RU2215290C2 - Method of estimating toxicity of pollutants in far east seas - Google Patents

Abstract

FIELD: analytical methods and environmental monitoring. SUBSTANCE: toxicity of pollutants is estimated by means of test object, in particular pre-larva of Japan anchovy (Engraulis japonicus). Invention can be used when projecting and predicting economical activity. EFFECT: enabled reliable estimation using appropriate test object. 1 tbl

Description

 The invention relates to the fishing industry, specifically to applied ecology and toxicology, and can be used to assess the toxicity of water pollutants in the Far Eastern seas when designing and predicting human economic activity.

A known method for the quantitative determination of organophosphorus pesticides (A. S. USSR 1111270, IPC 4 A 01 K 61/00, G 01 N 33/18), providing for the placement in the solution with the analyzed sample as test objects daphnia (Daphnia magna), accounting the duration of their survival at 34-36 ^o C and the subsequent determination of the amount of pesticide according to the calibration schedule.

 A known method for the detection of toxic metal cations in water (AS USSR 1168168, IPC 4 A 01 K 61/00, G 01 N 33/18), which consists in placing in the analyzed sample as a test object (Daphnia magna) and accounting continuing their survival.

 A device for the biological assessment of the toxicity of liquids (AS USSR 1242082, IPC 4 A 01 K 61/00), including a chamber made of a transparent material, with branched crustaceans placed in it, a system for bringing water to the chamber and removing it, block water supply, a light source and sequentially a photodetector, amplifier and pulse shaper.

 A device for the biological assessment of toxicity of water (AS USSR 946027, IPC 4 A 01 K 61/00, G 01 N 33/18), containing a reservoir for placing mollusks, filled with test water, and a device that records the movement of the mollusk flaps.

 A known method for determining the toxicity of aquatic environments (AS USSR 1270699, IPC 4 G 01 N 33/18, C 02 F 3/32), including the study of adaptation of aquatic organisms in them - gastropods (use either Lymnaea lagotis or Lymnaea auricularia, or Viviparus viviparus), preliminary calibration of the degree of sensitivity and resistance of aquatic organisms to a reference toxicant, carrying out test actions on experimental series of aquatic organisms by certain concentrations of the studied media, ensuring the constancy of external conditions and a stepwise increase in concentrations when exposed to each I will take from a subsequent series of aquatic organisms, recording the behavior of aquatic organisms, calculating motor activity for each concentration from it, and assessing the degree of toxicity of aquatic environments.

 A known method for determining the toxicity of aquatic environments (AS USSR 1112276, IPC 3 G 01 N 33/18, C 02 F 3/32) by studying the adaptation of aquatic organisms in them - gastropods (Lymnaea lagotis and Lymnaea auricularia). A preliminary calibration of the degree of sensitivity and resistance of aquatic organisms to a reference toxicant is carried out, test effects on experimental series of aquatic organisms are determined by determined concentrations of the studied media, ensuring the constancy of external conditions and stepwise increase in concentrations when each of the subsequent series of aquatic organisms is exposed, the reaction of the behavior of aquatic organisms is recorded, and their locomotor activity for each concentration and assess the degree of toxicity of aqueous media by means of Avnivaniya calculated on motor activity levels of survival of aquatic organisms of the control and experimental series.

 A known method of bioindication of toxicity of the aquatic environment (A. S. USSR 1546904, IPC 5 G 01 N 33/18, A 01 K 61/00), providing for the placement of mollusks Lymnaea stagualis in chambers with the test environment and clean water - experience, control, their keeping, introducing a radioactive label, extracting them and comparing the state of mollusks in the experiment and control and establishing the presence of contamination by deviating the true radioactivity of the mollusks in the experiment compared with the control.

 A known method of biotesting toxicity of wastewater (AS USSR 1573376, IPC 5 G 01 N 33/18) by registering an electrocardiogram in fish and changing RR intervals.

 A known method for assessing the toxicity of wastewater (AS USSR 1097947, IPC 3 G 01 N 33/18, C 12 Q 1/00) using microorganisms, where the strain Saccharomyces cerevisial 15B-P4 was used as a test object.

 A known method for the biological assessment of water toxicity and a device for the biological assessment of water toxicity (A. S. USSR 1068083, IPC 3 A 01 K 61/00, G 01 N 33/18), which provides for the separation of daphnia into two groups - the main and control, placement each group in the corresponding chamber, passage through the chamber with the control group of daphnia of pure water and through the chamber with the experimental group of the studied water, registration of the physiological state of daphnia in the groups and comparison of the results.

 A known method for determining the degree of toxicity of water (AS USSR 1405745, IPC 4 A 01 K 61/00, G 01 N 33/18) containing toxic compounds with membrane -olytic activity, providing for the introduction into the container with the incubation medium and a pigment-containing test the object of the studied water sample and determining the degree of toxicity of the compounds contained in it by controlling the change in the intensity of the release of pigments into the incubation medium under the influence of toxic compounds. Seawater is used as the incubation medium and Callithamnion red seaweed is used as the pigment-containing test object.

 A known method for determining the toxicity of chemicals (AS USSR 1564539, IPC 5 G 01 N 33/18, 33/00), including the cultivation of a strain of Saccharomyces cerevisial 15B-P4 on a liquid nutrient medium, the impact of the test substance on it, followed by evaluation of the obtained results.

