RU2029948C1 - Method of determining toxicity of natural water - Google Patents

Abstract

FIELD: testing equipment. SUBSTANCE: crustaceous-gammarus is used as test-object. If a toxin is presented in water the animal stops motion or changes direction of motion whereas in the normal fluid the animal moves in one direction over periphery of the tank. The presence and amount of the toxin can be judged by distortion of features of orientation behavior of the animals. EFFECT: simplified method. 2 cl, 3 dwg

Description

 The invention relates to methods for biotesting and can be used in toxicology, hydrobiology, ecology, zoology.

 A known method for determining the toxicity of natural waters by recording the motor activity of leeches and fixing their static poses [1].

 The method has the following disadvantages: a narrow range of bioassay, covering only fresh water; the need to bring the test object into a specific functional state.

 A known method for determining the salinity of natural waters by recording the locomotor activity of marine crustaceans and their selection of the optimal salinity gradient [2].

 The method has the following disadvantages: the duration of the method; uneconomical; insecurity.

 Closest to the claimed invention is a method for determining the toxicity of the aquatic environment by measuring the speed of fish in the optomotor installation [3]. An indicator of the effect of the toxicant is a decrease in the speed of movement of fish compared to the control group.

 The disadvantages of the prototype are the duration of the determination; uneconomical method - the need for large containers equipped with a purge or duct; the inaccuracy of the method is the inability to determine the concentration of a toxicant by the motor activity of fish.

 The goal is achieved as follows.

 As a test object, they use crustaceans (gammarus related to amphipods), widely distributed in marine and fresh water bodies. Gammarus, placed in small (18 to 29 cm in diameter) cylindrical vessels, usually float evenly along its walls (tigmotaxis), often at a constant speed. In most animals, predominantly unidirectional movement along the perimeter of the vessel (clockwise or against it) is observed, i.e., spatial asymmetry and tigmotaxis are manifested. Stopping and changing direction is extremely rare.

 In the presence of toxicants in the aquatic environment, for example, petroleum fuel (diesel fuel), these indicators of the behavior of amphipods change significantly. The movement of amphipods slows down, becomes chaotic, animals stop, change the initial direction of movement, the initial trajectory is violated, a characteristic turning along the vertical axis appears. The preferential choice of direction, characteristic of normal conditions, is reversed or the animal begins to equally likely move along and counterclockwise. High concentrations of the toxicant can cause complete cessation of movement and death of the animal. Thus, the normal physiological state of amphipods changes.

 Therefore, by measuring changes in motor activity, as well as assessing violations of the qualitative indicators of the orientational behavior of the studied animals (tigmotaxis and motor asymmetry), it is possible to determine the presence and magnitude of toxic effects. To achieve the goal, operations are performed in the following sequence.

 Caught animals (gammaruses) for adaptation are seated in containers in which conditions are created that are adequate to their environment in nature: sandy soil, stones at the bottom for shelter, algae, a layer of water from 10 to 20 cm. 1-2 days are enough for adaptation. The temperature regime is similar to the living conditions of the species. Planting density should not exceed 10 individuals per 1 liter of water. Change of water once a day.

 Before testing, amphipods are pre-planted one by one in vessels with a capacity of 500-700 ml with an amount of water of 200-300 ml, where they are kept for 1-1.5 hours to adapt to the experimental conditions in natural light.

 In FIG. 1-3 are diagrams and graphs illustrating the proposed method.

 Testing is as follows. Each individual is transferred to a vessel (crystallizer) with a diameter of 18-22 cm with a coordinate grid at the bottom and a height of a column of clean water of 3.5-5.0 cm, in which, after 1-1.5 minutes of adaptation, the total number and direction of movement of the animal is recorded ( clockwise and counterclockwise) every 20-30 s for 3-4 minutes (Fig. 1).

 A coordinate grid is applied to the bottom of the vessel, divided into three zones: peripheral (III), middle (II), central (I) and 8 sectors. For one movement take the transitions of the animal from any sector or zone to a neighboring sector or zone.

 After the first experiment (in pure seawater), water in vessels containing gammarus is replaced with a solution of a known toxicant of known concentration and test objects are again placed in them for 1-1.5 hours. Then, each animal is retested. This procedure is repeated 3-4 times with an interval of 1-2 hours (Fig. 2). The results of simultaneous testing of 2 groups: experimental and control (a and b) depending on the exposure time in the toxicant 1, 2, 3 hours and in pure water - control (15-20 individuals in each group) are presented in the graphs - changes average number of movements in each group depending on time (hours).

