Method for bioindication of water bodies

FIELD: physics.

SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.

EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.

3 ex

 

The invention relates to the field of physics and biology, can be used for environmental monitoring of water bodies.

It is known that for decision-making and implementation of measures for the protection of the natural environment, such as impaired under the influence of anthropogenic load, you must timely identification of violations for the implementation of environmental measures.

The well-known "Method of bioindication environment" [1]. The disadvantage of this method [1] is the poor reliability of the results of the assessment of the degree of pollution due to a low efficiency of actions aimed at the restoration of natural balance. In addition, the known method is time-consuming, and used indicator organisms have a limited distribution area. This substantially limits the possible applications of this method.

Known method of bioindication environment [2], including the choice of indicators, removing elements of the environment indicators, data processing and report on the cleanliness of the environment. The quality of the environment, as in the above ways, determined by one of the "show groups" ("taxa") and all "other groups" discovered organisms.

The disadvantages of this method [2] are insufficient for the practice of PR is changing the reliability of the result due to a formalized approach to the assessment of the diversity of the studied communities, referring to the fact that not all animals have the indicator value, and the absence of assessment indicative significance "of other groups of organisms.

The closest to the essence of the invention of the prototype - is a method of estimation of the mean saprobity (supranote - classification of organisms according to their resistance to pollution, organic load, lack of oxygen, the presence of compounds of hydrogen sulfide, as well as zoning of water bodies according to the degree of organic pollution of water bodies with the use of indicator organisms [3].

The disadvantage of [3] is the poor reliability of the results of the assessment of the degree of pollution of water bodies and as a result, low efficiency of actions aimed at the restoration of natural balance. In addition, the known method is very time consuming, its implementation requires the participation of staff is very high qualification, and used indicator organisms have a limited distribution area. This substantially limits the possible applications of this method.

General drawbacks and prototype are limited areas of their application due to insufficient versatility, the need to attract highly skilled professionals, the complexity and, as the trail is a journey - delays in obtaining information about the condition of water bodies and the delay in the adoption of environmental measures on the basis of obtained by monitoring the information.

The aim of the invention is to increase the reliability of the biomonitoring for use without restrictions territory, regardless of the geographic location of the studied reservoir.

Target the fact that monitoring is carried out by bioindication of water bodies in the study area. Monitoring includes sampling live in the pond planktonic organisms, the determination of the level of contamination by phylogenetic analysis of 18S rRNA planktonic organisms, evaluation of the analysis results.

The proposed method is carried out, for example, in the following ways.

The first example

1. From the study pond in a known manner [4, 5] select a sample of planktonic organisms. The number trapped in the sample of species of planktonic organisms on the reliability of the result is not affected. The complete absence of planktonic organisms in the sample in a particular situation is interpreted as an accomplished environmental catastrophe. In this case the example is not considered.

2. The organisms trapped in the sample, in a known manner, for example, [6], determine the nucleotide sequence of the gene 18S ribosomal RNA (further under the text of the 18S rRNA), for example - using universal eukaryotic primers.

3. Use the obtained nucleotide sequence of the gene 18S rRNA and perform molecular phylogenetic analysis of several, for example one or more different types of phyto - and zooplankton contained in samples of planktonic organisms.

4. Perform independent (for each of the analyzed organisms) search for homologous nucleotide sequences, for example using known data Bank [7], and define conservative sites stable indicator species.

5. Perform the multiple alignment (a comparison of every conservative site (nucleotide sequences of 18S rRNA) one planktonic organism with all the above found conservative sites stable indicator species of planktonic organisms, such as using the computer program ClustalW [8].

6. Nucleotide sequences of 18S rRNA design, for example - using the software package Phylip [9], molecular phylogenetic trees, for example, a well - known method of maximum savings.

7. Analyze phylogenetic trees constructed by conservative gene 18S rRNA and reveal evolutionary relationships of the studied organism with other Supramonte. Assess the value of the bout the chatter-cluster support, including the studied planktonic organisms and sustainable saprobity.

At high (>85%) is the bootstrap-cluster support concluded:

7.1. when combined into one cluster sustainable indicator organisms Xeno - and oligosaprobic (or exclusively xenochrony) reservoirs determines that the pond is in good ecological condition and the threat of adverse human impact is missing

7.2. when combined into one cluster sustainable indicator organisms oligo - and mesosaprobic (or exclusively oligosaprobic) reservoirs determines that the reservoir is in unstable (in the transition from advantaged to disadvantaged status) ecological condition, feels insignificant anthropogenic load, has the ability to heal itself and does not require the implementation of additional conservation measures,

7.3. when combined into one cluster sustainable indicator organisms meso - and polysaprobic (or exclusively mesosaprobic) reservoirs determines that the reservoir is in poor condition and has significant anthropogenic load, natural ability to heal itself and not enough water needs in the implementation of environmental protection measures,

7.4. when combined into one cluster Usto the sustainable indicator organisms polysaprobic bodies of water make a conclusion about presence of local ecological disaster and take immediate remedial measures, for example - combining biological and artificial means, using technical means purification, aeration.