 A known method for evaluating the toxicity of a liquid (AS USSR 1515105, IPC 4 G 01 N 33/18), which provides for the cultivation of a photosynthetic test object, lighting the test object with light and determining the luminescent characteristics, the change of which is used to judge the toxicity of the controlled liquid.

 A known method of bioindication of heavy metals in seawater (AS USSR 1479876, IPC 4 G 01 N 33/18), providing for placement in chambers with clean water and incubation of the test object - macroalgae, their adaptation to the background content of heavy metals, division chambers into two groups - experimental and control, adding test water to experimental chambers with macroalgae, measuring the physiological characteristics of algae, comparing them and concluding that the heavy metals in the test water are higher than the background when statistically available The difference in the physiological characteristics of the test object in the experiment compared with the control.

 A known method of bioindication of toxicity of wastewater (AS USSR 1578650, IPC 5 G 01 N 33/18, A 01 K 61/00), providing for the placement of a biological test object in samples with clean - control and test - water experience, aging in them, the registration of the physiological indicator of the test object and the judgment of the toxicity of the test water with differences in the physiological indicator in the experiment compared with the control, where a culture of dissociated sponge cells is used as the test object.

 A known method for the determination of cyanides in wastewater (AS USSR 1576860, IPC 5 G 01 N 33/18), including sampling water, incubating water plants in it, followed by determining the activity of the enzyme.

 A known method for determining the toxicity of aquatic environments (AS USSR 1328756, IPC 4 G 01 N 33/18, C 02 F 3/34), including the preliminary adaptation of aquatic organisms to conditions in a clean aquatic environment and calibration of the degree of sensitivity of aquatic organisms to a reference toxicant where fish, shellfish, crayfish, and higher aquatic plants are used as hydrobionts.

 A known method for determining the toxicity of liquids and a device for its implementation (A. S. USSR 1010557, IPC 3 G 01 N 33/18), comprising pre-culturing a photosynthetic test object, introducing the cells of the test object into the reaction chamber with a control liquid to determine physiological the state of the test object, the supply of a controlled fluid to the chamber, maintaining the test object and determining its physiological state, followed by a comparison of the physiological state of the test object in the controlled and control fluid x

 A known method of monitoring water for toxicity (AS USSR 1440729, IPC 4 A 01 K 61/00, G 01 N 33/18) by assessing the condition of the gill apparatus of carp fish.

 A known test object for determining the genotoxicity of water-soluble xenobiotics (AS USSR 1784147, IPC 5 A 01 K 61/00, G 01 N 33/18), where aquarium fish of the species Hotobranchius are used as a test object to determine the genotoxicity of water-soluble xenobiotics guntheri.

 A known method for assessing the toxicity of the aquatic environment (AS USSR 1784914, IPC 5 G 01 N 33/18), which provides for maintaining the biological test object in the analyzed medium, recording the physiological parameter of the test object and judging the toxicity of the analyzed medium by the value of the parameter, where as a biological test object using a segment of coleoptl plants.

 A known method for assessing the toxicity of the aquatic environment (AS USSR 1784915, IPC 5 G 01 N 33/18, A 01 K 61/00), providing for the introduction of crustaceans-filter in the experimental and control environment with a suspension of microalgae keeping them, parameter registration, characterizing the trophic activity of crustacean-filtering organisms, and assignment to toxic test water if experimental data differ from the control, where Epishura baicalensis crustacean is used as crustacean-filtering material.

 There is a method of assessing the toxicity of the aquatic environment (RF Patent 2003097, IPC 5 G 01 N 33/18, A 01 K 61/00), which includes placing benthic sponges in the experimental and control environment, keeping them, recording the physiological parameter of the sponges and judging the toxicity of the test environment according to the experimental data in comparison with the control.

 A known method of biological assessment of the toxicity of the aquatic environment (AS USSR 1837225, IPC 5 G 01 N 33/18), including exposure to a biological test object by direct electric current and subsequent toxicity assessment, where the neuromuscular muscle is used as a test object frog drug.

 A known method for determining the copper content in the aquatic environment (AS USSR 1835234, IPC 5 G 01 N 33/18), providing for the placement of fertilized bivalve mollusk eggs in samples with clean (control) and test (experiment) water, incubation, obtaining larvae and judgment on the presence of copper with differences in the physiological indicator in the experiment compared with the control.

 A known method of monitoring the toxicity of the aquatic environment (RF Patent 1814510, IPC 5 A 01 K 61/00, G 01 N 33/18), which provides for the cultivation of test objects - organisms of zooplankton-filtrators, placing them in the studied aqueous medium, supplying a test medium feed, which is a suspension of balls of polymer material, measuring a parameter that characterizes the filtration activity of organisms and correlating with their physiological state, and judging the toxicity of the environment based on the measurement.

 A known method for determining the toxicity of natural waters (RF Patent 2029948, IPC 6 G 01 N 33/18), including adaptation and measurement of motion parameters of aquatic animals, where marine crustacean gammarus are used as a test object.

 A known method for determining the toxicity of water (AS USSR 1762232, IPC 5 G 01 N 33/18), including incubating the sample with a test object, followed by determination, characterized in that as a test object use red blood cells.

