Determination of hydrocole daphnia magna straus sensibility to toxic effect of water-soluble chemicals

FIELD: toxicology, in particular determination of water flea sensibility to toxic effect of water-soluble chemicals.

SUBSTANCE: claimed method includes detection of water flea death time (min) caused by water-soluble chemicals, wherein concentration (C, mol/l) of chemical under consideration fluctuates according to logarithmic scale with interval of 0.1. Plot of Y versus X is made, wherein Y-axis represents average death time with scale of 1 point = 1 min; X-axis represents reverse concentration (1/C) of chemical under consideration; and scale is proportional to log increasing by 0.1. Water flea sensibility to toxic effect (tgα) is calculated according to equation: tgα = TL(min):1/KL = TL(min)xKl (I), wherein α is inclination of straight line to X-axis; TL(min) is death time (min) being determined according to point of hypothetical crosspoint of straight line with Y-axis; KL is lethality constant (mol/l) defined as chemical concentration wherein water flea death time is equal to 2TL(min).

EFFECT: Method allowing evaluation of toxic effect evolution dynamics and comparison of toxic effect of water-soluble chemicals in equal concentration ranges.

2 tbl, 1 ex, 1 dwg

 

The invention relates to medicine, in particular, toxicology, and in particular to methods of assessment of toxic effects of chemical substances.

In Toxicological studies, the basic concepts are “dose”, when the set number of chemicals penetrate directly into the body by injection intravenously, intramuscularly, subcutaneously, per os, and so on), and “concentration”when a chemical substance in a given volume is surrounding the body environment (water, air), but the exact amount penetrated the body, is not known. Toxic effects of chemical substances, as a rule, is “lethal” action (i.e., on the fact of death of the organism). Dose or concentration of the xenobiotic, causing death, are denoted as LD or LC with an index below, showing the percentage of fatal reactions. The standard way of determining the toxicity of a substance is the way to check-in 50% (medium) death of organisms taken in the experience (LD50, LC50). (Methods for determining the toxicity and hazards of chemicals. Under reducts. Iwinarcade, M, Medicine, 1970). This approach is based on the introduction of animals of increasing doses of chemical substances (or the presence of the organism in the environment with increasing concentrations of substances) with subsequent fixation of death over the period of the belts (Loit S.A., Savchenko F Preventive toxicology. A guide for toxicologists-experimenters - Irkutsk: Publishing house of Irkut. University, 1996, s-79). Closest to the claimed object is a method of determining the toxicity of water-soluble chemicals on Daphnia (Tocopy E, Zagrebin S.A., Sherstneva P.A., Kosmachev A.B. Journal evals. biochem. and Fiziol. - 1994, T.30. No. 3, s-391).

The main disadvantage of this method of determining the toxicity of a chemical is the fact that the method does not take into account toxicokinetics difference between substances and assess their effect dose or concentration at which there is a loss of 50% of individuals in one selected point in time, after 1 day after intoxication. If there are strong differences in the toxicokinetics of substances such method of comparison of their toxic action is not quite adequate.

The present invention is to develop a method of determining the sensitivity of Daphnia to the effects of water-soluble chemicals in experiments in vivo, which allows on the basis of the reception time of the death of aquatic organisms Daphnia magna Straus in different concentrations of aqueous solutions of chemicals to evaluate the dynamics of its toxic action.

To solve the problem determine the time of death of Daphnia under the influence of the each of the logarithmically increasing concentrations of the studied chemicals. Based on the obtained values of the average time of death of individuals (TL, min), at given concentrations of the chemical ([], M), from graph (see drawing) according to the time of the death of aquatic organisms Daphnia magna Straus on the value of the inverse molar concentration of the analyte, determine the minimum time of the death of Daphnia (TL(min)), a constant mortality (Land the tangent of the slope of the graph to the x-axis (tgα ). The analyzed concentration of toxicant change on a logarithmic scale with an interval of 0.1. The volume of solution per Daphnia, not less than 20 ml of the Real time of death of animals for each concentration of a chemical is determined visually.

