Test for the integrated assessment of the state of pollution of the marine and fresh water

 

(57) Abstract:

The invention relates to biotechnology and the pollution of natural waters. Developed an enzymatic test for the analysis of water pollution based on the use of meteozavisimost Gnkazy isolated from embryos or sea urchin egg. This enzyme retains 100% activity in the hydrolysis of substrate in sea water. He takes to evaluate the presence in water of heavy metals, pesticides and other toxicants on the inhibition of enzyme activity. The advantage of the test is its accuracy, sensitivity, simplicity and economy in use. 2 Il., table 1.

The invention relates to biochemistry, in particular to aquatic toxicology, and can be used for integrated rapid assessment of various toxicants in marine water.

Due to increasing anthropogenic impact on aquatic ecosystems are becoming ever more relevant to the problem of finding and developing a highly sensitive and simple methods for characterizing the quality of natural sea water. Widely used methods of physical-chemical and chemical control, although having high Chuvan in sea water complex pollutants /1/.

In this regard, over the last 15-20 years, the attention control services for water quality for timely protection measures increasingly attracts biotesting of the marine environment. Based on the study of changes in biological indicators, biological testing provides the ability to get integrated information about the quality of the environment and to predict the environmental consequences. In world practice, one of the most popular biotests, which is part of the system testing seawater, using gametes, embryos and larvae of sea urchin (Sea Urchin Test System"), is a method based on the analysis of the fertilizing capacity of sea urchin sperm, previously aged in the environment with toxic substances /2/. This Biotest has a number of advantages: relatively cheap, quick experiment (60 min). However, the accuracy and sensitivity of this method, and generally biotesting, not high enough (low reliability and poor reproducibility of results), as measured parameters livelihoods depend on the individual characteristics of the organism. However, there is a new approach to the development of biotests, combining SEB is snasti through the use of enzyme systems, responsible for any parameter the life of the body /3/. When biomonitoring natural seawater ability test systems to an integrated assessment of the toxicity of many substances (waste industrial and agricultural facilities, sewage and so on) is usually more important than their sensitivity to certain toxicants.

As is known, the rate of the enzymatic processes can largely depend on the presence of substances capable of specific to inhibit or activate the enzymatic reaction. Therefore, the introduction of a measured sample of sea water in the enzymatic test system, containing both the enzyme and its substrate, with subsequent registration of changes of system properties, allows to evaluate potential habitat for the implementation of the enzymatic reaction.

Previously developed rapid test systems trypsin - azocasein and amylase - modified starch, were used to assess the quality of the marine waters of the Black sea /4/. It was shown that some of the studied samples of water, detect the effects of inhibition as by and amylase activities.

It is known that natural seawater pH, and pH values depending on the time of year and the depth change in the interval from 7.5 to 8.1 /5/. Therefore, the monitoring of natural seawater is optimal enzymatic test system, in which the rate of hydrolysis of the substrate in buffer solution and in pure sea water is the same. Known as the test system have a lower enzymatic activity in clean sea water compared with the buffer solution (trypsin-azocasein up to 50%), which raises difficulties in the interpretation of the results. Possible protease inhibitors can be present in unpolluted natural waters metabolites produced by microorganisms. Therefore, the inhibition of the enzymatic activity in such cases, there may be an assessment of the degree of contamination of the environment. At the same time the amount of enzyme activity, in addition to polluting factors, is influenced by parameters such as different values of salinity, pH and salt concentration of metals present in natural sea water in natural background concentrations.

The objective of the invention is the development of the enzymatic test is suitable for the analysis of natural marine and fresh water, combining with jetty) with high precision analysis the simplicity and sensitivity; and expanding Arsenal of tests to assess pollution and quality of natural waters, including the sea.

The problem is solved by applying meteozavisimost deoxyribonuclease (Gnkazy), saves the activity in the hydrolysis of substrate in sea water, selected from, for example, embryos or sea urchin egg as a test for assessing the state of pollution of the marine and fresh water.

Selection and some physico-chemical properties of Gnkazy from embryos of the sea urchin Strongylocentrotus intermedius described in /6, 7/. The study of the properties and specificity of the enzyme showed that this Tnkase, as well as other highly specific Gnkazy, can be used in molecular biology and genetic engineering as a tool for studies of unusual DNA structures, as well as in biotechnology in obtaining biologically active substances. Due to the fact that the enzymes of this class (nucleases) have antiviral, antitumor and immunomodulatory effects, you can use this Gnkazy in medicine and veterinary medicine, the Use of Gnkazy as a test system for assessing the state of pollution of natural waters previously was not known. Away unique ability to break down the high-polymeric DNA in natural sea water (solution with high ionic strength) without loss of enzyme activity relative to the standard incubation mixture. Salts of various metals present in natural sea water background concentrations, as well as natural variations in water pH in the range of 7.6 to 8.2, do not affect the enzymatic activity, as the dilution of sea water distilled. This fundamentally distinguishes the test with we offer Dnazol from the known enzymatic tests.