 A generally accepted criterion for the action of a toxicant is used - statistically significant differences between control and experience. From the analysis of the relationship between concentration, exposure time and the number of movements in the toxicant solution (Fig. 2), a formula is derived that allows one to determine, based on the known (reference) concentration of oil, its unknown unknown concentration in the aquatic environment.

ln

ln

, (1) где С_i - концентрация нефти (солярка) в исследуемой среде;
С_эт. - эталонная концентрация, при которой проведены измерения движений;
N_эт. - количество движений при эталонной концентрации;
N_эт. (t_o) - количество движений группы, которая используется в опыте с эталонной концентрацией в чистой воде;
N_эт. (t_j) - количество движений в опыте с эталонной концентрацией после экспозиции в растворе токсиканта в течение времени t_j;
N_i(t_o) - количество движений исследуемой группы в чистой воде;
N_i(t_j) - количество движений исследуемой группы после экспозиции длительностью t_j в воде с неизвестной концентрацией нефти.C _i = C _et

ln

ln

, (1) where С _i is the concentration of oil (diesel fuel) in the studied medium;
With _fl . - reference concentration at which motion measurements were made;
N _floor - the number of movements at a reference concentration;
N _floor (t _o ) - the number of movements of the group that is used in the experiment with a reference concentration in pure water;
N _floor (t _j ) is the number of movements in the experiment with a reference concentration after exposure to a toxicant solution for a time t _j ;
N _i (t _o ) - the number of movements of the studied group in pure water;
N _i (t _j ) is the number of movements of the study group after exposure of duration t _j in water with an unknown oil concentration.

 An example of calculating an unknown concentration of a toxicant (oil) in an aqueous medium according to formula (I).

Take the reference (known) concentration of the toxicant (diesel fuel - diesel fuel), equal to 0.01% (C _floor ).

Register for 3 minutes the number of movements of animals of the reference group is normal (in pure water), get N _FL , (t _o ) = 100.

Animals of the reference group are transferred (one at a time) to a toxicant of the reference concentration (0.01%); after 2 hours again measure N _et . within 3 minutes Get N _et . (t = 2) = 80.

Take a new group of animals (experimental). Measure the number of movements in 3 minutes in the norm (in clean water); this measurement corresponds to the zero point. Get N _i (t _o ) = = 150.

The animals are transferred (one at a time) to a solution of a toxicant of unknown concentration (C _i ) and after 2 hours the number of movements is again measured for 3 min (t _i ). Get N _i (t = 2) = 75.

ln

ln

= 0,01

=
= 0,01

= 0,03%
Таким образом искомая концентрация нефтяного загрязнения равна 0,03%.Substitute the obtained results in the formula (I) and get the unknown unknown concentration (C _i ):
C _i = 0.01%

ln

ln

= 0.01

=
= 0.01

= 0.03%
Thus, the desired concentration of oil pollution is 0.03%.

 In addition to the main indicator - changes in the number of movements, a parameter of orientational behavior such as motor-spatial asymmetry is also used: unidirectional movement of gammarus clockwise or counterclockwise around the perimeter of the vessel with an almost constant speed.

 Under the influence of a toxicant, this indicator of behavior changes dramatically: the number of animals that maintain a certain direction of movement decreases. Gammarus begin to move randomly, repeatedly changing the direction of movement (Fig. 3). One can see a decrease in the number of animals with a reliably expressed choice of the direction of movement under conditions of oil pollution (a) and the control group without pollution (b).

 The proposed method is accelerated (20-24 hours) compared with the prototype (10-12 days), more economical, since it does not require large containers equipped with running water or air purge, is more accurate, since it allows determining the concentration by behavioral reactions toxicant, for example, oil pollution in the aquatic environment with an accuracy of 0.01%.

 PRI me R. Gammarus (Gammarus oceanicus, White Sea species) are placed in containers with sea water for adaptation, after which one individual is transferred into vessels with a capacity of 500 ml with a quantity of pure water of 200 ml, where they are kept for 1 hour under natural light. The experiments are carried out simultaneously in 2 groups: control and experimental (15-20 individuals in each).

