RU2245367C2 - Method for biotesting water and aqueous extract samples - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention is designated for biotesting water and aqueous extract samples. Daphnia are immersed firstly in solution or water for culturing to be tested and then daphnia are transferred into lightproof chamber with outlet hole for culturing and this chamber is immersed into water. Time passing out daphnia from the testing chamber to light is measured and toxicity of analyzed solution is estimated by difference time in daphnia passing out. Method allows carrying out the rapid control of water and aqueous extracts toxicity in laboratory and field conditions.

EFFECT: improved method for biotesting.

Description

The present invention relates to biology and is intended for biotesting water samples and water extracts.

Any pathology (including that caused by toxicants) develops as a result of a violation of homeostasis, which should be understood as a constant balancing of body functions with environmental fluctuations, i.e. as maintaining the structure and function of the body within normal limits.

Homeostasis is regulated by the nervous and neuroendocrine systems. The first has a decisive role, because it controls all the processes of the body. The ideas of nervousness developed by IP Pavlov were developed in many works by pathologists [2, 3, 4, 6]. The principle of nervous regulation extends to the toxicology of aquatic animals [7]. The action of toxic substances is based on their interference in the biochemical processes of the body, which ensure the functioning of certain organs and systems and determine the final response.

Knowledge of the physiological and biochemical principles of the action of toxic substances is extremely important for the development of measures for the diagnosis and classification of aquatic animal poisoning. Recently, they have become particularly relevant and practical in connection with the need to develop methods for bioassaying toxicants in the aquatic environment.

Behavioral reactions in animals are one of the means of adaptation to the environment and its changes. They differ from tropisms in plants by the participation of effectors — special organs or cells that realize signals received from receptors [1].

Against the background of light rhythms, hydrobionts develop certain rhythms of vital activity. This includes not only activity rhythms, but also others: reproductive (mating, egg laying), ontogenetic (molting, hatching juveniles), metabolic (nutrition, breathing), biochemical, etc. The presence of these rhythms in aquatic invertebrates indisputably proves that in In an aquatic environment, light plays the same important role as on land, changing the quantitative indicators of vital processes in its inhabitants.

The simplest example of a behavioral reaction is taxis, or a motor reaction carried out without the participation of the central nervous system in response to an external stimulus. In cases where this stimulus is light, phototaxis, photosynthesis, and photophobic reaction are distinguished. Phototaxis is an oriented response to a light source. Photosynthesis, or a change in the speed of movement depending on the intensity of lighting, can be considered both in relation to the whole organism, and in relation to some organs, for example, the filtration apparatus in sea acorns. Photophobic reactions are to avoid illumination (in protozoa).

The most widely studied effect on the phototaxis of light intensity. A change in the sign of phototaxis from positive to negative when exposed to the maximum (within the natural interval) values of illumination in invertebrates [8, 9, 11]. In some cases, a phototaxis threshold was noted [12], expressed by a certain amount of illumination intensity. Such a threshold has been established for cyclops and shrimp [10].

Assessment of toxicity of pollutants for aquatic organisms is based on the use of tests that most fully and reliably reflect the well-being of the body. These are the survival, reproduction, fertility and quality of the offspring. However, the study of such biological processes is laborious, expensive and possible only on a limited number of species. Therefore, lately, more and more attention has been paid to physiological, biochemical and behavioral reactions, with the help of which it is possible to identify the pathology caused by toxicants long before the violation of the body's integral responses [7].

For widespread use, as “environmentally significant” indicators, physiological and biochemical parameters should be used that reflect violations of the body's integral reactions.

The effect of water quality on the distribution of aquatic animals in relation to light was found. As a prototype, the method of determining the relation to light in aquatic animals was chosen, which consists in the use of alternative chambers, where the animal has to choose between darkness and light. Moreover, the animals are placed in the camera of choice, from which there is a sufficiently large exit into the illuminated and unlit chamber [7]. If the toxicant inhibits or, conversely, enhances the mobility of animals, their distribution in the chambers becomes uniform regardless of their relationship to light.

The aim of the invention is to provide a simple and affordable testing method based on the behavioral response of phototaxis.

This goal is achieved thanks to the biotesting method, based on the assessment of the time of daphnia exit from the “dark” chamber to the light. The difference of the proposed method lies in the fact that the animals do not have an alternative choice, they are all placed in a dark chamber, from which there is only one exit to a source of scattered light. With this method, a fundamentally different orientation mechanism, consisting in finding the direction to the sources of stimulation (light) and movement in this direction. Due to the limitation of the diameter of the outlet at the dark chamber, redistribution of daphnia depending on changes in motor activity when exposed to toxic substances is practically eliminated.