In model experiments we have shown that the additional introduction as in the standard incubation mixture and in pure sea water of different concentrations of divalent ions of copper, zinc, cadmium, lead, iron is almost completely inhibit the enzyme activity at concentrations of 0.1 - 3.0 mg/L. Sensitivity Dnesday activity to the presence in sea water, pesticides (DDT, isopen, chlorophos, lindane), detergents (powder Lotus, sodium dodecyl sulphate) and hydrocarbon oil affects less and largely depends on the type of connection. This testing was conducted to generate baseline data on the Toxicological effects of various reagents on the proposed model object to determine its sensitivity and versatility in the analysis of pollution of the marine environment.

Also shown is the kami of the waters of the Amur and Ussuri gulfs (sea of Japan), that is, the sensitivity of the enzyme to the integrated effects of toxicants. Biological testing conducted on the presence in sea water of heavy metals and some pesticides on the activity indicators Gnkazy suggests that the proposed method is comparable in its sensitivity with Biotest, using as the test object, the sperm of sea urchins /8/ (see table).

The proposed test combines a number of advantages, characteristic biochemical methods (accuracy and reproducibility, short-term analysis, the relative simplicity and economy), with some advantages of Biotest based on the analysis of the fertilizing capacity of sea urchin sperm (obtaining the integral characteristics of the toxicity of the environment). However, unlike the latter, the use of Dnesneho test does not depend on the time of year and season of spawning sea urchins. Used standard and is quite stable in the preparation of the enzyme, possibly testing of toxicants in marine water of varying degrees of salinity, up to fresh. Thus, Tnkase from embryos or sea urchin egg is a convenient model object and can be used in the test system in the La for her condition,

Example 1 of embodiment of the invention.

Previously conducted Toxicological testing of the test system, with the aim of establishing baseline data on the Toxicological effects of various reagents on the proposed model object. In pure sea water (0.9 ml) add 5 units of the act. Gnkazy and 300 g DNA (0.05 ml) and known quantities of specific chemicals (0.05 ml). The standard incubation mixture in a total volume of 1 ml contains 300 mg of DNA, 5 units of the act. Gnkazy, 5 mm Tris-HCl buffer, pH 7.5, 5 mm MgCl2and 0.1 mm CaCl2. The mixture is incubated at 37oC for 30 minutes Enzymatic activity determined by the number formed in the hydrolysis of DNA to acid-soluble oligonucleotides according to the standard methodology /7/.

When assessing the toxicity of a known reagent determine the concentration at which achieved 50% inhibition Dnesday activity (IC 50). 100% accept the enzyme activity determined in the standard incubation mixture minus control. The results of parallel experiments differ by 2-5%. Tnkase from embryos of the sea urchin Strongylocentrotus intermedius allocate method /6/. The enzyme was stored in 50 mm Tris-HCl buffer, pH 7.5, 50% glycerol for 6 months without >/P>Example 2.

Conduct direct testing of pollution of sea water to measure the total toxicity of unknown chemicals. To 0.9 ml sample of water, add 0.1 ml 300 msdnc 5 per act. Gnkazy followed by incubation for 30 min at 37o.

Example 3.

All stages are identical to example 2, except that instead of Gnkazy from embryos of the sea urchin Strongylocentrotus intermedius, use Tnkase from eggs of the sea urchin Strongylocentrotus intermedius.

Example 4.

All stages are identical to example 2, except that instead of Gnkazy from embryos of the sea urchin Strongylocentrotus intermedius use Tnkase of the sea urchin egg Tripheustes ventaicosus.

Example 5.

All stages are identical to example 2, except that instead of Gnkazy from embryos of the sea urchin Strongylocentrotus intermedius use Tnkase from eggs of the sea urchin Lytechinus variegatus.

Example 6.

All stages are identical to example 2, except that the sample is pure natural seawater diluted in 2, 3, 4, 5 times with distilled water, the salinity varies from 16 to 6 promilla, while the enzyme activity is retained. (Fig. 1 and 2).

Example 7.

All stages are identical to example 2, except that instead of Mor MgCl2and 0.1 mm CaCl2< / BR>
Literature

1. Environmental regulation of human activity //Ecology. 1992. N 6. C. 3-12.

2. Improved methodology for a sea urchin sperm cell bioassay for marine waters //Arch. Environ. Contam. Toxicol. 1987. V. 16. P. 23-32.

3. Luciferase biotesting: biophysical bases, methods and application. Diss. Prof. Biol. Sciences. Krasnoyarsk, 1994.

4. The use of enzymatic test kits for monitoring the state of the marine waters of the Black sea //Ecology. 1996. N 5. C. 589-597.

5. Heavy metals in natural waters. M: Peace. 1987. 288 S.

6. Isolation and some properties of Ca, Md-dependent deoxyribonuclease from embryos of the sea urchin Strongylocentrotus intermedius // Biochemistry. 1976. So 41. N 11. C. 2007-2014.

7. The influence of ions of divalent metals on the enzymatic activity of Ca, Mg-dependent Gnkazy from embryos of the sea urchin Strongylocentrotus intermedius//Biochemistry. 1980. So 45. N 3. C. 544-553.

8. Comparative sensitivity of sea urchin sperm bioassays to metals and pesticides //Arch. Environ. Contam. Toxicol. 1989. V. 18. P. 748-755.

Application meteozavisimost deoxyribonuclease (Gnkazy), isolated from embryos or sea urchin egg, as a test for the integrated assessment of the state of pollution of the marine and fresh water.

 

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