roads, as well as kitchen gardens and an enterprise whose main production is the extraction and purification of natural sodium sulfate, ammonia nitrogen, nitrates, nitrites, lithium and petroleum products. The biological determination of the main toxicants and the degree of general toxicity of the test water sample was carried out by the first variant of the method. As test organisms, individuals were taken from those grown for the purpose of biotesting by closely related crosses at a constant maintenance regime in 20-100-liter aquariums (constant light and temperature conditions, constant aeration and feeding regimes) on artificial pure reference water and feed from the laboratory culture of hydrobionts . Initially, aquatic organisms were purchased at a pet store and were a perennial aquarium culture living in the river. Amazonian species Ampullaria gigas. The laboratory culture of the test organisms was considered to be individuals of the second and subsequent generations grown by the above method in the laboratory conditions of an aquarium culture. Test organisms of the indicated laboratory cultures of test organisms are adapted to live in fresh, weakly and medium saline, as well as slightly saline waters, therefore, they are suitable as test organisms for the biological determination of the toxicity of an aqueous sample taken for testing.

 For initial determination in locomotor activity units and the behavioral spectrum of each test organism for initial determination, 12 test organisms of the first part of the laboratory culture of test organisms are placed in a set of the following model toxicants differing in the concentration of the series of solutions: ammonium nitrogen, nitrates, nitrites, lithium, crude oil, ferric iron, cadmium, sulphate, zinc, lead and nickel and into fresh, artificial pure reference water, as in non-toxic control water, at the same temperature as uyu has water to contain the laboratory culture of test organisms. Moreover, each model toxicant is used in the form of four solutions differing in concentration - 0, 5, 2, 7, and 50 MPCT for cultural, domestic and drinking water (according to [3]) on fresh artificial pure reference water, and test organisms placed once in three in one-liter vessels. After the defensive-defensive reaction to the process of transfer from one vessel to another vessel disappears, locomotor activity - LA (the number of completed divisions applied to the bottom and walls of the vessels) and the behavioral spectrum are determined as constant for each test organism in arbitrary units a set of values, each of which corresponds to the degree of manifestation of a particular behavioral reaction of the test organism in arbitrary units. For each test organism, 10 reactions A, B, C, D, E, F, G, H, K, L are recorded, among which: the number of falls - A, the number of normal respiratory acts - F, death of the animal (1 - yes, 0 - no) - L.

 Then the test organisms of the first part of the laboratory culture of the test organisms are kept in a set of different concentration series of solutions of each of the model toxicants and in fresh artificial pure reference water for a time sufficient for physiological adaptation to new aqueous media and the vessel, which in this case is 2 days, in other one-liter vessels under the regime of the laboratory culture of the test organisms, and each test organism is kept in that solution of the model toxicant and in fresh Artificial pure reference water, which is primarily determined by its locomotor activity and the spectrum of behavioral reactions. Then, the locomotor activity and the spectrum of behavioral reactions are secondly determined for each test organism of the second part of the laboratory culture of the test organisms in fresh artificial pure reference water at a temperature and in vessels identical to the temperature and vessels used to determine the locomotor activity and the spectrum of behavioral reactions of test organisms the first part of the laboratory culture of test organisms in fresh, artificial, pure reference water, during the time interval and in arbitrary units that were used Ali to determine the locomotor activity and the spectrum of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms in fresh, artificial, pure reference water and a set of series of solutions of model toxicants differing in concentration. Moreover, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the process of transfer from a vessel to another vessel.

After that, for each test organism of the first part of the laboratory culture of test organisms, normalized primary and secondary defined spectra of behavioral reactions are calculated by dividing the values of each behavioral reaction of all spectra of behavioral reactions by the values of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the magnitude of which for a given laboratory culture of test organisms, a given set of behavioral reactions of the determined spectra of behavioral reactions of test organisms, and arbitrary units in which the values of behavioral reactions change, are respectively equal to k ₁ = 0, 01 and k ₂ = 50.

Then, average values (X ₁ ) and standard errors of average values (Y ₁ ) are calculated separately for normalized primary and secondary defined spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of each of the toxicants differing in concentration in fresh artificial pure reference water, by calculating the mean values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. As an example, in table. 1 presents the results obtained for fresh artificial pure reference water, i.e. for non-toxic control water.

 Then put 6 test organisms of the second part of the laboratory culture of test organisms, three in two one-liter vessels, identical to the vessels for primary and secondary determination of locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of test organisms, with a sample of the test water the same temperature as water for the maintenance of the laboratory culture of test organisms, and after the disappearance of their defensive-defensive reaction to the process of transfer from one vessel to another vessel, the primary for each test organism, the locomotor activity and the spectrum of behavioral reactions are allocated in the same arbitrary units that were used for the primary and secondary determination of the locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of the test organisms.

 After that, test organisms of the second part of the laboratory culture of test organisms are kept in the same sample of test water in other one-liter vessels identical to those for keeping in fresh artificial pure reference water and a set of different concentration of series of solutions of model toxicants of test organisms of the first part of the laboratory culture of test organisms, at a temperature of the content of laboratory culture of test organisms for 2 days. Then the test organisms of the second part of the laboratory culture of the test organisms are transferred to one-liter vessels identical to the vessels for the primary and secondary determination of the locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of the test organisms with fresh artificial pure reference water of the same temperature that the temperature of the water used to maintain the laboratory culture of the test organisms, and after the disappearance of the defensive-defensive reaction of for each test organism of the second part of the laboratory culture of test organisms, locomotor activity and the spectrum of behavioral reactions in the same arbitrary units that were used for the primary and secondary determination of locomotor activity and the behavioral spectrum of each test organism are secondarily determined from one vessel to another vessel the first part of the laboratory culture of test organisms.

After that, the normalized primary and secondary determined spectra of behavioral operations of each test organism of the second part of the laboratory culture of test organisms are calculated in the same way as they were done for test organisms of the first part of the laboratory culture of test organisms. Then calculate the average values (X ₂ ) and standard errors of the mean values (Y ₂ ) separately for the normalized primary and secondary defined spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms. The results obtained in this case are presented in table. 2.

Then, the obtained average values of the normalized primary and secondarily determined spectra of behavioral reactions of test organisms of the second part of the laboratory culture are compared with the average values of the corresponding normalized primary and secondarily determined spectra of behavioral reactions of the first part of the laboratory culture of test organisms using the standard statistical analysis formula [4] :
t _st = (X ₁ -X ₂ ) / (Y $\genfrac{}{}{0ex}{}{\text{2}}{\text{1}}$ + Y $\genfrac{}{}{0ex}{}{\text{2}}{\text{2}}$ ) ^0.5 , (1)
where in this case x ₁ and y ₁ are the average value and standard error of the average value of each behavioral reaction of the normalized primary or secondary specific spectrum of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms, x ₂ and y ₂ are the average value and standard error of the average value of the corresponding behavioral reaction of the normalized corresponding spectrum of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms.