The construction schedule (see drawing) allows you to define values: minimum time of occurrence of death, TL(min)equal to the cut length, cut a continuation of the straight line on the y axis, constant mortality, KLequal concentration of toxicant at which time the loss is equal to two minimum, and the slope of a straight line to the x-axis (tgα =TL(min):1/KL=TL(min)·ToL). For the correct calculation and, further, comparison between tgα different chemicals need the chart to stick on both axes of a single scale: x-axis - change concentric and (mol/l) is proportional to the increase in log 0.1, expressed an inverse value on the ordinate axis 1 division 1 minute, TLmin

The definition of the tangent of an angle α it is necessary to obtain quantitative sensitivity characteristics of a living organism to the investigated substance, taking into account both the concentration of the toxicant and the dynamics of the time of onset of effect (death). Therefore, tgα can be called a quantitative measure of the sensitivity of Daphnia to toxic chemical substances in the concentration range corresponding to the graph a straight line segment. The tangents of the angle α it is advisable to compare in the same concentration ranges of the compounds under investigation.

The principal difference from the previously used method for the assessment of toxic effects of chemical substances (line with an50) is that the proposed method takes into account not only the effective concentration of the analyte in one arbitrarily chosen point in time, but also the dynamics of the effect of the death of aquatic organisms, which allows for comparison of toxicity of different substances in the same concentration ranges.

Example.

Provides a procedure for the determination of toxic effects of hydrogen peroxide in the experiments on aquatic organisms Daphnia magna Straus, bioobject, widely used in toxi is ideological practice (international standard ISO 6341-82. The water quality. Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea). - 1987). Record the time of the death of Daphnia stays in solution (mol/l) hydrogen peroxide in concentrations varying in proportion to the increase in the log of 0.1, and calculate the values back to the used concentration (1/[S]) (table 1). Based on the results presented in table 1, construct a graph of the average time of death of Daphnia from concentrations of hydrogen peroxide, presents the inverse value (1/[S], M) (see drawing). Graphically determine the value of KLTL(min)and I hope tgα .

The value of TL(min)found, continuing straight part of the graph to the intersection with the ordinate axis (TL(min)=1 min), lay on the graph y-axis 2TL(min)(2TL(min)=2 min), from the point of intersection values 2TL(min)with the schedule omit perpendicular to the intersection with the axis 1/[S], at this point of intersection to find the value of 1/KL(1/KL=0,32), we get KL=3.1 mol/l tg Value of angle α calculated by the formula:

tgα =TL(min):1/KL=TL(min)·KL=1× 3,1=3,1.

The same way you have defined settings for the sensitivity of Daphnia to toxic action karbaholina and arecoline (table 2).

the table 1
The average time of death of Daphnia when exposed to decreasing concentrations of hydrogen peroxide (concentration are on a logarithmic scale with the interval 0,1)
Concentration [C] (M)1/[S]The average time of death of Daphnia (min)
11of 3.46±0,2
0,81,34,22±0,2
0,631,64,72±0,5
0,525,94±0,7
0,42,57,27±0,9
0,323,28,44±0,9
0,25411,5±0,8
0,2515,0±1,3
0,166,317,5±1,8
0,13822,5±2,1
0,11029,5±2,8
0,081341,5±3,7
0,0631649±5,4

Table 2
Determination of the sensitivity of aquatic organisms Daphnia magna Straus to toxic effect of hydrogen peroxide, karbaholina and arecoline
ChemicalsThe minimum time of the death of Daphnia (TL(min)), minConstant mortality (L), MA measure of the sensitivity of Daphnia to chemical substance (tgα )
The peroxide1,03,13,1
Karbaholin0,26,7· 10-31,3· 10-3
Arcolin0,48,3· 10-33,3· 10-3

The method of determining the sensitivity of Daphnia to toxic action of water-soluble chemicals, including registration of the death of Daphnia under the action of chemical substances, characterized in that record the time of death in minutes when changing on a logarithmic scale with an interval of 0.1 concentration (C) in mol/l analyte with the subsequent construction of the graphical dependence of the averaged time of the death of the ordinate scale 1 division - 1 min from the values of the inverse concentration (1/S) of the analyte on the x-axis, and the scale is proportional to the increase in the log of 0.1, and the calculation of the sensitivity of Daphnia to toxic effects (tgα ) by the formula

tgα =TL(min):1/KL= TL(min)·KL(I)

where α - the angle of the direct plot on the graph to the x-axis,

TL(min)the time of occurrence of death in minutes, determined by the point gipoteticheskogo intersection of a segment of a straight line with the y-axis,

KL- constant mortality rate in mol/l is defined as the concentration of a chemical at which time the death of Daphnia equal to 2TL(min).



 

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