The second example

1. From the study pond in a known manner [4, 5] select a sample of planktonic organisms. The number of species of planktonic organisms caught in the test for the reliability of the result is not affected. The complete absence of planktonic organisms in the sample in a particular situation is interpreted as an accomplished environmental catastrophe. In this case the example is not considered.

2. Determine the species composition of samples obtained on the basis of morphological characters.

3. Search the nucleotide sequences of 18S rRNA caught in a sample of planktonic organisms, such as is known in the database [7].

4. Moving on to item 3 of the first Example and consistently perform all paragraphs 3-7.

Third example :

Made bioindication of the reservoir BULAK, located on the territory of Kazan Republic of Tatarstan [10]. Bioindication performed by using a known method [3]. Work (bioindication) with 5 specialists performed with expenditure of 450 man-hours. The result of bioindication: pollution level of the reservoir corresponds to the alpha mesosaprobic zone, the water belongs to the fourth class quality contaminated. The presence of fish not detected. The result of bioindicator is no assessment of the ability of the reservoir BULAK to heal itself. There are no recommendations for the implementation of environmental protection measures in the pond.

Re bioindication of the same reservoir BULAK made by the claimed method, two assistants, consuming 40 man-hours of working time. Sequence of actions:

1. Take samples of plankton.

2. From the sample produce samples of planktonic organisms.

3. Determine the primary nucleotide sequence of a gene 18S rRNA.

4. Perform molecular phylogenetic analysis of gene 18SpPHK indicator species of plankton.

5. Perform independent software search for homologous sequences in the international database of GenBank. Nucleotide sequences of the gene 18S rRNA caught in a sample of organisms identified as Brachionus calyciflorus, Keratella cochlearis, Moina brachiata, Scapholeberis mucronata (a unique number in the international database of nucleotide sequences, respectively GQ503607.1, GQ503608.1, GQ503606.1, GQ503605.1 [7]).

6. Perform the multiple alignment was found homologous sequences.

7. Design and analyze phylogenetic trees constructed by conservative gene 18S rRNA, and reveal the evolutionary relationships of the studied organism with other Supramonte.

8. Assess the value of the bootstrap support. The clusters containing the studied planktonic organisms and the sustainable saprobity, have a high (>85%) bootstrap support.

The result of bioindication by the claimed method showed that the reservoir BULAK on the level of pollution refers to alpha-mesosaprobic zone, which coincides with the estimate by a known method. Additionally (to the assessment by a known method) identified the cause of the pollution experienced by the reservoir of significant anthropogenic pressures; the reservoir is in a dysfunctional state's natural ability to heal itself and not enough water needs in the implementation of environmental protection measures. Water belongs to the fourth class quality contaminated. To reduce water pollution, it is recommended (water) aeration. Water fountains, promote aeration of the water in the pond appeared fish, and on the shore - anglers on the water body appeared migratory birds. The implementation of the recommendations of the bioindication claimed process has led to a positive result.

Comparison of the results of bioindication known and declared methods shows a full match assessments of water quality as "polluted". Assess the level of contamination is known and stated methods are the same and are characterized as "alpha mesosaprobic zone. When bioindication in a known manner there is no assessment of the capacity of the reservoir BULAK to heal itself and recommended the promotion to the implementation of environmental protection measures in the pond. When bioindication of the claimed method to estimate the capacity of the reservoir BULAK to heal itself. Bioindication by a known method performed with the expenditure of 450 man-hours of working time, the claimed method for bioindication spent 40 man-hours.

The result of instrumental bioindication by the claimed method has a higher accuracy, because free from the subjectivity of visual assessment of the condition of the pond known method [3] is by evaluating the saprobity index planktonic organisms, i.e. the classification of organisms according to their resistance to adverse conditions of environment.

Required for carrying out the method of the genes characteristic of all planktonic organisms, regardless of geographic location exposed biomonitoring areas. The applicability of the inventive method is not limited to the example planktonic organisms used in the analysis of gene 18S rRNA exists and other planktonic organisms. So for bioindication and other applicable than shown in the example of planktonic organisms. This factor eliminates the territorial limits of applicability of the claimed method of bioindication of water.