Then choose from a set of model toxicants and fresh artificial pure water, as non-toxic control water, those toxicants for which the average values of all behavioral reactions of the normalized spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms do not statistically significantly differ from the average values of the corresponding behavioral reactions of normalized spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms, i.e. those for which the absolute value of t _st does not exceed 2.0 for each behavioral reaction of the primary or secondary defined spectra of behavioral reactions of test organisms. Selected model toxicants and their concentrations are presented in table. 3.

 Then, for each selected toxicant, its average concentration is found by calculating the geometric mean of all its selected concentrations and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant. The results are presented in table. 4.

 The degree of general toxicity of the test aqueous medium is determined by comparing certain major toxicants of the test aqueous medium with the standards applicable to the test aqueous medium for toxicological indicators and the degree of exceeding the maximum permissible concentrations. Since the tested aqueous medium refers to the type of surface water for cultural, domestic, and drinking purposes, the toxicity of which is evaluated in the MPC for water for cultural, domestic and drinking purposes (according to [3]), in which the main toxicants defined according to this variant of the method are expressed of the tested aqueous medium, the general toxicity of the tested aqueous medium is determined by choosing the highest of the calculated average concentrations of certain basic toxicants. It is equal to 18.7 MPCT, which, according to the norms of the degree of exceeding the maximum permissible concentrations [5], corresponds to the range of degrees of exceeding the MPCt from 10 to 100, the general toxicity of which is defined as "extremely high".

 Example 2. In August 1995, a sample of sea water from the White Sea was taken for the biological determination of the main toxicants and the degree of general toxicity in the Chupinskaya Bay area near the working village and local industrial enterprise. The purpose of the test was to determine the possible presence of heavy metals in the tested sample of the aquatic environment in unacceptable MPCT according to the standards for marine water bodies used for living, reproduction and migration of aquatic organisms [6], and to determine its degree of general toxicity. The biological determination of the main toxicants and the degree of general toxicity of the sample of the test aqueous medium was carried out by the first variant of the method. As test organisms, individuals were taken from the laboratory culture of test organisms. The laboratory culture of the test organisms was obtained by preliminarily maintaining long-term aquatic organisms living in the White Sea of the species Littorina littorea, taken from one ecologically clean region of the White Sea, using artificial pure reference sea water and feed under constant conditions in the laboratory.

 For initial determination in arbitrary units of locomotor activity and the behavioral spectrum of each test organism, 12 test organisms of the first part of the laboratory culture of test organisms are placed in a set of the following model toxicants differing in the concentration of the series of solutions: copper sulfate, iron, cadmium, zinc, lead and nickel and into fresh artificial pure reference sea water, as into non-toxic control water, with the same temperature as the water has for the maintenance of laboratory culture of test organisms . Moreover, each model toxicant is used in the form of four solutions differing in concentration - 0, 5, 2, 7 and 50 maximum concentration limits for marine water bodies used for living, reproduction and migration of aquatic organisms (according to [6]) on fresh artificial pure reference sea water , and test organisms are placed once in three in one-liter vessels. After the defensive-defensive reaction to the process of transfer from one vessel to another vessel disappears, locomotor activity - LA (the number of completed divisions applied to the bottom and walls of the vessels) and the behavioral spectrum are determined as constant for each test organism in arbitrary units a set of values, each of which corresponds to the degree of manifestation of a particular behavioral reaction of the test organism in arbitrary units. For each test organism, 6 reactions A, B, C, D, E, F are recorded, among which: the number of falls - A, the death of the animal (I-yes, 0-no) - F.

 Then the test organisms of the first part of the laboratory culture of test organisms are kept in a set of different concentration series of solutions of each of the model toxicants and in fresh, artificial pure reference sea water for a time sufficient for physiological adaptation to new aquatic environments and the vessel, which in this case is 3 days, in other one-liter vessels under the regime of laboratory culture of test organisms, each test organism is kept in that particular model toxicant solution and fresh artificial pure reference sea water, in which its locomotor activity and the spectrum of behavioral reactions were primarily determined. Then, locomotor activity and the spectrum of behavioral reactions are secondly determined for each test organism of the second part of the laboratory culture of the test organisms in fresh artificial pure reference sea water at a temperature and in vessels identical to the temperature and vessels used to determine the locomotor activity and the spectrum of behavioral tests organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference sea water, over a time interval and in arbitrary units, to torye used to determine the locomotor activity and the spectrum of behavioral responses of test organisms, the first part of the laboratory culture of test organisms in fresh artificial sea water clean reference and a set of differing concentrations of a series of solutions of model toxicants. Moreover, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the process of transfer from one vessel to another vessel.

After that, for each test organism of the first part of the laboratory culture of test organisms, normalized primary and secondary defined spectra of behavioral reactions are calculated by dividing the values of each behavioral reaction of all spectra of behavioral reactions by the value of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the magnitude of which for a given laboratory culture of test organisms, a given set of behavioral reactions of the determined spectra of behavioral reactions of test organisms and arbitrary units in which values of behavioral reactions are measured, are equal to k ₁ = 0, 05 and k ₂ = 20.

Then, average values (x ₁ ) and standard errors of average values (y ₁ ) are calculated separately for normalized primary and secondary determined spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of each of the toxicants differing in concentration in fresh artificial pure reference sea water by calculating mean values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. As an example, in table. 5 presents the results obtained for fresh artificial pure reference sea water, i.e. for non-toxic control water.

 Then put 6 test organisms of the second part of the laboratory culture of test organisms, three in two one-liter vessels, identical to the vessels for primary and secondary determination of locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of test organisms, with a sample of the test water the same temperature as water for the maintenance of the laboratory culture of test organisms, and after the disappearance of their defensive-defensive reaction to the process of transfer from one vessel to another vessel, the primary for each test organism, the locomotor activity and the spectrum of behavioral reactions are defined in the same arbitrary units that were used for the primary and secondary determination of the locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of the test organisms.

