RU2482474C2 - Method for biotesting toxicity of water and aqueous solutions - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves exposing the analysed sample of a plant object to intermittent light of high intensity of 100-250 W/m² from blue light-emitting diodes with luminous excitation of said sample and detection of after-glow in the red spectral region using a photodetector between light pulses passing through a red light filter, wherein by cutting duration of light periods of luminous excitation while simultaneously increasing dark intervals in between, conditions are created for low light exposure, and by increasing the ratio of duration of light periods to dark periods, conditions are created for high light exposure; intensity of delayed fluorescence of the analysed sample is measured in high light exposure mode at the beginning of each damping curve; intensity in low light exposure mode is measured at the end of each damping curve, and toxicity is determined from their ratio.

EFFECT: high accuracy and sensitivity of determination.

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Description

The present invention relates to biophysical methods for determining the toxicity of water and aqueous solutions and can be used for operational biotesting of the toxicity of natural and waste waters, as well as for establishing the hazard class of various wastes when microalgae and other plant organisms are used as a test object.

A known method for determining the phytotoxicity of water, based on recording changes in the intensity of the millisecond photoinduced afterglow (delayed fluorescence) of chloroplasts or chlorella cells under the action of chemical compounds contained in the tested samples [ed. testimonial. USSR No. 492805, G01N 33/00, publ. 11.25.75 g.].

The disadvantage of this method is that it does not provide high accuracy in measuring the degree of exposure to toxic substances in the analyzed waters, since the intensity of the recorded delayed fluorescence (PF) will depend not only on the degree of toxicity of the sample, but also on the turbidity and color of the tested water or solution, and also the amount of test organism taken.

The closest technical solution for the method for determining the content of phytotoxic substances, selected as a prototype, is [RF patent No. 2069851, G01N 21/64, G01N 33/00, publ. November 27, 1996], based on the determination of the toxicity of various waters and aqueous solutions by measuring the ratio of the intensities of the ZF of the test organism at the induction maximum at high and low exciting light. This relative parameter of the SF does not depend on the turbidity and color of the analyzed samples, as well as the amount of the test organism taken, since the change in the glow intensity will in all cases be proportional to both light conditions for the excitation and registration of the SF and therefore does not affect the value of its relative parameter.

The disadvantage of this method is the difficulty in selecting the intensity of low exciting light, providing maximum sensitivity and accuracy of this method. The situation is aggravated by the fact that this method does not use the possibility of component-wise recording of PF intensity, which also has a significant effect on the sensitivity of plant test organisms to toxicants.

The technical result of the invention is to increase the accuracy and sensitivity of the method for measuring toxicity during biotesting of water and aqueous solutions by creating new conditions for recording the delayed fluorescence of chlorophyll in plant objects.

The technical result is achieved by the fact that in the method of biotesting the toxicity of water and aqueous solutions, including irradiating the test sample of a plant object with high-intensity intermittent light of 100-250 W / m ² from blue light-emitting diodes with excitation of the delayed fluorescence of this sample and its registration at the induction maxima in red the spectral region of the photodetector between light pulses, moreover, by reducing the duration of the light periods of the excitation of the glow with a simultaneous increase in At intervals between them, low light exposure conditions are created, and by increasing the ratio of light to dark periods, high light conditions are created, the delayed fluorescence intensity of the test sample is measured in the high light exposure mode at the beginning of each attenuation curve, and the intensity in the low light exposure mode is measured at the end of each attenuation curve, and toxicity is judged by their ratio.

The inventive method for biotesting water and aqueous solutions is characterized by an increase in the response range of the relative delayed fluorescence of chlorophyll of the plant test organism when exposed to pollutants, which provides greater accuracy and sensitivity of the method for measuring the toxic effect.

Thus, the claimed method of biotesting the toxicity of water and aqueous solutions meets the criterion of "novelty."

The features distinguishing the claimed technical solution from the prototype are not identified in other technical solutions when studying data and related areas of technology and, therefore, provide the claimed solution with the criterion of "inventive step".

The invention is illustrated by drawings. Figure 1 shows the dependence of the SF intensity of a chlorella suspension excited in high (ZFv) and low (ZFn) light modes on the concentration (in moles / liter) of diuron, a toxicant that suppresses photosynthetic electron transport. Figure 2 shows the concentration dependences of the parameters PF algae chlorella in the presence of cadmium ions. Figure 3 shows the dependence of the relative indicator of ZF on the concentration (percentage dilution) of the tested wastewater.

The essence of the invention lies in the fact that the toxic effect on test organisms is evaluated on the basis of recording the intensity of the delayed fluorescence of the chlorophyll contained in them using a device for recording the chlorophyll PF of plant objects. When determining the toxicity of the tested waters by the relative indicator (OD) of the SF at the induction maximum, the luminescence intensity of the test organism when excited by high intensity light is measured at the beginning of each attenuation curve, and in the low-light excitation mode, at its end. In this case, low light exposure conditions are created by reducing the duration of the light excitation pulses while increasing the duration of the dark intervals between them. In turn, the formation of high light exposure conditions is ensured not only by using high-intensity exciting light, but by increasing the average level of light exposure of the test organism by increasing the duration of light periods and reducing dark ones.