 A known method for detecting fluid toxicity (AS USSR 1702304, IPC 5 G 01 N 33/18), which consists in introducing a test object into the control and test media with the subsequent change in the electrochemical response of the electrodes placed respectively in the control and test media, different by the fact that the alga of the genus Cylorella is used as a test object.

 A known method for determining the aquatic environment (AS USSR 1698757, IPC 5 G 01 N 33/18), which provides for the determination by the method of dark and light vials of the amount of oxygen consumed jointly by autotrophic and heterotrophic organisms in clean and test water and the classification of the test water as toxic when the deviation of the data obtained in the experiment from the control.

 A known method for determining pesticidal intoxication of fish (AS USSR 1693548, IPC 5 G 01 N 33/18), which provides for the separation of fish into experimental and control lots, placing the experimental fish batch in the test environment, taking blood samples from fish of both parties, washing and erythrocyte hemolysis, determination of hemoglobin concentration, treatment with sodium nitrite, determination of hemoglobin half-oxidation time by recording the dynamics of solution extinction, comparison of experimental and control data, and establishment of pesticidal intoxication in fish of the experimental batch in the case of deviations of the experimental data from the control.

 A known method for determining the concentration of N-tributylstannyl-4-chlorophthalimide in an aqueous medium (AS USSR 1689847, IPC 5 G 01 N 33/18) by introducing dilutions of the substance into the wells of nutrient agar seeded with a test culture, followed by determining the concentration of the substance by the size of the zones of growth inhibition of the test culture, characterized in that in order to increase the accuracy of determination, a bacterial strain Bacillus sphaericus 386 is used as a test culture.

 A known method for determining the toxic effects of chemicals contained in the aquatic environment on the culture of planktonic aquatic organisms (AS USSR 168816147, IPC 5 G 01 N 33/18), providing for the cultivation of planktonic aquatic organisms in experimental and control thermostatic chambers, the introduction of experimental chambers of the studied medium, maintaining the culture of hydrobionts in the presence of the studied medium, measuring while maintaining the average speed of movement over the ensemble of hydrobionts in the experiment and control by the correlation-Doppler spectroscopy method ii, evaluation of relationship of moving aquatic organisms in a culture to the number of fixed respectively to the experiment and control, the evaluation value from the data on the motion energy of aquatic organisms and judgment of toxic effects of the investigated medium largest energy in the movement of aquatic organisms in the experiment.

 A known method of analyzing a liquid medium for the presence of melatonin (A.S. USSR 1645892, IPC 5 G 01 N 33/18), comprising placing tailless amphibians as a test object in a culture medium, adding a test sample to it, keeping the test object, recording the nature of the distribution of pigment in the pigment cells (melanophores) of the test object and determining the presence of melatonin in the test sample by the degree of clarification of the test object, characterized in that in order to increase the accuracy and simplify the analysis, as well as expand the function According to the method’s potential, isolated skin areas previously adapted to the dark background for at least 45 minutes use tailless amphibian larvae as a test object, and the test object with the test sample is kept in the light for at least 30 minutes, while the cultural volume medium is from 1 to 2 ml.

 A known method for determining the toxicity of water and aqueous solutions containing biologically active substances (A. S. USSR 1751669, IPC 5 G 01 N 33/18), by exposing the studied sample to plant cells with subsequent evaluation, characterized in that in order to increase sensitivity of the method, the effect is carried out by introducing a part of the test sample into the leaf leaves of duckweed.

 A known method for determining the toxicity of industrial wastewater (AS USSR 1751670, IPC 5 G 01 N 33/18), which involves sampling microorganisms of activated sludge, their incubation in environments with wastewater components and control clean water, subsequent centrifugation of the media, disintegration , determination of protein fractions by electrophoresis in flat blocks of polyacryl polyamide gel, staining of protein fractions with the establishment of molecular forms of the enzyme and their activity in experimental and control media, and classification of the experimental medium as toxic in case of changes Nia activity of molecular forms of the enzyme in the test environment as compared to the control.

 There is a method of determining water quality in coastal recreational areas of the sea (A. S. USSR 1751671, IPC 5 G 01 N 33/18, A 01 K 61/00), which provides for sampling water in the study area, assessing the abundance of two groups of test microorganisms and determining the water quality by the number of these test microorganisms, where heterotrophic halotolerant and saprophytic microorganisms are used as test microorganisms.

 There is a method of determining the degree of acidification of water bodies (AS USSR 1741068, IPC 5 G 01 N 33/18, A 01 K 61/00), which involves taking samples from a reservoir with a biological test object, analysis of the biological characteristics of the test object and judgment the degree of acidification of the reservoir according to this characteristic, characterized in that, in order to simplify the method, perches are used as a test object, and the presence and nature of the pattern of the center of the bookmark of scales taken in the dorsal fin region is used as a biological characteristic.

 A known method for assessing the toxicity of aquatic environments (AS USSR 1730581, IPC 5 G 01 N 33/18, A 01 K 61/00), providing for the placement of a biological test object in samples with a clean control and studied - experimental environment, keeping in them, the subsequent registration of the motor activity of the test object and the classification of the test medium as toxic in the absence of motor activity of the test object in the experiment as compared with the control, characterized in that sponge larvae are used as a test object to increase the accuracy of the assessment.