 After that, the test organisms of the second part of the laboratory culture of the test organisms are kept in the same sample of test water in other one-liter vessels identical to those for keeping in fresh artificial pure reference sea water and a set of different concentration of series of solutions of model toxicants of test organisms of the first part laboratory culture of test organisms, at a temperature of the content of laboratory culture of test organisms for 3 days. Then the test organisms of the second part of the laboratory culture of the test organisms are transferred into one-liter vessels identical to the vessels for the primary and secondary determination of the locomotor activity and the behavioral spectrum of each test organism of the first part of the laboratory culture of the test organisms with fresh artificial pure reference sea water of the same temperatures as the temperature of the water used to maintain the laboratory culture of the test organisms, and after the disappearance of the defensive-defensive reaction in test organisms to The transfer process from one vessel to another vessel is secondarily determined for each test organism of the second part of the laboratory culture of test organisms by locomotor activity and the spectrum of behavioral reactions in the same arbitrary units that were used for the primary and secondary determination of locomotor activity and the spectrum of behavioral reactions of each test organism of the first part of the laboratory culture of test organisms.

After that, the normalized primary and secondary determined spectra of behavioral reactions of each test organism of the second part of the laboratory culture of test organisms are calculated in the same way as they were done for test organisms of the first part of the laboratory culture of test organisms. Then calculate the average values (x ₂ ) and standard errors of the average values (y ₂ ) separately for the normalized primary and secondary defined spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms. The results obtained in this case are presented in table. 6.

 Then, the obtained average values of the normalized primary and secondarily determined spectra of behavioral reactions of test organisms of the second part of the laboratory culture are compared with the average values of the corresponding normalized primary and secondarily determined spectra of behavioral reactions of the first part of the laboratory culture of test organisms using the standard formula (1) of Example 1 .

Then choose from a set of model toxicants and fresh artificial pure sea water, as non-toxic control water, those toxicants for which the average values of all behavioral reactions of the normalized spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms do not statistically significantly differ from the average values of the corresponding behavioral reactions of the normalized spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms, i.e., those for the cat On the other hand, the absolute value of t _st does not exceed 2.0 for each behavioral reaction of the primary or secondary determined spectra of behavioral reactions of test organisms. Selected model toxicants and their concentrations are presented in table. 7.

 Then, for each selected toxicant, its average concentration is found by calculating the geometric mean of all its selected concentrations and the presence of each main toxicant in the tested aqueous medium is judged by the found average concentrations of the corresponding toxicant. The results are presented in table. eight.

 The degree of general toxicity of the test aqueous medium is determined by comparing certain major toxicants of the test aqueous medium with the standards applicable to the test aqueous medium for toxicological indicators and the degree of exceeding the maximum permissible concentrations. Since the tested aquatic environment is a type of seawater for fishery purposes. the toxicity of which is evaluated in the MPCT for marine water bodies used for living, reproduction and migration of aquatic organisms (according to [6]), in which the main toxicants of the tested aquatic environment defined according to this variant of the method are expressed, the general toxicity of the tested aquatic environment is determined by choosing the highest from calculated average concentrations of certain major toxicants. It is equal to 1.9 maximum permissible concentrations, which, according to the norms of the degree of exceeding the maximum permissible concentrations [5], corresponds to the range of degrees of excess of the maximum permissible concentration from 1.0 to 3.0, the general toxicity of which is defined as “moderately toxic”.

 Example 3. In August 1995, a sample of the filtered aqueous extract of soil from the area of the weather station located in the territory of the complex environmental review of the production activities of the chemical plant and monitoring the state of surface and ground waters, as well as soils was taken for biological determination of the main toxicants and the degree of general toxicity. in the Altai Territory. The sample volume was 2 liters. It was known that the aqueous extract is a weakly mineralized fresh or slightly saline aqueous medium, which may contain heavy metals, ammonia nitrogen, nitrites and nitrates in unknown concentrations. The biological determination of the main toxicants and the degree of general toxicity of the sample of the tested aqueous extract of the soil was carried out by the second method variant. As test organisms, individuals were taken from the test organisms grown as described in the first example of a laboratory culture. Test organisms of the indicated laboratory culture of test organisms are adapted to live in fresh, weakly, medium-mineralized, and also slightly salty waters; therefore, they are suitable as test organisms for the biological determination of toxicity of the filtered aqueous extract of soil taken for testing.

 The average time interval necessary for the grown test organisms of the laboratory culture of the test organisms is determined to find at the distance of 15 cm the same type of feed identical to one of the feeds of the laboratory culture of the test organisms. It is 20 minutes.

 After this, 12 test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of the following model toxicants are kept for a sufficient time for physiological adaptation to new aqueous media and the vessel, which in this case is 2 days, in this case: ammonium nitrogen, nitrates, nitrites, lithium, crude oil, ferric iron, cadmium, vitriol, zinc, lead and nickel and in fresh artificial pure reference water, as in non-toxic control water, with such the same temperature that water has to maintain the laboratory culture of test organisms. Moreover, each model toxicant is used in the form of four solutions differing in concentration - 0.5, 2, 7, and 50 MPCT for cultural, domestic and drinking water (according to [3]) on fresh artificial pure reference water, and test organisms placed once in three in one-liter vessels. Then, locomotor activity and a behavioral spectrum similar to the behavioral spectrum described in Example 1 are determined in arbitrary units at the same time in fresh artificial pure reference water at the same temperature as the water used to maintain the laboratory culture of the test organisms. In this case, the determination is made in the same vessels, smaller in volume than the vessels for the maintenance of the laboratory culture of the test organisms, after the protective organisms react to the process of transfer from one vessel to another vessel disappear.

 Next, the test organisms of the first part of the laboratory culture of test organisms are divided into groups, each of which contains 6 test organisms, previously conditioned in the same solution from a set of series of solutions of model toxicants and fresh artificial reference water. Then, each group of test organisms of the first part of the laboratory culture of test organisms is placed in other vessels smaller in volume than the vessels for containing laboratory culture of test organisms, with fresh artificial pure reference water at the same temperature as the water for containing the laboratory culture test organisms, at a distance of 15 cm from the same dose of the same feed in the vessel, identical to the dose and feed used to determine the time interval the feed was spent by test organisms of laboratory cultures s in vessels for their maintenance. After the defensive reaction of the test organisms to the transfer procedure from one vessel to another vessel disappears, the percentage of test organisms that find the same dose of food after 20 minutes is determined in each group.

After that, for each test organism of the first part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated by dividing the values of each behavioral reaction of all spectra of behavioral reactions determined previously for each test organism by the value of the following expression:
k ₁ • (k ₂ + LA),
where k ₁ and k ₂ are experimentally determined coefficients, the magnitude of which for a given laboratory culture of test organisms, a given set of behavioral reactions of the determined spectra of behavioral reactions of test organisms and arbitrary units in which values of behavioral reactions are measured, are equal to k ₁ = 0, 01 and k ₂ = 50.