The novelty of the proposed method of bioindication of water bodies is to use molecular Phil the genetic analysis in assessing the extent of contamination. The method allows to determine the state of the aquatic environment through the use of organisms indicator regardless of geographic location (location) of the studied reservoir. The reliability of the result, the method does not depend on the species composition of organisms-indicators in the sample. Therefore, the proposed method for the biomonitoring of the molecular basis of universal, has a sensitivity at the level of the highest achievements of molecular genetics, and the results of the application of the method is more accurate compared to the results of the use of analogues.

The proposed method allows to monitor the impact on the environment as anthropogenic factors and natural changes, such as the impact of changes in planktonic organisms, recording the cumulative effect of their (anthropogenic factors and environmental changes) of actions in real field conditions. The method allows timely and properly make decisions to improve the state of natural communities, such as environmental systems, including water bodies, to assess the results of the adopted environmental measures.

Examples of application of the present invention showing its usefulness for environmental monitoring, such as environmental, agricultural, recreationalist. The application of the proposed method facilitates the identification of detrimental human impacts in the early stages, allows timely and accurately make decisions on improving the status of water bodies, with the least damage to the environment.

The present invention satisfies the criteria of novelty, as in the prior art is not detected, the tool having characteristics identical (i.e. the same executable their function and form complete these signs all signs listed in the claims, including the characteristics of the destination.

The method of bioindication involves an inventive step, because it is not identified technical solutions that have the signs consistent with the distinguishing characteristics of this invention, and is not installed fame of influence of distinctive features on the specified technical result.

The claimed technical solution can be implemented on an industrial scale for environmental, agricultural, recreational activities (recreation), activities of health care organizations, and through the use of known standard of technical devices and equipment. This corresponds to the criterion "industrial applicability", presented to the inventions.

Sources of information

1. PA is UNT EN 2213350, MPC G01N 33/24; G01N 33/18. Priority from 24.12.2001. Applicants Segnali and other Description of the patent.

2. The Makrushin AV Biological analysis of water quality. - Leningrad: Academy of Sciences of the USSR, 1974. - P.46-47

3. Sladechek V. System of water quality from the biological point of view. Arch. Hydrobiol. Ergeb. Limnol,, 1973. - S-191.

4. Ed. Winberg GG, Lavrentieva HE was Methodical recommendations on the collection and processing of materials at the hydrobiological studies on freshwater ponds. Zooplankton and its products. - L.: GosNIORKh, ZIN an SSSR, 1982. - 33 S.

5. Ed. Winberg GG, Lavrentieva HE was Methodical recommendations on the collection and processing of materials at the hydrobiological studies on freshwater ponds. Phytoplankton and its products. - L.: GosNIORKh, ZIN an SSSR, 1984. - 32 S.

6. Watts D., MacBeath J.R. Automated fluorescent DNA sequencing on the ABI PRISM 310 Genetic Analyzer. Methods Mol Biol. 2001. - S, 153-170.

7. http://www.ncbi.nlm.nih.gov (international database of nucleotide sequences of GenBank/EMBL/DDBJ)

8. http://www.ebi.ac.uk/Tools/clustalw2/index.html

9. http://evolution.genetics.washington.edu/phylip.html

10. Ecology of the city of Kazan (collective monograph). - Kazan: Publishing house "SCIENCE" of the Academy of Sciences, 2005. - 576 S.

The method of bioindication of water bodies, including sampling live in the pond planktonic organisms, the determination of the level of contamination by analysis and evaluation of the results of the analysis, wherein the determination of the level of contamination is carried out by filogenetic the RCM analysis of genes ribosomal RNA (18S rRNA) of planktonic organisms in the sample, analyze phylogenetic trees constructed by conservative gene 18S rRNA and reveal evolutionary relationships of the studied organism with other Supramonte, and evaluation of the results of the analysis is as follows:
at high (>85%) is the bootstrap-cluster support, including the studied planktonic organisms and sustainable saprobity, make the following conclusions:
when combined into one cluster sustainable indicator organisms Xeno - and oligosaprobic (or exclusively xenochrony) reservoirs and studied planktonic organism determines that the pond is in good ecological condition and the threat of adverse human impact is absent
when combined into one cluster sustainable indicator organisms oligo - and mesosaprobic (or exclusively oligosaprobic) reservoirs and studied planktonic organism determines that the reservoir is in unstable (in the transition from advantaged to disadvantaged status) ecological condition, feels insignificant anthropogenic load, has the ability to heal itself and does not require the implementation of additional conservation measures,
when combined into one cluster sustainable indicator organisms meso - and polysaprobic (or iscrucial is but mesosaprobic) reservoirs and studied planktonic organism determine the reservoir is in poor condition and has significant anthropogenic load, natural ability to heal itself and not enough water needs in the implementation of environmental protection measures,
when combined into one cluster sustainable indicator organisms polysaprobic reservoirs and studied planktonic organism conclude there is a local environmental disaster and the need for urgent remedial measures.



 

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