 A known method for determining the toxicity of water (AS USSR 1709212, IPC 5 G 01 N 33/18), including incubating the test organism in a translucent cell filled with test water, in the dark and in the light, followed by establishing the value of photosynthesis by measuring using oxygen sensor of oxygen concentration in the water under study in the light phase, characterized in that higher water plants are used as a test organism.

 There is a known method of biotesting for monitoring coastal zones, where embryos of echinoderms and bivalves are used as a test object (I.V. Khodakov. Method for using early embryos of the Black Sea mussel for biotesting when monitoring coastal zones of the northwestern shelf // Theses of the All-Russian Conference "Ecosystems" Russian seas under anthropogenic press (including fishing) ", Astrakhan, September 20-22, 1994. - Astrakhan, 1994. - P.352-353), providing for the cultivation of test objects, where the beginning of the experiment was etsya time premises inseminated eggs in the test environment. The criterion for the influence of the test medium on the development of embryo culture is a significant difference between the distributions of embryos according to the crushing stages in the control and the test medium (chi-square); approximately 500 embryos in each repetition of the control and test sample after formalin fixation are calculated for its calculation. Comparison of the degree of toxicity of samples is carried out using indicators of synchronization of fragmentation separately for groups of one-, two- and four-cell embryos taken in relation to the control.

 There is a method of judging water toxicity by determining the developmental abnormalities in Atlantic fish when exposed to pollutants (Pollution and development abnormalities of Atlantic fishes / Longwell Arlene Crosby, Chang Sukwoo, Habert Andrew, Hughes Jamts B., Perry Pean // Environ. Biol. Fish . - 1992. -35, 1. - P.1-21). On pelagic caviar of mackerel, Scomber scombrus, it has been shown that water pollution causes mortality, developmental abnormalities, and abnormal chromosome division in embryos. Adult individuals of this species, as well as rhombus, Scophthalmus acquosus and winter flounder, Pseudopleuronectes americanus, from the most polluted coastal areas have a greater proportion of mitotic anomalies than fish from less polluted areas cf. and sowing. Atlantic. The development of genetic and epigenetic resistance to the effects of pollution can depend on fluctuations in the environment, climate change and fishing intensity.

 A known method for determining the toxic effect of wastewater on embryos and larvae of marine fish (Effects of sewage sludge on marine fish embryos and larvae / Costello Mark J., Gamble John C. // Mar. Environ, res. - 1992. - 33, 1, -P.49-74), which consists in identifying a significant toxic effect of wastewater on the survival of embryos of herring (Clupea harengus) and cod (Gadus morhua).

Known work to identify the influence of copper and lead on embryos and larvae of carp at two temperatures (Effect of copper and lead on common carp embryos and larvae at two temperatures / Lugowska kanarzyna, Jezierska Barbara // Folia Univ. Agr. Stetin. Pisc. - 2000 26. - P.29-38). The effects of Cu at a concentration of 0.2 and 0.25 mg / L and Pb at a concentration of 2.0 mg / L at a temperature of 20 and 26 ^o C on embryonic development, as well as on the quality and further development of carp larvae, were investigated.

 A well-known work is comparing the responses of pike and roach larvae to low pH and aluminum: sodium influx, development and activity (Comparison of the responses of the yolk-sac fry of hike (Esox lucius) and roach (Rutilus rutilus) to low pH and aluminum: Sodium influx, development and activity / Keinanen Marja, Peuranen Seppo, Nikinmaa Mikko, Tigerstedt Christina, Vuorinen Pekka J. // Aquat. Toxicol. -1999. - 47, 3-4. - P.161-179), which considers the effect of acidic water and A1 on newly hatched larvae of an acid-tolerant pike and acid-sensitive roach during a 1-10 day experiment, and the effect of a decrease in the level of metabolism in A1-containing e prelarvae pike on their survival.

 Known work to determine the biological effects of the combined effects of petroleum products and OCPs on the sturgeon embryogenesis (Varobieva A. A. Biological consequences of the combined effects of petroleum products and OCPs on the sturgeon embryogenesis // Abstract, 1st scientific-practical conference. Problems modern commodity sturgeon breeding ", Astrakhan, March 24–25, 1999. Astrakhan, 1999. - S.120-121). The work was carried out on eggs and hatching sturgeon and stellate sturgeon embryos. It was experimentally established that the combination of a mixture of organochlorine pesticides (OCP, DDT) with petroleum products (diesel fuel L-0.5 GOST305-82) is more dangerous for developing eggs and embryos than each individual ingredient, since petroleum products increase the solubility of pesticides in water. The mixture caused their death in a shorter time and at lower concentrations (1.5-2 times). In all cases, embryos at the hatching stage and early larvae are more vulnerable in comparison with eggs.

 Known work to determine the effects of developmental stages and duration of exposure on the embryotoxicity of diazinon (The role of development and duration of exposure to the embryotoxicity of diazinon / Hamm JT, Hinton DE // Aquat. Toxicol. - 2000.-48, 4. - C. 403-418). The effect of diazinon at a concentration of 1-26 mg / l on the development of medaka was studied. A daily macroscopic study of embryos and larvae revealed the main morphological manifestations of toxicity - swelling of the yolk veins, pericardial edema, spinal deformity, insufficient filling of the swimming bladder.