Then, average values (x ₁ ) and standard errors of average values (y ₁ ) are calculated for the normalized specific spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of each of the toxic toxicants and fresh artificial pure reference water, by calculating the mean values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. As an example, in table. 9 presents the results obtained for fresh artificial pure reference water, i.e. for non-toxic control water.

After that, average values for each population of all groups of test organisms of the first part of the laboratory culture of test organisms aged in fresh artificial pure reference water and in each of the solutions of a set of series of solutions of model toxicants are calculated, percent values (p ₁ ) and standard errors of average percent values (z ₁ ) test organisms that find the same dose of feed after 20 minutes. As an example, in table. 10 shows the results obtained for fresh artificial pure reference water, i.e. for non-toxic control water, and for a series of solutions of model toxicant of copper sulfate.

 After that, 12 test organisms of the second part of the laboratory culture of the test organisms in the test aqueous medium are kept for 2 days under the regime of the content of the laboratory culture of the test organisms, placing the test organisms three in one-liter vessels.

 After that, in the same arbitrary units and during the same time interval as for the test organisms of the first part of the laboratory culture, the locomotor activity and the spectrum of behavioral reactions of the test organisms of the second part of the laboratory culture in fresh, artificial, pure reference water at the same temperature are determined and in the same vessels that were used to determine the lacomotor activity and the spectrum of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms. In this case, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the process of transfer from one vessel to another vessel.

 Next, test organisms of the second part of the laboratory culture of test organisms are divided into groups, each of which contains 6 test organisms. Then, each group of test organisms of the second part of the laboratory culture of test organisms is placed in other vessels, with fresh artificial pure reference water of the same temperature as the water for maintaining the laboratory culture of test organisms, at a distance of 15 cm from the same dose of one and the same feed, and the vessels, dose and feed are identical to the vessels, dose and feed used to determine the time interval the feed was spent by the test organisms of the first part of the laboratory culture. After the defensive reaction of the test organisms to the transfer procedure from one vessel to another vessel disappears, the percentage of test organisms that find the same dose of food after 20 minutes is determined in each group.

After that, normalized spectra of behavioral reactions are calculated for each test organism of the second part of the laboratory culture of test organisms in the same way as it was done for test organisms of the first part of the laboratory culture of test organisms. Then calculate the average values (x ₂ ) and standard errors of the average values (y ₂ ) of the normalized specific spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms. The results obtained in this case are presented in table. eleven.

 Then, the obtained average values of the normalized specific spectra of behavioral reactions of test organisms of the second part of the laboratory culture are compared with the average values of the corresponding normalized specific spectra of behavioral reactions of the first part of the laboratory culture of test organisms using the standard formula (1) of example 1.

Then choose from a set of model toxicants and fresh artificial pure reference water, as non-toxic control water, those toxicants for which the average values of all behavioral reactions of the normalized spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms do not statistically significantly differ from the average values of the corresponding behavioral reactions of normalized spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms, i.e., those for The absolute value of t _st does not exceed 2.0 for each behavioral reaction of the spectra of behavioral reactions of test organisms. Selected model toxicants and their concentrations are presented in table. 12.

After that, the average percent values (P ₂ ) and standard errors of the average percent values (z ₂ ) of test organisms that find the same feed dose after 20 minutes are calculated for each population of all groups of test organisms of the second part of the laboratory culture of test organisms. They are equal, respectively, p ₂ = 83 and z ₂ = 11. Then compare the average values of the percentage of test organisms for all groups of the second part of the laboratory culture of test organisms that found the same dose of food for the same time interval (P ₂ ), with the average values for each set of all groups of test organisms of the first part of the laboratory culture of test organisms percent of test organisms that find the same dose of food at the same time interval (P ₁ ) using the standard formula (1) of example 1. Select from all model toxicants, including fresh, artificial pure etal test water, as non-toxic control water, those toxicants and their concentrations for which the average percentage of test organisms of the first part of the laboratory culture of test organisms that find the same dose of food after the same time interval do not statistically significantly differ from the corresponding average values of the percentage of test organisms of the second part of the laboratory culture of test organisms, i.e. those for which the absolute value of t _st does not exceed 2.0. In this case, it is control water and copper sulfate at a concentration of 0.5 MAC. In this case, the average value is 0.5 MPCT.

 Then, a preliminary value of the toxicity of the test aqueous medium is determined first by comparing the main toxicants of the test aqueous medium determined as a result of the first comparison with the current standards for toxicological parameters and the degree of exceeding the maximum permissible concentrations. Since, under the terms of the environmental review, the tested water medium is a type of surface water for cultural, domestic, and drinking purposes, the toxicity of which is estimated in the MPC for water for cultural, domestic and drinking purposes (according to [3], in which certain according to this To the method variant, the main toxicants of the tested aqueous medium, then the preliminary toxicity value of the tested aqueous medium is determined first by choosing the highest of the average concentrations of the main toxicants determined as a result of the first comparison. It is equal to 0.5 MACT. Then the preliminary value of the toxicity of the tested aqueous medium is determined by choosing the highest concentration of the main toxicants determined as a result of the second comparison. It is 0.5 MPCt. After that, it is finally determined the degree of general toxicity of the test aqueous medium as the largest of the two predefined degrees of general toxicity. It is equal to 0.5 MPCt, which, according to the norms of the degree of exceeding the maximum permissible concentrations [5], corresponds to the range of the degrees of excess of the MPCt from 0.1 to 1.0, the general toxicity of which is defined as "permissible".

 Example 4. In June 1995, when conducting comprehensive monitoring of the state of surface water bodies in St. Petersburg, a sample of water from a reservoir located in the city of St. Petersburg was taken for the biological determination of the main toxicants and the degree of general toxicity. The sample volume was 2 liters. It was known that the water taken belongs to fresh, slightly mineralized waters and may contain heavy metals, nitrites, nitrates, lithium, iron, ammonia nitrogen and petroleum products. The biological determination of the main toxicants and the degree of general toxicity of the sample of the tested aqueous extract of the soil was carried out by the second method variant. As test organisms, there were individuals from the laboratory culture of the test organisms grown as described in Example 1. Test organisms of the indicated laboratory culture of test organisms are adapted for living in fresh, weakly and moderately mineralized waters and are therefore suitable for testing taken water.

 We determine the average time interval required for the grown test organisms of the laboratory culture of the test organisms to find the same type of feed at a distance of 15 cm, identical to one of the feeds of the laboratory culture of the test organisms. Oe is 20 minutes.