Example 1

4 cm ^{3 of a} suspension of chlorella algae with an optical density of 0.040, measured at a wavelength of 560 nm and a layer thickness of 2 cm, are introduced into 6 cuvettes from a material with a low level of afterglow (ZF). 0.1 cm ^{3 of} distilled water is added to the first one ( control), and the remaining 0.1 cm ^{3 of a} solution of diuron of various concentrations. After this, the cuvettes are loaded into the device for recording the delayed fluorescence of chlorophyll of plant objects and the measurement of SF in two light modes is performed. Figure 1 shows the dependence of the SF intensity of a chlorella suspension excited in high (ZFv) and low (ZFn) light modes on the concentration (in moles / liter) of diuron, a toxicant that suppresses photosynthetic electron transport. The first mode is achieved by the fact that relatively long pulses (20 ms) of high-intensity exciting light (100-250 W / m ² ) are alternated with relatively short dark intervals (5 ms), during which, in the first millisecond, the SF is recorded. The low light mode is obtained due to the fact that an equally bright excitation pulse is supplied for 1-4 ms, followed by a longer dark interval (50 ms). In this case, the intensity of the SF is not measured immediately after the end of the light excitation, but with a delay of 20-30 ms. Since fast components will completely decay during this interval, only slow components remain and are recorded at the end of the damped glow curve.

Due to the fact that the intensity of these components in the control test culture of algae in the absence of toxicants is several hundred times lower than that of the fast components, when switching to low light mode, the sensitivity of the registration system automatically increases by 100 times. Therefore, for a real idea of the magnitude of the OP ZF its values, shown in figure 3, must be increased 100 times. So that the luminescence intensity can be measured at its induction maxima, the light pulses of excitation, alternating in periods, are recorded by the SF, and they are supplied for 2-3 seconds.

The data presented in figure 1 show that the intensity of the PFV of the test culture of chlorella algae decreases with increasing concentration of the toxicant. Under these conditions, the ZFn increases equally. In this case, the relative index of the SF, in the form of the ratio of the intensities of the glows in the regimes of high and low exciting light, decreases by a factor of 100 or more.

Example 2

In cuvettes similar to Example 1, instead of diuron, various concentrations of cadmium ions are introduced. According to the recommendations [RF patent No. 2222003, G01N 21/64, publ. January 20, 2004] the tested samples, for a greater manifestation of the action of this heavy metal on algal cells, are exposed to light exposure for 1 hour. The intensity of light irradiation of the cell with a test culture of algae is 60 W / m Figure 2 shows the concentration dependences of the SF indicators of chlorella algae in the presence of cadmium ions, measured under conditions similar to those described in example 1. It is clearly seen that this toxicant also causes a significant decrease in the intensity of the RFF and an increase in the APF. The magnitude of the ratio of these indicators of SF also decreases many times.

Example 3

3.6 cm ^{3 of} wastewater from a pulp and paper mill (PPM) and wastewater at the outlet of a municipal treatment plant (OS) diluted with distilled water in a multiple of two are added to cuvettes similar to Example 1. Distilled water is used as a control. 0.4 cm ³ of chlorella algae culture with an optical density of 0.4 is added to each wastewater dilution. After a 1-hour light exposure with a light irradiation intensity of 60 W / m ² , the delayed fluorescence intensity is measured in high and low light irradiation modes. Figure 3 shows the dependence of the relative indicator of SF on the concentration (percentage dilution) of the tested wastewater (measurement parameters of SF, as in example 1). Wastewater after treatment plants practically does not cause a change in the relative indicator of SF of algae culture, which indicates its non-toxicity. Conversely, the effluents of the pulp and paper mill show rather high toxicity, which is removed only after their 8-fold dilution with clean water.

The advantage of the proposed method for biotesting the toxicity of water and aqueous solutions is to increase the accuracy and sensitivity of measuring the toxicity of water during biotesting due to an increase in the response range of the relative delayed fluorescence of chlorophyll of the plant test organism when exposed to pollutants, by creating new conditions for recording the delayed fluorescence of chlorophyll of the plant test -organisms.

Claims (1)

A method for biotesting the toxicity of water and aqueous solutions, including irradiating a test sample of a plant object with high-intensity intermittent light of 100-250 W / m ² from blue light-emitting diodes with excitation of the luminescence of this sample and recording the afterglow in the red spectral region of the photodetector between light pulses transmitted through the red filter moreover, by reducing the duration of the light periods of the excitation of the glow with a simultaneous increase in the dark intervals between them, conditions of low light exposure, and by increasing the ratio of the duration of light periods to dark, create conditions for high light exposure, measure the delayed fluorescence intensity of the test sample in the high light exposure mode at the beginning of each attenuation curve, the intensity in the low light exposure mode is measured at the end of each curve attenuation, and toxicity is judged by their ratio.

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