Some data are known on the physiological and biochemical reactions of rainbow trout during early development to dicyclopentadiene poisoning (Kurzykina L.G., Artemyeva N.V., Sudakova N.M. Some data on the physiological and biochemical reactions of rainbow trout during early development to poisoning dicyclopentadiene, // 8th Scientific Conference on Ecology, Physiology, and Biochemistry of Fish, September 30 – October 3, 1992: Abstracts, vol. 1 / Kar. Scientific Center of the Russian Academy of Sciences. Institute of Biol. - Petrozavodsk, 1992. - S.181-182), which shows that dicyclopentadiene (DCPD) at a concentration of 500-1000 mg / l inhibits the formation of the circulatory system in trout embryos, even at a concentration of 5-10 mg / l, the capillary tissue of the embryos was reduced. After the action of DCPD for 62 days (concentration of 0.01-5 mg / l), the amount of cholesterol and lipids in trout larvae was reduced by 17-22%, respectively, the effect of DCPD on the intensity of consumption of O _{2 was} shown.

Known work to determine the subchronic toxicity of formaldehyde baths for eggs and juvenile fish depending on the hardness of the water (Subchronische Fischtoxizitat von Formaldehydbadern auf Eier und Brut in Abhangigkeit von der Wasserharte / Meinelt T., Stuber A. // Fisher und Meichwirt. -1992. 43, 8. - P. 294-295 Toxicity of long formalin baths depending on water hardness was studied on caviar and larvae of striped zebrafish (Brachydanio rerio) .The total water hardness in the studied solutions of 37% formalin was 308.8 and 62, 5 mg CaCO ₃ / L. Evaluation of the results of the experiments was carried out every 24 hours with a total duration of and 144 h. It was found that in both soft and hard water, the formalin concentration of 0.264 μg / L did not affect the survival of eggs and hatching of juveniles.The survival of hatched larvae in hard water was slightly reduced, and in soft water it was already sharply reduced at formalin concentration 0.198 μl / L. In the control, the eggs were affected by fungi: in hard water groups, mushrooms developed on dead caviar, and in soft water groups on dead caviar and the nearest live eggs in the first 24 hours. In all cases with fungal formalin caviar was not noted. The number of deformed larvae in groups with formalin was slightly higher than in the control. To prevent the departure of juveniles, it is recommended to stop formalin treatment before hatching.

 Known is the work on determining the response of the cyclase system of rainbow trout caviar to the action of heavy metals (Mikhkieva BC, Shishkova N.A., Zalicheva I.N. Response of the cyclase system of rainbow trout caviar to the action of heavy metals // Biological Resources of the Baltic Sea Basin: Abstracts 23rd Scientific Conference on the Study of the Bodies of the Baltic States / USSR Academy of Sciences, - Petrozavodsk, 1991. P.102). The activity of enzymes synthesizing cyclic nucleotides in trout eggs was studied under the action of salts of Zn and Pb. On caviar survival, a conclusion was drawn on the toxicity of all salts used in the experiment. It is suggested that metabolic changes in the developing trout roe under the action of heavy metal salts, leading to significant loss of material, can be associated with fluctuation in the activities of the components of the cyclase system.

 A known technique for assessing the effect of water pollutants on fish embryogenesis (Kostrov B.P., Dokholyan V.K. Methodology for assessing the effect of water pollutants on fish embryogenesis // Methods of ichthyotoxicological studies: Abstracts of the First All-Union Symposium on ichthyotoxicological research methods Leningrad, October 1987 , - L., 1987. S. 65-67). The effect of toxic substances on the embryonic development of fish was determined by elucidating the mutagenic ability of pollutants. Studies on caviar and larvae of Kutum, silver carp, loach, sturgeon, salmon under the influence of toxicants of organic and inorganic nature revealed a significant increase in the level of abnormal mitoses in concentrations of toxicants that do not cause embryogenesis disturbances.

A known method for determining the toxic effects of various pollutants (dissolved petroleum products, polychlorinated biphenyls, lead, zinc, mercury, copper) on fish in the early stages of ontogenesis. (Patin S. A. Influence of pollution on biological resources and productivity of the oceans, Moscow: Food Industry, 1979, pp. 157-160), where caviar and larvae of Trachurus trachurus, Acipenser guldenstadti, Salmo are used as test objects trutta caspius, Hypophtalmichthys molitrix, Cyprinus carpio, Rutilus frisii kutum. The characteristics of the effect of toxicants LC ₅₀ (in μg / l) on the developing eggs and larvae of some marine and freshwater fish were determined (the duration of the experiments was 2-7 days).