 After that, 12 test organisms of the first part of the laboratory culture of test organisms in a set of different series of solutions of the following model toxicants are kept for a sufficient time for physiological adaptation to new aqueous media and a vessel, which in this case is 2 days, in this case: ammonium nitrogen, nitrates, nitrites, lithium, crude oil, ferric iron, cadmium, vitriol, zinc, lead and nickel and in fresh artificial pure reference water, as in non-toxic control water, with th same temperature as that of the water for maintenance of laboratory culture of test organisms. Moreover, each model toxicant is used in the form of four solutions differing in concentration - 0.5, 2, 7, and 50 MPCT for cultural, domestic and drinking water (according to [3]) in fresh artificial pure reference water, and test organisms are placed once three in one-liter vessels. Then, locomotor activity and a behavioral spectrum similar to the behavioral spectrum described in Example 1 are determined in arbitrary units at the same time in fresh artificial pure reference water at the same temperature as the water used to maintain the laboratory culture of the test organisms. the determination is made in the same vessels, smaller in volume than the vessels for the maintenance of the laboratory culture of the test organisms, after the protective organisms react to the process of transfer from one vessel to another vessel. Next, the test organisms of the first part of the laboratory culture of test organisms are divided into groups, each of which contains 6 test organisms, previously conditioned in the same solution from a set of series of solutions of model toxicants and in fresh artificial reference water. Then, each group of test organisms of the first part of the laboratory culture of the test organisms is placed in other vessels smaller in volume than the vessels for containing the laboratory culture of the test organisms, with fresh artificial pure reference water at the same temperature as the water for containing the laboratory culture test organisms, at a distance of 15 cm from the same dose of the same feed in the vessel, identical to the dose and feed used to determine the time interval the food was spent by the test organisms in the laboratory cult s in the vessels to their content. After the defensive reaction of the test organisms to the transfer procedure from one vessel to another vessel disappears, the percentage of test organisms that find the same dose of food after 20 minutes is determined in each group.

After that, for each test organism of the first part of the laboratory culture of test organisms, the normalized spectra of behavioral reactions are calculated by dividing the values of each behavioral reaction of all spectra of behavioral reactions determined previously for each test organism by the value of the following expression:
k ₁ • (k ₂ + LA),
Where
k ₁ and k ₂ are experimentally determined coefficients, the magnitude of which for a given laboratory culture of test organisms, a given set of behavioral reactions of the determined spectra of behavioral reactions of test organisms, and arbitrary units in which values of behavioral reactions are measured are equal to k ₁ = 0, 01 and k ₂ = 50. Next, average values (x ₁ ) and standard errors of average values (y ₁ ) for normalized specific spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms in n a set of different series of solutions of each of the toxic toxicants and fresh artificial pure reference water differing in concentration by calculating the average values and their standard errors for each behavioral reaction of the indicated normalized spectra of behavioral reactions. As an example, see table. 11 (example 3.).

After that, the average values for each population of all groups of test organisms of the first part of the laboratory culture of test organisms aged in fresh artificial pure reference water and in each solution of a set of series of solutions of model toxicants are calculated, percent values (P ₁ ) and standard errors of average percent values (z ₁ ) test organisms that find the same dose of feed after 20 minutes. As an example, see table. 10 (example 3).

 After that, 12 test organisms of the second part of the laboratory culture of the test organisms in the test aqueous medium are kept for 2 days under the regime of the content of the laboratory culture of the test organisms, placing the test organisms three in one-liter vessels.

 After that, in the same arbitrary units and during the same time interval as for the test organisms of the first part of the laboratory culture, the locomotor activity and the spectrum of behavioral reactions of the test organisms of the second part of the laboratory culture in fresh, artificial, pure reference water at the same temperature are determined and in the same vessels that were used to determine the locomotor activity and the spectrum of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms. In this case, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the process of transfer from one vessel to another vessel.

 Next, test organisms of the second part of the laboratory culture of test organisms are divided into groups, each of which contains 6 test organisms. Then, each group of test organisms of the second part of the laboratory culture of test organisms is placed in other vessels with fresh artificial pure reference water of the same temperature as the water for maintaining the laboratory culture of test organisms, at a distance of 15 cm from the same dose of one and one in the vessel the same feed, and the vessels, dose and feed are identical to the vessels, dose and feed used to determine the time interval for the feed by the test organisms of the first part of the laboratory culture. After the defensive reaction of the test organisms to the transfer procedure from one vessel to another vessel disappears, the percentage of test organisms that find the same dose of food after 20 minutes is determined in each group.

After that, normalized spectra of behavioral reactions are calculated for each test organism of the second part of the laboratory culture of test organisms in the same way as it was done for test organisms of the first part of the laboratory culture of test organisms. Then calculate the average values (x ₂ ) and standard errors of the average values (y ₂ ) of the normalized specific spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms. the results obtained in this case are presented in table. 13.

 Then, the obtained average values of the normalized specific spectra of behavioral reactions of test organisms of the second part of the laboratory culture are compared with the average values of the corresponding normalized specific spectra of behavioral reactions of the first part of the laboratory culture of test organisms using the standard formula (1) of example 1.

Then choose from a set of model toxicants and fresh artificial pure reference water, as non-toxic control water, those toxicants for which the average values of all behavioral reactions of the normalized spectra of behavioral reactions of test organisms of the first part of the laboratory culture of test organisms do not statistically significantly differ from the average values of the corresponding behavioral reactions of normalized spectra of behavioral reactions of test organisms of the second part of the laboratory culture of test organisms, i.e., those for The absolute value of t _st does not exceed 2.0 for each behavioral reaction of the spectra of behavioral reactions of test organisms. Selected model toxicants and their concentrations are presented in table. fourteen.

Then calculate the average percent values (P ₂ ) for each population of all groups of test organisms of the second part of the laboratory culture of the test organisms and the standard errors of the average percent values (Z ₂ ) of the test organisms that found the same dose of food after 20 minutes. They are equal, respectively, P ₂ = 42 and Z ₂ = 14. Then compare the average percentage of test organisms for all groups of the second part of the laboratory culture of test organisms that found the same dose of food for the same time interval (P ₂ ), with the average for each set all groups of test organisms, the first part of the lab culture test organisms percent values of test organisms that have found the same dose via the same feed time interval (P ₂₎ using the standard formula (1) of example 1. Select the model from all toxins, including of fresh artificial pure reference water, like non-toxic control water, those toxicants and their concentrations for which the average percentages of test organisms in the first part of the laboratory culture of test organisms that find the same dose of food after the same time interval do not statistically significantly differ from the corresponding average the percentage of test organisms of the second part of the laboratory culture of test organisms, i.e. those for which the absolute value of t _st does not exceed 2.0. In this case, it is copper sulfate in concentrations of 7.0 and 50.0 MAC. Then find the average concentration of copper sulfate by calculating the geometric mean of all selected concentrations. It is equal to 18.7 MAC.