 After an hour-long adaptation, the animals' motor activity in pure water is determined (zero point), for which amphipods are transferred one at a time for testing into a cylindrical vessel 18 cm in diameter with a coordinate grid at the bottom, with a clear water column height of 3.5 cm, where adaptation to new conditions for 1 min, then record the total number of movements and the number of movements clockwise and counterclockwise when moving around the perimeter of the vessel every 20 s for 3 minutes. After that, the clean water in the vessels is replaced with a solution of the toxicant 0.01% in water, the test objects are returned to them and kept for 1 hour. Then each individual is again transferred to a vessel with a coordinate grid, where the number and direction of movements are recorded (testing is performed in water, containing toxicant).

 After testing, the amphipod is returned to the vessel with the toxicant solution. Similarly, testing is done after 2 and 3 hours of exposure to the toxicant. Thus, after testing all amphipods in groups, a calibration curve is obtained that shows the total number of movements versus exposure time in the toxicant (Fig. 2). In FIG. 2 shows that in the experimental group (a) there is a sharp decrease in motor activity as a result of the toxic effects of the aquatic environment; at the same time, in the control group (b) this indicator practically does not change. The differences between the values of the momentum in the control and experimental groups are statistically significant.

 PRI me R 2. Gammarus one by one adapt in a container with sea water, and then transferred to vessels with a capacity of 700 ml with a quantity of pure water of 200 ml, where they are kept for 1.5 hours under natural light. Then each individual is transferred to a cylindrical vessel with a diameter of 21 cm with a grid at the bottom and a height of a column of pure water of 5 cm, where adaptation is carried out for 1.5 minutes, then the number and direction of gammarus movements along the vessel perimeter are recorded every 30 s for 4 minutes . After that, the clean water in the vessels is replaced with a solution of natural water with an unknown concentration of toxicant (oil pollution), the gammarus are returned to them and kept for 1.5 hours. Then, one by one is transferred to a vessel with a coordinate grid, where the number and direction of movements are re-recorded. In the experimental group (in natural water), a statistically significant decrease in motor activity was observed compared with the control group (by 75%). After that, the calculation is carried out according to the formula (I) using a calibration curve (Fig. 2), an unknown concentration of oil pollution equal to 0.04% is obtained.

 PRI me R 3. Gammarus adapt in containers with sea water, and then transferred to vessels with a capacity of 300 ml with a quantity of pure water of 200 ml, kept for 0.5 h, then transferred to a vessel with a coordinate grid at the bottom, s a height of a column of pure water of 2.5 cm, the number and direction of movements are recorded, and then kept in a toxicant solution for 0.5 hours. Gammarus detect a relatively large number of movements, which in the control group is not statistically different from the experimental group. The test concentration is not determined.

Claims (2)

 1. METHOD FOR DETERMINING THE TOXICITY OF NATURAL WATERS, including adaptation and measurement of movement parameters of aquatic animals, characterized in that marine crustacean gammarus are placed in adaptation vessels with a capacity of 500 - 700 ml, with an amount of water of 200 - 300 ml and with a stone, as shelter, where they are kept for 1 - 1.5 hours, after which each individual is transferred to a cylindrical vessel with a diameter of 18 - 21 cm with a coordinate grid at the bottom and a height of a column of clean water of 3.5 - 5 cm, withstand 1 - 1.5 minutes, then register the number and direction of movement of the hammar and every 20 - 30 s for 3 - 4 minutes, after which the water in the adaptation vessels is replaced with the test solution, where the gammarus are returned and kept for 1 - 1.5 hours, after which the number and direction of movements in the vessel with the coordinate are measured again the grid. 2. The method according to claim 1, characterized in that the unknown concentration of toxicant in water is determined by the formula

where C is the concentration of oil (diesel fuel) in the test medium;
C _{e t} - reference concentration at which measurements of movements were made;
N _{e t} - the number of movements at a reference concentration,
N _{e t} (t ₀ ) - the number of movements of the group, which is used in the experiment with a reference concentration in pure water;
N _{e t.} (t _j ) is the number of movements in the experiment with a reference concentration after exposure to a toxicant solution for a time t _j ;
N _i (t ₀ ) is the number of motions of the studied group in pure water;
N _i (t _j ) is the number of movements of the study group after exposure of duration t _j in water with an unknown oil concentration.

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