 Until now, juveniles and adult mysids - (Cherkashin S.A., Reaction of hydrobionts (juvenile fish and crustaceans) of some toxicants: Abstract of Cand. Sci. Biol. Sciences - Sevastopol, 1986. - were used to assess the toxicity of pollutants in the Far Eastern waters). 17 pp.), Copepods (Cherkashin SA Using the method of functional loads to assess the state of zooplankton and the quality of the waters of the Far Eastern seas. // All-Russian conference "Ecosystems of the Russian seas in anthropogenic press (including fishing)": Abstract. okl. Kaliningrad. 1994. S.541-543), shrimp (Blinova N.K., Cherkashin S.A. Effect of phenol on the chemoreception of antennas of grassy chillim // Biological Sciences, 1987. 2. P.44-48, Ternovenko V .A. Biotesting of the marine environment with the help of behavioral reactions of crustaceans: Abstract of thesis, Candidate of Biological Sciences. - Sevastopol, 1989. - 24 pp.), Echinoderms at various stages of embryonic development - (Vashchenko MA Effect of water-soluble hydrocarbons of the lung diesel fuel for the development of germ cells and the quality of offspring in the sea urchin Strongylocentrotus nudus // Marine Biology. 1980, 4. P.68-73, Moyseichenko G.V., Cherkashin S.A. The use of gametes, embryos and sea urchin larvae to assess the toxicity of drilling fluids and their components. // Biology of the sea, Vol.21, 6, 1995. P. 419, Moiseichenko G.V., Scheglov V.V., Lukyanova O.N. Biological effects of heavy metals contained in drilling fluids // Fishery Issues. 2000. Vol. 1 2-3, part 2. - P.59-60), mussels - (Karpenko A.A., Tyurin A.N. Influence of sublethal effects of some environmental factors on the behavior of bivalves and biotesting issues // Biology of the shelf zones of the World Ocean: Abstracts of the All-Union Conference on of marine biology. Vladivostok, 1982, part 3, pp. 121-123, Khristoforova NK, Bioindication and monitoring of pollution of sea water by heavy metals. L .: Nauka, 1989. - 192 p., Chelomin V.P., Belcheva N.N., Zakhartsev M.V. Biochemical mechanisms of adaptation of the mussel Mytilus trossulus to cadmium and copper ions // Marine Biology. - 1998. - T.24, 5. - P.319-325), rowing ki - (Karpenko A.A., Tyurin A.N. Influence of sublethal influences of some environmental factors on the behavior of bivalves and biotesting issues // Biology of shelf zones of the World Ocean: Abstracts of the All-Union Conference on Marine Biology. Vladivostok, 1982, p. .3, P.121-123, Kholodenko G.M., Moisenchenko G.V., Cherkasova I.V. Histochemistry of the digestive system of juvenile scallop yearlings during zinc intoxication // V All-Union Conference on Aquatic Toxicology, Odessa, April 18-22 1988, Odessa, 1988. - P.150, Scheglov V.V., Moiseichenko G.V., Cherkashin S.A., Wow Rodnikova A.A. General patterns of changes in the stability of different stages of ontogenesis of marine aquatic organisms to the toxic effects of heavy metals and petroleum products // Y All-Union Conference on Aquatic Toxicology. Thes. dokl., Odessa, April 18-22, 1988, Moscow: Rotprint VNIRO, 1988. - P.86), tunics - (Tyurin AN, Khristoforova N.K. Assessment of the suitability of the tunic Lepidozona albrechti for bioassay of marine pollution environment with heavy metals and synthetic detergents // Biological Sea. 1993a. 3. P.97-105, Tyurin AN, Khristoforova NK Evaluation of the suitability of Ischnochiton hakodadensis chiton for biotesting marine pollution with heavy metals and detergents // Biol . of the sea. 1993 5-6. S. 100-106), juvenile fish - (Cherkashin S. A. Reaction of avoidance by hydrobionts (juvenile fish and crustaceans) of some toxicants: abstract of dissertation of Candidate of Biological Sciences - Sevastopol, 1986. - 17 pp., Moiseichenko GV, Scheglov VV, Lukyanova O. N. Biological effects of heavy metals contained in drilling fluids // Fishery Issues . 2000. T.1 2-3, part 2. - S.59-60).

 It has been established that small hydrobionts are the most vulnerable to pollution and other changes in environmental conditions. They also include caviar and fish larvae, which are part of ichthyoplankton. The large specific surface area associated with the small size of the body is one of the determining reasons for their reduced resistance to environmental changes. Another reason, obviously, is the specificity of the morphophysiological processes taking place in a rapidly developing organism, and the absence of developed protection systems (Patin S.A. Influence of pollution on biological resources and productivity of the oceans. M: Food industry, 1979. - P. 111-157), therefore, the search for test objects in the Sea of Japan and the Sea of Okhotsk was aimed at finding fish larvae that could serve as a test object for determining the toxicity of marine water pollutants in the Far East.

Known work on the comparative acute sensitivity of the larvae of Atherinops affinis and Menidia beryllina to the effects of 11 chemical compounds (Comparative acute sensitivity of larval topsmelt, Atherinops affinis, and inland silverside, Menidia beryllina, to 11 chemicals / Hemmer Michael J., Middaugh Douglas P., Comparetta Valerie // Environ. Toxicol. And Chem. - 1992. - 11, 3. - P. 40-40). Separate incubation of A. affinis, smelt argentina larvae and M. beryllina menidia for 96 h in the presence of 11 chemicals, the ratio of high and low values (LC ₅₀ ) for endosulfate, methoxychlor, carbofenothione, chlorpyrifos, terboufos, phenvalerate, permethrin, 4- nitrophenol and lauryl sulfate were <2. A. affinis larvae were more sensitive to azinphosmethyl and to 2,4-dinitrophenol, while their sensitivity to chemicals was similar to that of the 2 most sensitive freshwater fish species. It is concluded that A. affinis can be successfully used in toxicological studies instead of M. berullina.