 Then, a preliminary value of the toxicity of the test aqueous medium is determined first by comparing the main toxicants of the test aqueous medium determined as a result of the first comparison with the current standards for toxicological parameters and the degree of exceeding the maximum permissible concentrations. Since the tested aquatic environment refers to the type of surface water for cultural, domestic, and drinking purposes, the toxicity of which is estimated in the MPC for water for cultural, domestic and drinking purposes (according to [3]), in which the main toxicants defined according to this method variant are expressed of the tested aqueous medium, then the preliminary value of the toxicity of the tested aqueous medium is determined first by selecting the highest of the average concentrations of the main toxicants determined as a result those of the first comparison. It is equal to 7.0 MAC. Then, the preliminary toxicity value of the test aqueous medium is determined by selecting the largest of the average toxicants determined as a result of the second comparison. It is equal to 18.7 MAC. After that, the degree of general toxicity of the test aqueous medium is finally determined as the largest value of the two predefined degrees of general toxicity. It is equal to 18.7, which, according to the norms of the degree of exceeding the maximum permissible concentrations [5], corresponds to the range of the degree of exceeding the maximum permissible concentration from 10 to 100, the general toxicity of which is defined as "extremely high".

Literature
1. International Standard Organization (ISO). 6341: 1989 (E). "Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladoceta, Crustacea). International Standatd Otganization Publications, London, Great Britain.

 2. Copyright certificate of the USSR N 1270699, cl. G 01 N 33/18, C 02 F 3/22 "Method for determining the toxicity of aqueous media".

 3. Bespamyatnov G.P. and Krotov Yu.A. Maximum allowable concentrations of chemicals in the environment. M .: Chemistry, 1985, p. 528.

 4. G.F. Lakin. Biometrics. - M.: Higher School, 1973, p. 343.

 5. Guidelines for determining the real load on a person of chemicals coming from atmospheric air, water and food. - M.: Ministry of Health of the USSR, 1986, p. 46.

 6. Regulatory data on the maximum permissible levels of pollution by harmful substances of environmental objects. SPb., "AMECOS", 1994, p. 234.

Claims (2)