The order of testing is as follows: a gas cage containing 5 daphnia is placed in the test solution. After being in the test solution for 60 minutes, the animals are placed in a “dark” chamber, which is previously immersed in a 500 ml beaker filled with water for cultivation. The camera is a cube with dimensions of 60 × 60 × 60 mm, made of black organic glass with a lid of the same material. In the center of the lateral surface is a hole through which animals can leave the “dark” chamber. The diameter of the hole depends on the size of the daphnia used for biotesting. So, when using diurnal juveniles in the experiment, the Daphnia diameter of the outlet should be 1 mm, and mature individuals - 2.5 mm. The diameters of these holes are determined by the size of the animals. The emptied juveniles are 0.7-0.9 mm long, by the time of maturity the females reach 2.2-2.4 mm, and the males 2.0-2.1 mm. During the experiment, the liquid level in the beaker and the test chamber should be the same. In this case, the outlet must be in the liquid layer. In parallel, an experiment is set up in which instead of the test solution, water is used to cultivate Daphnia.

The test function is the response time to a light stimulus. The test tool is a “dark” chamber, and the toxicity criterion is a significant difference in the time of the behavioral reaction (phototaxis) of the experimental sample compared to the control sample.

When processing the results of the experiment, a comparison is made between the experimental and control daphnia. The reliability of deviations from control is determined by the methods of variation statistics [5]. In this case, calculate:

M - the arithmetic mean value of the indicator

where Vi is the individual value of the indicator; n is the number of repetitions;

Σ is the summation sign; δ is the standard deviation

m is the error of the arithmetic mean

Δ - confidence interval

Where t _St - tabular value, student criterion. In this case, for its determination, a significance level of P = 0.05 and a degree of freedom of n-1 are adopted; t ₄ - criterion of reliability of the difference

where M ₁ , M ₂ - the compared average values (in control and tested samples); m $\genfrac{}{}{0ex}{}{\text{2}}{\text{one}}$ , m $\genfrac{}{}{0ex}{}{\text{2}}{\text{2}}$ - squares of errors; t _St is the standard value of student criterion.

In this case, for its determination, a significance level of P = 0.05 and a degree of freedom n ₁ + n ₂ -2 are taken.

If the calculated value of t4 is greater than or equal to the value of the Student criterion (t ₄ > t _St ), the deviation of the biotesting results from the control is significant. Based on this, it can be concluded that the tested water sample has a toxic effect on daphnia.

The method is intended for quick control of the degree of integral toxicity of samples of solutions of ecotoxicants, water and aqueous extracts of various environmental objects in laboratory and field conditions.

The method was used in biotesting water samples and aqueous extracts containing anticholinesterase compounds (organophosphorus and carbamates) in the range of average lethal concentrations of these substances 1 · 10 ^-2 -1 · 10 ^-5 mg / l, which approximates the determination of anticholinesterase compounds using these hydrobionts using the proposed biotesting method to the sensitivity of gas-liquid chromatographic analysis methods.

Literature:

1. Willy K., Datier W. Biology, 1974.

2. Horizons P.D. Issues of pathological physiology in the works of I.P. Pavlov. M., 1952.- 314 p.

3. Horizons P. D. Resistance and defeat. Questions of general pathology // Pathological physiology of experimental conditions. M., 1973. S. 7-35.

4. Ivanov-Smolensky A.G. Essays on the pathophysiology of higher nervous activity (according to I.P. Pavlov and his school). M., 1952. - 387 p.

5. Plokhinsky N.A. Mathematical methods in biology / educational method. allowance. - M: Moscow State University, 1978, 340 p.

6. Speransky A.D. Elements of the construction of the theory of medicine / Izb. works. M., 1955. - 404 p.

7. Flerov B.A. Ecological and physiological aspects of the toxicology of freshwater animals. - L .: “Science”, 1989.

8. Aiken D., Hailman G. // Can. J. Zool. 1978. Vol. 56, No. 4. P.708-711.

9. Bourdilion A., Castelbon Ch. // Vision invertebres, Paris, 1984. P.202-209.

10. Burba A., Maksimov Yu., Plyarpa A. // Acta hydrobiologia Lituanica. 1977, No. 6. S.93-100.

11. Rimet V // Bull. Sci. Zool. France 1979. No. 2-3. R. 342-357.

12. Swift V., Forward R. // J. Plancton Res. 1983. Vol. 5, No. 3. P.407-415.

Claims (1)

A method of biotesting water samples and aqueous extracts, including assessing the toxicity of the test solution based on the phototaxis reaction of Daphnia magna Straus, characterized in that the daphnia of the experimental and control groups is placed for 60 minutes in the test solution or water for cultivation of daphnia, respectively, and then the animals of each the groups are transferred to a Daphnia impervious camera immersed in water for cultivation with an outlet from 1 to 2.5 mm in size, selected depending on the age sizes of Daphnia used for biota stirovaniya further measured output time of the daphnia test chamber for the light and the difference in output time daphnia test and control groups assess toxicity of the test solution.