 The closest technical solution is the method for assessing the toxicity of water pollutants described in A. S. USSR 1234770, IPC 4 G 01 N 33/18, C 02 F 3/34, including keeping the test object - daphnia - in the test solution, registration physiological response, including the indicator of death, fertility, mutagenicity, etc., and a comparison of the results.

 The problem solved by the invention is the expansion of the number of test objects for assessing the toxicity of pollutants of marine waters of the Far Eastern seas.

 The problem is solved in that as a test object for assessing the toxicity of pollutants of marine waters of the Far Eastern seas, the prelarvae of Japanese anchovy (Engraulis japonicus) are used.

 The essence of the invention is as follows: as a test object use the larvae of the Japanese anchovy (Engraulis japonicus), which are obtained by incubating eggs.

 In order to reduce death due to possible injury to the object at various stages of work and to improve the reproducibility of the experimental results, prelarvae aged 16-22 hours are used.

The proposed prelarvae of Japanese anchovy (Engraulis japonicus) were investigated as a test object by placing and keeping them in a marine aquatic environment into which zinc salts (ZnCl ₂ ) were introduced as a toxicant in the concentration range of Zn ⁺ ions from 0.01 to 1 , 0 mg / l, and also set the control (without the introduction of toxicants) and the reference series.

 The studied range of concentrations should be selected so that the observed lethal toxic effect is greater and less than 50%, for this, before the main experiment, a preliminary experiment is carried out with a concentration step in one order. The number of concentrations in the preliminary experiment, as well as the number of preliminary experiments themselves, varies depending on the need. Based on preliminary experiments, a range of five test concentrations is established for the main experiment. Preliminary experience includes a control series, that is, a series with observance of all experimental conditions, only without introducing the test toxicant.

 The main experiment is set with the concentration step of the test pollutant selected based on the results of the preliminary experiment. The main experience includes a control series, that is, a series with observance of all experimental conditions, only without introducing the test toxicant.

 In parallel with the experiments, two concentrations of the reference toxicant are investigated. And the experimental, and control, and reference groups are placed in triplicate.

 The toxicity parameter is taken as the number of dead individuals, which after death acquire a milky-white color almost immediately, but in general death is judged by the absence of the body's reaction to contact with a glass rod. Readings are taken every 12 hours, while dead prelarvae are immediately removed from the glass. The experiment lasts for 96 hours (during this time the yolk sac of the prelarvae is almost completely absorbed), in the case of death of the larvae in the control group more than 50%, the experiment is repeated.

At the end of the experiment, the degree of toxicity of the pollutant is judged by toxicometric parameters - LK ₀ , LK ₁₆ , LK ₅₀ , LK ₈₄ and LK ₁₀₀ .

 The invention is illustrated by example.

 Example 1

The proposed prelarvae of Japanese anchovy (Engraulis japonicus) were investigated as a test object by placing and keeping them in a marine aquatic environment, into which zinc salts (ZnCl ₂ ) were introduced as a toxicant in the concentration range of metal ions - from 0.01 to 1, 0 mg / l, and also set the control (without the introduction of toxicants) and the reference series.

 The prelarvae of Japanese anchovy (Engraulis japonicus) are obtained as follows.

Caviar is taken in June - August from the natural environment in areas with an environmentally friendly environment using an anchored surface plankton network such as Jedi. The collected planktonic material is immediately delivered to the laboratory, where 50-100 pieces of Japanese anchovy caviar are planted in separate 1000 ml vessels filled with settled and filtered seawater. and wait for the hatching, which occurs approximately 2-8 hours after the transplant is completed at a temperature of 15-21 ^o C. 5-10 hours after the completion of the mass hatching of the larvae, 5 pcs are planted. into tall graduated glass glasses (> 100 ml) with clean, settled and filtered seawater, a little less than 100 ml. Settled larvae are kept in these glasses for 11-12 hours to exclude individuals injured during transplantation from the experiment. Thus, from the moment hatchlings hatch to the moment the toxicant is introduced, it takes 16-22 hours, and the larvae injured at different stages of the preparatory work are excluded from the experiment, that is, only deliberately viable larvae aged 16-22 hours fall into the experiment.

 Toxicant solutions are prepared as follows.

Zinc salts (ZnCl ₂ ) are dissolved in double-distilled water to obtain mother liquors with a metal ion concentration of 1 g / l. For the convenience of preparing the studied concentrations of the pollutant, intermediate solutions are prepared by diluting the mother liquor 10-100 times with clean, settled and filtered seawater, to obtain solutions containing 10 mg / L and 100 mg / L of metal ions.

 The range of the studied concentrations of the toxicant is established on the basis of the results of preliminary experiments, and it is also necessary to choose such concentration values that they are slightly higher and lower than the values established by the MPC.

 Conducting an experiment.

 Preliminary experience is as follows.

 Prepare 18 graduated glass glasses with a volume of more than 100 ml with clean, settled and filtered water, in each of which 5 prelarvae of Japanese anchovy (Engraulis japonicus) are placed at the age of 5-10 hours and kept in these glasses for another 11-12 hours to exclude from experiment of possible injured when transplanting larvae. Thus, only viable non-injured individuals aged 16-22 hours fall into the experiment. Then, the amount of toxicant necessary to obtain the calculated concentration of the pollutant is added, thereby bringing the amount of solution to the required volume of 100 ml.

