RU2069851C1 - Process of detection of content of phytotoxic substances - Google Patents

Abstract

FIELD: phytobiology, ecology. SUBSTANCE: invention is related to biophysical methods of detection and quantitative analysis of phytotoxic compounds in aqueous and other solution and may be used by environmental control services for timely monitoring of toxicity of natural water and sewage as well as in analytic and toxicology monitoring laboratories for detection and subsequent determination of content of chemical substances displaying phytotoxical activity. In agreement with process amplitude of induction maximum of slow fluorescence (3F) of alga of chlorella is registered in millisecond damping interval during excitation with light first of high (50-100 W/sq.m) and then of low (5-10 W/sq.m) intensities to increase accuracy and sensitivity of process of measurement of degree of phytotoxicity of chemical compounds and determination of their content. Ratio of registered values of 3F under specified conditions referred to test sample of alga having no toxicity is used as indicator of phytotoxicity. EFFECT: increased accuracy and sensitivity of process. 2 dwg, 1 tbl

Description

 The invention relates to biophysical methods for the detection and quantitative analysis of phytotoxic compounds in aqueous and other solutions and can be used in nature protection services for the operational control of the toxicity of natural and waste waters, as well as in analytical and toxicological laboratories for the detection and subsequent determination of chemical content substances with phytotoxic activity in various environments.

 Closest to the invention is a method for determining the phytotoxicity of drugs, based on recording changes in the intensity of a millisecond photoinduced afterglow (delayed fluorescence) of chloroplasts or chlorella cells under the influence of chemical compounds.

 However, this method does not provide high accuracy in measuring the content of these substances in the analyzed samples, since the intensity of the recorded delayed fluorescence (PF) will depend not only on the magnitude of the toxic effect of the sample, but also on the turbidity and color of the analyzed solution.

 The technical result of the invention is to increase the accuracy and sensitivity of the method for measuring the degree of phytotoxicity of chemical compounds and determining their content in the analyzed aqueous solutions.

This is achieved in that the phytotoxicity of substances detected by recording the maximum amplitude of the induction 3F algae Chlorella attenuation in the millisecond range when excited by light _{in the} first high Polar Division, then a low intensity Polar Division _n.

 It is known that the quantum yield and, consequently, the intensity of SF in high light, at which the fast damping component dominates, decreases after the addition of phytotoxic substances. Conversely, in low light, when the glow is represented by a slow component, the toxic effect manifests itself in the form of an increase in the intensity of the PF.

Figure 1 shows the dependences of the intense delayed fluorescence of a chlorella suspension excited by high (1-55 W / m ² ) and low (2-6 W / m ² ) light intensities, the ratio of their intensities (3), and the toxicity coefficient (4) on the concentration of promethrin in the medium.

In the proposed method, improving the accuracy of measuring toxicity is achieved by using as a ratio indicator the intensity of the SF and induction maxima at high (50-100 W / m ² ) and low (5-8 W / m ² ) exciting light. This parameter does not depend on the turbidity and color of the analyzed samples, since the attenuation of the SF intensity by an impurity will be proportional to both light conditions for the excitation of the glow.

An increase in the sensitivity of the method is achieved due to the fact that an increase in the SF intensity in low light (slow component) is observed at significantly lower concentrations of the toxicant than its decrease in high. As a result, with the help of the PF index _in / PF _{n, it is} possible to detect a lower degree of phytotoxicity and, consequently, lower concentrations of its substances than when registering one fast component of the glow.

(1)
где (ЗФ_в/ЗФ_н)_{контроль} показатель, зарегистрированный для пробы водоросли, в которую не вводились токсические вещества, а (ЗФ_в/ЗФ_{н опыт} показатели проб той же суспензии тест-объекта, в которые добавлены образцы, проверяемые на фитотоксичность. С увеличением последней данный коэффициент изменяется от нуля до величин, близких к единице, независимо от типа токсиканта и исходного состояния биотеста.Since the absolute values of this indicator vary significantly depending on the growing conditions and the subsequent content of the algae culture, a data rationing procedure is introduced to compare the results of different series of experiments. It consists in calculating the relative toxicity coefficient.

(one)
wherein (DF _in / Polar Division _{n) control} indicator registered sample algae, which were not introduced toxic substances, and (DF _in / Polar Division _{n experience} samples figures the same suspension of the test object, in which the added samples are checked for phytotoxicity. C by increasing the latter, this coefficient varies from zero to values close to unity, regardless of the type of toxicant and the initial state of the biotest.

 Example 1. 2 ml of a suspension of chlorella with an optical density of 0.3-0.4 at a wavelength of 670 nm and a layer thickness of 1 cm are added to several cuvettes from a material with a low level of afterglow. 2 ml of distilled water is added to the first one, and in others, the same number of standard solutions of prometrine herbicide, which has high phytotoxicity in relation to photosynthetic processes.

By sequentially installing the cuvettes in the device for recording the intensity of the millisecond delayed fluorescence and turning on the exciting light, first with an intensity of 50 W / m ² , then after a few seconds with an intensity of 5 W / m ² , the maximum values of ZF _in and ZF _n, respectively, are measured. After calculating their ratio for each cuvette, toxicity factors K _{t are} calculated.