 1. A biological method for determining the degree of general toxicity and the main toxicants of the aquatic environment, based on a comparison of physiological parameters of the functional state of the test organisms of aquatic organisms in the test sample of the aquatic environment and a model solution of the toxicant, characterized in that they are pre-grown on artificial clean reference water and feed with constant laboratory culture of test organisms by closely related crossing of individuals of one species of hydrobionts, the natural environment is inhabited which, according to the salinity interval, corresponds to the salinity interval of the tested aqueous medium, then they are initially determined in arbitrary units over the same time interval in vessels smaller in volume than vessels for the maintenance of the laboratory culture of test organisms, locomotor activity and the spectrum of behavioral reactions as a constant set of values, each of which corresponds to the degree of manifestation of a particular behavioral reaction of the test organism in arbitrary units, for each test organism of the first part is grown th laboratory culture of test organisms in fresh artificial pure reference water of the same temperature at which the laboratory culture of test organisms is contained, and determination is made after the protective organisms react to the process of transfer from one vessel to another vessel, after which withstand test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water in vessels of a smaller volume than vessels for the maintenance of laboratory culture of the test organism s, during the time of adaptation to a new aquatic environment and vessel under the regime of maintaining the laboratory culture of the test organisms and secondly determine the locomotor activity and the spectrum of behavioral reactions for each test organism of the first part of the laboratory culture of the test organisms in fresh artificial pure reference water at a temperature in vessels identical, respectively, to the temperature and vessels used for the initial determination of locomotor activity and the spectrum of behavioral reactions for each test organism in the first hour the laboratory culture of test organisms in fresh artificial reference water for the time interval and in arbitrary units that were used to initially determine the locomotor activity and behavioral spectrum for each test organism of the first part of the laboratory culture of test organisms in fresh artificial pure reference water moreover, the determination is made after the disappearance of the test-organisms of the defensive-defensive reaction to the transfer process from one vessel to another vessel, then it is first determined locomotor activity and a spectrum of behavioral reactions for each test organism of the second part of the laboratory culture of test organisms in a set of different concentration series of solutions of each of the model toxicants corresponding to possible toxicants of the test aqueous medium at a temperature and in vessels identical to temperature and vessels, respectively used for the initial determination of locomotor activity and the spectrum of behavioral reactions for each test organism of the first part of the laboratory culture anisms in fresh artificial pure reference water, during the time interval and in arbitrary units that were used to determine the locomotor activity and behavioral spectrum for test organisms of the first part of the laboratory culture of test organisms in fresh artificial pure reference water, all solutions of model toxicants prepared on fresh artificial pure reference water, each test organism is placed only once in a model toxicant solution, and determination is made after disappearance I test organisms have a defensive-defensive reaction to the process of transfer from a vessel to another vessel, after which the test organism of the second part of the laboratory culture of test organisms is maintained in a series of solutions of model toxicants for a time interval and in vessels identical to the time interval and vessels used to withstand the test organisms of the first part of the laboratory culture of the test organisms in fresh artificial pure reference water, under the conditions of the laboratory culture of the test organisms, and each test organism is kept in that model toxicant solution in which its locomotor activity and the spectrum of behavioral reactions were first determined, then the locomotor activity and behavioral reaction spectrum for each test organism of the second part of the laboratory culture of test organisms in a fresh, artificial pure reference water at a temperature and in vessels identical to the temperature and vessels used to determine the locomotor activity and the spectrum of behavioral reactions of the test organism the first part of the laboratory culture of the test organisms in fresh artificial pure reference water, for the time interval and in arbitrary units that were used to determine the locomotor activity and the behavioral spectrum of the test organisms of the first part of the laboratory culture of test organisms in the fresh artificial pure reference water, moreover, the determination is made after the disappearance of the test-organisms defensive-defensive reaction to the process of transfer from a vessel to another vessel, and the spectrum of behavioral reactions died their test organisms are considered to be equal to zero in all values, after which the obtained primary and secondary values of the locomotor activity of the test organisms of the first and second parts of the laboratory culture of test organisms are conditionally divided into several intervals and for each interval they are calculated for their locomotor activity this interval of test organisms means and standard deviations from the average values of the spectra of behavioral reactions characterizing the functional state of test organisms in a series of solutions of model toxicants and artificial pure reference water, by calculating the average values and standard deviations from the average values for each behavioral reaction of the indicated spectra of behavioral reactions, then locomotor activity and the spectrum of behavioral reactions for each test organism of the third part of the laboratory culture of test organisms are first determined in a test sample of an aqueous medium at a temperature and in vessels identical to the temperature and vessels used to determine loco, respectively motor activity and the spectrum of behavioral reactions for each test organism of the first and second parts of the laboratory culture of test organisms in fresh, artificial pure reference water and a series of solutions of model toxicants, for the time interval and in arbitrary units that were used to determine locomotor activity and the spectrum of behavioral reactions of the first and second parts of the laboratory culture of test organisms in fresh artificial pure reference water and series of solutions of model toxicants, each of which The t-organism is used for testing only once and the determination is made after the protective organisms react to the transfer process from the vessel to another vessel after the test organisms disappear, after which the test organisms of the third part of the laboratory culture of the test organisms in the test aqueous medium are kept for an interval time and in vessels identical, respectively, to the time interval and vessels used to withstand the test organisms of the first and second parts of the laboratory culture of test organisms in fresh clean reference water and a series of solutions of model toxicants under the regime of laboratory culture of test organisms, then the locomotor activity and the spectrum of behavioral reactions of each test organism of the third part of the laboratory culture of test organisms in fresh artificial pure reference water for a time interval and vessels identical to the time interval and vessels used to determine the locomotor activity and the spectrum of behavioral reactions of each test organism of the first and second Astey laboratory culture of test organisms in fresh artificial pure reference water and a series of solutions of model toxicants, over a time interval and in arbitrary units, which were used to determine the locomotor activity and the spectrum of behavioral reactions of each test organism of the first and second parts of the laboratory culture of test organisms in fresh artificial pure reference water and a series of solutions of model toxicants, and the determination is made after the disappearance of the test organisms defensively th reaction to the transfer process from a vessel to another vessel, after which the corresponding primary and secondary defined spectra of the behavioral reactions of each test organism of the third part of the culture of test organisms are compared with the calculated average values of the spectra of behavioral reactions of the test organisms of the first and second parts of the laboratory culture test organisms having the same intervals of locomotor activity, calculating the differences between the values of each behavioral reaction of the spectra of behavioral reactions of each t of an organism of the third part of the laboratory culture of test organisms and the calculated average values of behavioral reactions of the spectra of behavioral reactions of test organisms of the first and second parts of the laboratory culture of the test organisms, divide them into calculated standard deviations of the average values of the corresponding behavioral reactions of the spectra of behavioral reactions of the test organisms of the first and the second part of the laboratory culture of test organisms and are selected from a set of model toxicants, including artificial pure reference water to toxicant in permissible non-toxic concentrations, those toxicants for which the sum of the absolute values of the differences between the values of each behavioral reaction of the spectra of behavioral reactions of each test organism of the third part of the laboratory culture of the test organisms and the calculated average values of the behavioral reactions of the spectra of behavioral reactions of the test organisms of the first and second parts of the laboratory culture of test organisms divided by the calculated standard deviations of the average values of the corresponding behavioral reactions spectra of behavioral reactions of test organisms of the first and second parts of the laboratory culture of test organisms, for each spectrum of behavioral reactions of each test organism of the third part of the laboratory culture of test organisms does not exceed twice the number of behavioral reactions in each spectrum of behavioral reactions of test organisms of the first and second parts of the laboratory culture of the test organisms, then for each selected toxicant, its average concentration is found by averaging all selected as a result of the comparison its concentration for all test organisms of the third part of the laboratory culture of test organisms, while its concentration is taken equal to zero for each test organism, the behavioral response spectra of which were not revealed when comparing the presence of this toxicant, and judge the presence in the test aqueous medium of each main toxicant according to the found values of the average concentrations of the corresponding toxicant, and the degree of general toxicity of the tested aqueous medium is determined by comparing certain basic toxic ntov aqueous test medium with the test current in an aqueous environment regulations of Indicators and toxicological powers exceeding the maximum allowable concentrations.  