3 * 1 Art. under the concentration of Zn + - 0.001 mg / l;
3 * 1 Art. under the concentration of Zn + - 0.01 mg / l;
3 * 1 Art. under the concentration of Zn + - 0.1 mg / l;
3 * 1 Art. under the concentration of Zn + - 1 mg / l;
3 * 1 Art. under the concentration of Zn + - 10 mg / l;
3 * 1 Art. - control series.

 Each concentration is prepared three times, the number of concentrations is five. Also, in triplicate, a parallel series with a control group that differs from the experimental one only in the absence of a toxicant is put.

 Readings on the number of dead prelarvae are taken every 12 hours, dead prelarvae are immediately removed from the glasses. Based on the results obtained, a narrower and more accurate range of concentrations is selected for the main experiment.

 The main experience is as follows.

 Prepare 18 graduated glass glasses with a volume of more than 100 ml with clean, settled and filtered water, in each of which 5 prelarvae of Japanese anchovy (Engraulis japonicus) are placed at the age of 5-10 hours and kept in these glasses for another 11-12 hours to exclude from experiment of possible injured when transplanting larvae. Thus, individuals aged 16-22 hours fall into the experiment. Then, the amount of toxicant necessary to obtain the calculated concentration of the pollutant is added, thereby bringing the amount of solution to the required volume of 100 ml. Each concentration is prepared three times, the number of concentrations is five.

3 * 1 Art. under the concentration of Zn + - 0.01 mg / l;
3 * 1 Art. under the concentration of Zn + - 0.02 mg / l;
3 * 1 Art. under the concentration of Zn + - 0.06 mg / l;
3 * 1 Art. under the concentration of Zn + - 0.5 mg / l;
3 * 1 Art. under the concentration of Zn + - 1.0 mg / l;
3 * 1 Art. - control series.

 Also, in triplicate, a parallel series with a control group that differs from the experimental one only in the absence of a toxicant is put.

 Readings on the number of dead prelarvae are taken every 12 hours, dead prelarvae are immediately removed from the glasses. The results are analyzed, taking into account the Abbot amendment (Methodological recommendations for establishing maximum permissible concentrations of pollutants for water in fishery reservoirs. M: VNIRO, 1986, 88 pp.). Materials for the study of cadmium toxicity and calculations of toxicometric parameters are given in the table.

The experiment with the reference toxicant (Cd ⁺ ) is set both in preliminary and in the main experiments with the toxicant, the known concentrations of which cause a known pronounced toxic effect to determine the degree of suitability of this sample of the test object in the experiment. The experiment is carried out as follows.

Prepare 9 graduated glass cups with a volume of more than 100 ml with clean, settled and filtered water, in each of which 5 prelarvae of Japanese anchovy (Engraulis japonicus) are placed at the age of 5-10 hours and kept in these glasses for another 11-12 hours to exclude from experiment of possible injured when transplanting larvae. Thus, individuals aged 16-22 hours fall into the experiment. Then, the amount of the reference toxicant necessary to obtain the calculated concentration of the pollutant is added, thereby bringing the amount of solution to the required volume of 100 ml. Each concentration is duplicated three times, the number of concentrations is two. Also, in triplicate, a parallel series is set up with a control group that differs from the reference group only in the absence of a reference toxicant:
3 * 1 Art. under the concentration of Cd ⁺ - 0.02;
3 * 1 Art. under the concentration of Cd ⁺ - 0.1;
3 * 1 Art. - control series.

Thus, the proposed test object can be successfully used to assess the degree of toxicity of marine pollutants in the design and forecasting of human activities. The prelarvae of Japanese anchovy (Engraulis japonicus) can be more recommended for toxicological studies than other well-known marine test objects in the early stages of individual development. The values of LK ₀ and LK ₅₀ obtained as a result of our experiments were for Zn: LK ₀ - 0.01, LK ₅₀ - 0.219 mg / l, while when studying the effect of Cd, Zn and other toxicants on eggs and larvae of various fish species of the Far East in the region, LK ₅₀ values for Zn ranged from 25 to 80 μg / L (Patin S.A. Influence of pollution on biological resources and productivity of the World Ocean. M: Food Industry. - 1979. - 304 p.).

 A comparison of the results of our studies with the approved Russian MPCs for Zn for sea waters showed that the approved MPCs for Zn are five times higher than our safe concentrations for this metal (List of fishery standards: maximum permissible concentrations (MPCs) and tentatively safe exposure levels ( SHOES) of harmful substances for water of water bodies of fishery importance Compiled by Shilenko N.A., Sokolova S.A., Anisova S.N., Lesnikov L.A., Lebedev A.T., Semenova I.V. - M.: VNIRO, 1999. - 304 p.).

 Based on this, the larvae of the Japanese anchovy (Engraulis japonicus) were proposed by us as a new test object for inclusion in the "Methodological recommendations for establishing ecological and fishery standards for pollutants for sea waters". In addition, it is planned to submit to the NTS of the Glavrybvod of the State Committee for Fisheries of Russia a proposal to clarify the MPC of some metals for the waters of the Far Eastern seas.

Claims (1)

 A method for assessing the toxicity of pollutants in the waters of the Far Eastern seas, including keeping the test object in test solutions, recording the physiological response and comparing the results, characterized in that Japanese anchovy larvae (Engraulis japonicus) are used as the test object.

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