The results presented in Figure 1 show that with increasing herbicide concentration as a result of increase of the toxic effect is reduced _in relation Polar Division / Polar Division _n and increases the coefficient K _t. In this case, in contrast to the absolute values of PF _in and PF _{n, the} dependence between the relative toxicity indicators and the logarithm of the concentration of herbicides, as a rule, is linear.

, (2)
где С_б ближайшая, большая по величине концентрации гербицида, чем С_пр; l_пр длина отрезка от С_б до точки пересечения перпендикулярна с осью абсцисс; l_o линейная длина концентрационного шага на ось абсцисс; Н его величина.Using the concentration dependence of K _t as a calibration graph, it is possible to determine the content of promethrin in the studied samples. To do this, you need to postpone the toxicity coefficient of the studied solution on the ordinate axis of the graph and draw a line parallel to the abscissa axis until it intersects with the calibration curve. Having lowered the perpendicular from this point to the abscissa axis, the concentrations of the herbicide in the analyzed sample are found by the formula

, (2)
where C _{b is the} closest, higher concentration of herbicide than C _pr ; l _straight length of a segment of C _b is perpendicular to the intersection with the abscissa axis; l _{o the} linear length of the concentration step on the abscissa axis; H is its magnitude.

For FIG. 1 l _ave is 6.3 mm, that for l _o = 15 mm corresponds to a concentration in the sample prometryn: C _ave = 1,85 • 10 ^-8 mol / l.

Example 2. Cells taken in example 1, with standard formaldehyde solutions and the sample under study, are first irradiated with 100 W / m ² PAR light, and then 8 W / m ² PAR, registering the amplitudes of the SF _in and the SF of _n , respectively. After calculating the ratio of PF _in / PF _n and the toxicity coefficient, build a calibration graph (Fig. 2). Figure 2 presents the dependence of the intensity of the SF of the suspension of chlorella excited by high (1-100 W / m ² ) and low (2-8 W / m ² ) light intensity, the ratio of their intensities (3), toxicity coefficient (4) on the formaldehyde concentration in the environment.

Since the value of l _CR for the analyzed sample is 7.5 mm, the concentration of formaldehyde in it according to formula (2) is 3.7 • 10 ^-4 mol / L.

,
где С_пр, С₁ и С₂ концентрации токсиканта в пробе и в двух стандартных растворах соответственно (С₂>C₁; Кт пр, Кт₁ и Кт₂ коэффициенты токсичности этих растворов.The calculation of the content of formaldehyde and other phytotoxic substances can be made not only graphically, but also analytically. For this, a formula is used, which is derived on the basis of the existence of a wide linear section on the curve of the dependence of fluorescence toxicity indicators on the logarithm of the concentration of phytotoxicant:

,
where C _pr , C ₁ and C _{2 are the} concentrations of the toxicant in the sample and in two standard solutions, respectively (C ₂ > C ₁ ; Cp pr, Ct ₁ and Ct ₂ toxicity factors of these solutions.

Example 3. In cuvettes with 2 ml of biotest, 2 ml of wastewater of a pulp and paper mill (PPM) and a combine plant in several dilutions are added. The SF _in and ZF _{n are} measured at 80 and 7 W / m ^2, respectively, and the coefficient K _{t is} calculated. As can be seen from the table, the wastewater of the pulp and paper mill containing a large amount of organic impurities has a high phytotoxicity, which becomes small only with significant dilution. And vice versa, the phytotoxicity of industrial wastes of the combine plant, in which minerals predominate, is small, even without first diluting them with clean water.

Thus, using the dilution factor along with the coefficient K _t , we can conduct a comparative assessment of the phytotoxicity of industrial wastewater of various enterprises and organize current express control over their effluents.

It should be noted that in order to obtain the maximum accuracy in determining phytotoxicity, it is necessary to strictly preserve the selected mode of recording PF _in and PF _n when analyzing control (standard) and prototypes. For the same purpose, all biotest samples for each measurement session should be taken from a single suspension of algae. And, finally, when working with non-aqueous solutions of phytotoxic substances (for example, alcohol, acetone, hexane and other extracts), to compensate for the effect on the biotest of the solvent itself, the latter is introduced into the control cell in the same volumes as in the cells with the toxicant.

Claims (1)

A method for determining the content of phytotoxic substances, comprising irradiating light with cells of chlorella algae in the presence of an analyte of toxic substance and measuring the intensity of photoinduced delayed fluorescence of algae in a millisecond damping interval under the influence of a toxic substance, characterized in that the intensity of delayed fluorescence is measured at the induction maxima of light after first activating the excitation at last the intensity value 50 100 W / m ^2, and for values of ii excitation intensity May 10 W / m ^2, and as an index of phytotoxicity is used the ratio of the measured values, normalized to the corresponding values measured for control samples, the content of toxic substances is calculated by the indicator value phytotoxicity using pre-constructed calibration curve exponent toxicity from logarithm of the concentration of the substance.

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