Method of determining water medium toxicity

FIELD: chemistry.

SUBSTANCE: invention can be applied for bioindication and biotesting of polluted waters and separate pollutants and can be applied as an additional method to biotests of obligatory application in determination of water quality, in which (representative) dominant species is sponge (Spongia). A method includes placement of a sponge cell suspension into a tested solution, standing for a day with the following calculation of bubble-shaped cells under a microscope, a conclusion about toxicity is made by reliable increase of the bubble-shaped cells in the tested solution in comparison with the control one.

EFFECT: considerable increase of sensitivity of the method with the minimal terms of the experiment performance.

2 ex, 1 dwg

 

The present invention relates to aquatic toxicology and can be used for bioindication and biotesting of polluted water and individual pollutants and can be used as an additional method to biotests mandatory application in the determination of water quality, in which (representative) dominant species is Spongia.

When conducting toxicometric pollutants at the present time, it is recommended to use not only the required biotests included in the Federal register, but the tests developed as an additional, representative for the studied pond organisms. Especially it concerns the protection of the waters of this unique lake Baikal. Freshwater Baikal sponges (Lubomirskiidae) - endemic organisms that predominate in biomass among littoral benthic animals, little studied in terms of ecotoxicology.

Known methods for determining toxicity using benthic organisms, unable to move and get away from the zone of contamination, in particular sponges.

There is a method of assessing the toxicity of aquatic environments / Auth. St. USSR №1730581, G01N 33/18, AK 61, 1992/, based on the immobilization of larvae Baikal sponges Other bacillifera in a toxic environment.

The method is quite sensitive, but it has the disadvantage of limiting the period of study because the reproduction of larvae occurs only twice a year.

There is a method of determining the toxicity of water repulsion clouds of dye water, throw away sponge from the region / Patent RF №2003096 G01N 33/18, AK 61/00 from 15.11.93 1993/. Preliminary animal exhibit in the test solutions in aquariums, then, using a micropipette, at a distance of 3-5 cm from the animal's body make the dye solution in harmless concentrations, such as fluorescein. In case of unsatisfactory physiological state of the cloud slowly dropping the sponge in the control aquaria sharp repulsion cloud fluoresceine a stream of water from the region.

The reaction is insensitive, not reproducible, and requires the use of a large number of intact specimens of animals, but convenient when monitoring their status directly in native conditions, which is carried out by scuba divers using a syringe.

There is a method of biotesting toxicity aquatic environment / Patent RF №2462707, G01N 33/18, AK 61/00, 2012 / as the test object using the rotifer where environmentally corresponds to the mineral composition of the water environment. To determine the degree of toxicity of the water environment having a series of dilutions of a measured sample of water taken from the background net area of the water body. The degree of toxicity of the water among the s is estimated by the degree of dilution with clean water to remove abortions, moreover, the dilution of the sample to 1:1 analyzed water refers to non-toxic; up to 1:25 to slightly toxic; up to 1:50 - moderately toxic; up to 1:100 to strategiczny; up to 1:500 and more - very toxic.

Known biological method of determining the degree of General toxicity and key toxicants in water protection /Patent RF №2110067, G01N 33/18, AK 61/00, 1998/ as test organisms once individuals use laboratory culture of the test organisms grown by inbreeding organisms at a constant regime on artificial pure reference water and feed. Determine and compare resulting in the set of solutions of model toxicants artificial pure reference water and test the water environment spectra behavioral responses of test organisms, their locomotor activity.

The known method with the use of active dyes to determine the physiological state of the animal, based on the property proteonomix dyes to form a strong covalent bond with the substance of the sponge skeleton /Patent RF №2003097 G01N 33/18, AK 61/00, 15.11.93/. The degree of staining of the skeleton can be judged on the trophic activity of the animal, and you can use only a portion of the sponge body. Experimental and control of the aquatic environment is placed benthic sponges, able to record a physiologically the cue parameter sponges and judge the toxicity of the investigated medium on the obtained experimental data in comparison with the control, and additionally in the experimental and control environment contribute procianoy dye. Keeping sponges in environments should be performed within 8 hours, then cook the sliced sprouts sponges, and as a physiological parameter register the presence or absence of coloration of the body sponges, to include toxic experienced the environment in the absence of color on the cut bone sponge, containing in the experimental environment.

When using expensive dye sensitivity is not high enough.

The closest analogue is the method of bioindication of wastewater toxicity /A. St. No. 1578650 from G01N 33/18, AK 61/00 .1988,/. The sponge has a unique ability to regenerate from dissociated cells of the animal, forming a conglomerate of various forms. This property underlies the method of biotesting. The toxicity criterion is the absence of an Assembly of cells in the conglomerates in the test solutions.

The test is simple, rapid (Assembly time 1 day), allows the use of a small portion of an animal body, but is not sensitive enough.

Object of the present invention is to provide a method that improves its sensitivity with minimum terms of the experiment.

This object is achieved in that in the method of biotesting of water using a test OS is consistent on the reaction cell suspension sponge, including the location of the suspension in the test solution, keeping within a certain time, followed by bubble-counting cells under a microscope, on the toxicity judged by a significant increase of cells in the test solution in comparison with the control.

The method is as follows.

In Petri dishes with 20 ml of Baikal water or solution of toxicant add 1 ml of the cell suspension sponge and placed in a cooling chamber with a temperature of 8°C. the next day the resulting conglomerates with dispenser add 5 ál of the cell suspension are placed on a glass slide and counted under a microscope at magnification 20×18 number of bubble cells (diameter of about 16 microns) in the Goryayev camera or the field of view. In the control, usually 3-10 cells in field of view.

Example 1

Investigated the toxicity of para-Besnainou, pollutant, formed by the oxidation of diphenols contained in the waste waters of the Baikal pulp and paper plant and the decay of lignin - waste production BPPM. Figure 1 shows the dependence of the number of bubble cells on the concentration of para-benzoquinone and mercury chloride (II).

Figure 1 shows that the test solution at a concentration of 10-1-10-4mol/l has acute toxicity and suspension cells of the sponge does not have time to react to it is ricotta release of bubble cells and dies, as evidenced by the absence of the conglomerates of the assembled cells.

If the listed test analogs are toxic concentrations of para-benzoquinone 10-5-10-6mol/l, the test for the emergence of the bubble cells 10-10mol/L.

Example 2

The obtained cell suspension sponges incubated in solutions of HgCl2through the day in each sample calculate the number of bubble cells under a microscope.

Figure 1 shows that concentrations of 10-1-10-6. mol/l disastrous for the suspension and the cells are not formed.

When using the closest analogs non-toxic for sponges can be considered as the concentration of HgCl2up to 10-7mol/l, whereas the test of occurrence of bubble cells such is the concentration of 10-12mol/l, which indicates its high sensitivity in comparison with analogues.

Effect : a significant increase in sensitivity with a minimum period of the experiment.

The method of determining the toxicity of the aquatic environment using a test based on the reaction of a suspension of cells of sponges, including the location of the suspension in the test solution, keeping during the day with the subsequent bubble-counting cells under a microscope, characterized in that on the toxicity judged by a significant increase of cells in the studied solution p is compared with the control.



 

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2 tbl, 1 ex, 1 dwg

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2 cl, 2 tbl

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3 ex, 4 tbl

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5 cl, 4 tbl

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EFFECT: higher efficiency of investigation.

1 tbl

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