2. The biological method for determining the degree of general toxicity and the main toxicants of the aquatic environment, based on a comparison of the physiological parameters of the functional state of aquatic organisms in the test sample of the aquatic environment and a model solution of the toxicant, characterized in that they are pre-grown on artificial pure reference water and feed with a constant laboratory culture of test organisms by closely related crossing of individuals of one species of hydrobionts, whose natural habitat is the salinity interval corresponds to the salinity interval of the test aqueous medium, and the average time interval required for the grown test organisms of the laboratory culture to find the same dose of the same type of feed at a constant distance is determined, and the type of feed is identical to one of the feeds of the laboratory culture of the test organisms, after what the first part of the test culture organisms of the laboratory culture can withstand in fresh, artificial, pure reference water, and the second part in a set of different solution concentration series in each of the model toxicants corresponding to possible toxicants of the test aqueous medium, in vessels of a smaller volume than vessels for the maintenance of laboratory culture of test organisms, for the same time interval sufficient to adapt to new environments and vessels, in the laboratory culture of test organisms, and each test organism is placed only once in a model toxicant solution, then locomotor activity and sp are determined in arbitrary units for the same time The behavioral reaction spectrum, which is a constant set of values, each of which expresses the degree of manifestation of certain behavioral reactions of the test organism in arbitrary units, for each test organism of the first and second parts of the hydrobiont culture in fresh, artificial, pure reference water, at the same temperature as water used to contain the laboratory culture of the test organisms in the same vessels, smaller in volume than the vessels for the maintenance of the laboratory culture of the test organisms, and the definition of after the disappearance of the defensive-defensive reaction in the test organisms to the procedure of transferring from one vessel to another vessel, then the test organisms of the second part of the laboratory culture of the test organisms are divided into groups, each of which contains the same number of test organisms, previously maintained in one in the same solution from a set of series of solutions of model toxicants, the test organisms of the first part of the laboratory culture of test organisms are also divided into groups, each of which contains the same number of test organisms as each group of test organisms of the second part of the laboratory culture of test organisms, and locomotor activity and a spectrum of behavioral reactions for each test organism of each group of the first and second parts of the laboratory culture of test organisms in fresh, artificial units are determined in arbitrary units for the same time clean reference water at the same temperature as the water for the maintenance of the laboratory culture of the test organisms, after placing each group of test organisms in the same separate vessels, smaller in volume than For the maintenance of the laboratory culture of test organisms, and the disappearance of test defenses in the test organisms to the transfer procedure from one vessel to another vessel, then each group of test organisms of the first and second parts of the laboratory culture of test organisms is placed in other vessels smaller in volume than vessels for maintaining laboratory culture of test organisms, with fresh artificial pure reference water of the same temperature as water for maintaining laboratory culture of test organisms at the same distance from the same dose of the same feed in the vessel, identical to the dose and feed used to determine the time interval between the test organisms of the laboratory culture in the vessels for their maintenance, and after the test organisms disappear a defensive-defensive reaction to the transfer procedure from the percentage of test organisms in each group that determine the same dose of food after the same time interval equal to the previously determined average time interval required by the test organisms of a laboratory culture to find an identical dose of food at an identical distance in the vessels for maintaining the laboratory culture of the test organisms, then certain locomotor activity values of all test organisms of the first and second parts of the laboratory culture of the test organisms are conventionally divided into several intervals and for each interval are calculated for falling in the value of their locomotor activity in this interval of test organisms, the average values and standard deviations from the average values of the spectra of reactions characterizing the functional state of test organisms in fresh artificial pure reference water after they have been kept for the same time necessary for test organisms to adapt to new environments and vessels, in fresh artificial pure reference water and in solutions from a set of series solutions of model toxicants, then the average for each set of all groups of test organisms of the first and second parts of the laboratory culture of test organisms, aged in fresh, artificial, pure, is calculated. water and in each of the solutions of a set of series of solutions of model toxicants, the percentage of test organisms that find the same dose of food after the same time interval equal to the previously determined time interval required by the test organisms of the laboratory culture of the test organisms to find an identical dose of food on an identical the distance in the vessels for the maintenance of the laboratory culture of the test organisms, after which a third of the test organisms of the laboratory culture of the test organisms in the test aqueous medium are maintained de containing soil elements during the regime of the laboratory culture of the test organisms during the time interval and in vessels identical to the time interval and the vessels used to withstand the test organisms of the first and second parts of the laboratory culture of the test organisms in a fresh artificial clean reference water and solutions from a set of series of solutions of model toxicants, and each test organism is used only once and the number of test organisms of the third part of the laboratory culture must be a multiple The test organisms of one group of the first and second parts of the laboratory culture of the test organisms are then determined in the same arbitrary units and during the same time interval as for the test organisms of the first and second parts of the laboratory culture, locomotor activity and the spectrum of behavioral reactions test organisms of the third part of the laboratory culture in fresh artificial pure reference water at the same temperature and in the same vessels that were used to determine the locomotor activity and the spectrum of behavioral reactions th test organisms of the first and second parts of the laboratory culture of test organisms, and the determination is made after the protective organisms react to the process of transfer from one vessel to another vessel after the test organisms disappear, after which test organisms of the third part of the laboratory culture of test organisms are divided into groups equal in number of test organisms to the groups of the first and second parts of the laboratory culture of the test organisms, and place each group of test organisms of the third part of the laboratory culture in a separate vessel with fresh art clean reference water of the same temperature as the temperature of the water for maintaining the laboratory culture of the test organisms, at the same distance from the same dose of the same feed, and the vessels used, the distance and the feed are identical to the vessels, distance and feed used to determine percent of test organisms of the first and second parts of the laboratory culture of test organisms that found the same dose of food for the same time interval, and after the disappearance of the test organisms defensively reactions to the transfer procedure from one vessel to another vessel, determine in each group of test organisms of the third part of the laboratory culture of the test organisms the percentage of test organisms that find the same dose of food over a time interval equal to the previously determined time interval required by the test culture of the laboratory culture test organisms to find an identical dose of food at an identical distance in the vessels to contain the laboratory culture of the test organisms, and then calculate the average percentage of the test organism in for all groups of the third part of the laboratory culture of the test organisms that found the same dose of food for the same time interval, then the spectra of behavioral reactions of each test organism of the third part of the laboratory culture are compared with the calculated average values of the spectra of behavioral reactions of test organisms of the first and second parts of the laboratory culture test organisms having the same intervals of locomotor activity, calculating the differences between the values of each behavioral reaction of the spectra of behavioral reactions to each test organism of the third part of the laboratory culture of test organisms and the calculated average values of the behavioral reactions of the spectra of behavioral reactions of the test organisms of the first and second parts of the laboratory culture of the test organisms and dividing them by the calculated standard deviations of the average values of the corresponding behavioral reactions of the spectra of behavioral reactions of the test organisms the first and second parts of the laboratory culture of test organisms, and choose from all model toxicants, including artificial pure reference ode as a toxicant in permissible non-toxic concentrations, those toxicants for which the sum of the absolute values of the differences between the values of each behavioral reaction of the spectra of behavioral reactions of each test organism of the third part of the laboratory culture of the test organisms and the calculated average values of the behavioral reactions of the spectra of behavioral reactions of the test organisms of the first and the second part of the laboratory culture of test organisms divided by the calculated standard deviations of the average values of the corresponding behavioral reactions of the spectra of behavioral reactions of test organisms of the first and second parts of the laboratory culture of test organisms, for each spectrum of behavioral reactions of each test organism of the third part of the laboratory culture of test organisms does not exceed twice the number of behavioral reactions in each spectrum of behavioral reactions of test organisms of the first and second parts of the laboratory culture of the test organisms, then for each selected toxicant find its average concentration by averaging all selected as a result of a comparison of its concentrations for all test organisms of the third part of the laboratory culture of test organisms, while its concentration is taken equal to zero for each test organism, the behavioral response spectra of which were not revealed during the comparison of the presence of this toxicant, and judge the presence in the test water medium of each major toxicant according to the found values of the average concentrations of the corresponding toxicant, then compare the average values of the percentage of test organisms for all groups of the third part of the laboratory test organisms that found the same dose of food for the same time interval, with average percentages of test organisms that found the same dose of food after the same time interval for each set of all groups of test organisms of the first and second parts of the laboratory culture of test organisms, and choose of all model toxicants, including artificial pure reference water as a toxicant in acceptable non-toxic concentrations, those toxicants that correspond to the closest average percentages, then determine first, the toxicity of the test aqueous medium is determined by first comparing the toxicological parameters and the degree of exceeding the maximum permissible concentrations determined as a result of the first comparison of the main toxicants of the test aqueous medium with the toxicity indicators, and then by comparing the basic toxicants of the test aqueous determined as a result of the second comparison media with toxicological standards for the tested aquatic environment and the degree of exceeding the maximum permissible concentrations, after which the degree of general toxicity of the test aqueous medium is finally determined as the largest value of the two predefined degrees of general toxicity.

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