RU2595867C2 - Method of detecting and selecting organisms-biosensors for operative bioindication and biomonitoring of sea and fresh water, including drinking and waste water - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: invention relates to ecology, particularly, to environmental protection, and can be used for operative bioindication and biomonitoring of sea and fresh water, including drinking and waste water. For this purpose organism-bioindicator is selected by determining distribution and abundance of type (types) and assessment of their availability, application of test effects to selected organisms. Selection criteria for organisms-biosensors as rapid bioindicators are determination of their survival rate, technological effectiveness, reactivity and assessment of reaction adequacy. First assessment of technological effectiveness of using organisms-potential biosensors-by checking their ability to transfer manipulation in experiments, transportation, drying, temperature difference and possibility to use it in certain technical system for monitoring operational bioindication. Then selected organisms-potential biosensors-on are checked on reactivity by determining speed and stability of physiological and behavioral reactions on various limiting and non-toxic factors of medium effect with determining response capacity for each type of test effect. Based on derived data final selection of organisms-biosensors is performed. Based on recurrence of certain reactions with identical by force effects of medium parameters of monitoring biosensors are selected.

EFFECT: invention provides continuous biological monitoring in operative biological assessment (display) of quality for both sea and fresh water, including drinking and waste water in natural or artificial conditions in real time mode.

3 cl, 11 dwg

Description

The invention relates to the field of ecology and environmental protection and can be used both for continuous biological monitoring and for operational biological assessment (indication) of the quality of both sea and fresh waters, including drinking and wastewater in natural or artificial conditions in real time.

Modern biological monitoring systems should operate in a continuous automatic mode and, practically, in real time, detect and determine the degree of environmental hazard of pollution, generate alarms and transmit them to the appropriate tracking centers. This is important not only for the water supply systems of cities and towns, but also for large economic and industrial facilities that inevitably pollute the environment. Therefore, it is important to exercise such control both in the zones of the exhaust manifolds of cities and industrial enterprises, and in the places of development and transportation of oil and gas fields, especially in conditions of vulnerable ecosystems in the Arctic.

Continuous water quality control (operational biomonitoring) is based on the use of a group of bioindicator organisms that have the properties of biosensors, that is, bioindicators that quickly respond to dangerous / significant changes in the state of the environment. For these purposes, in Europe, for example, early warning systems for the occurrence of hazardous concentrations of pollutants in the aquatic environment, Musselmonitor and Dreissenamonitor, are widely used. When selecting mollusk biosensors for use in these systems, they rely on previously known data on the biology, ecology, physiology, and behavior of these animals. However, this approach is insufficient, limited only to the field of known knowledge. This is especially important in cases when new species of animals are used in conditions of poorly studied ecosystems - indicators (biosensors), the ecology and physiological reactions of which are poorly studied or not known at all.

The use of selected animals as indicator organisms - biosensors of the state of the aquatic environment without proven biological justification (testing) and analysis of their capabilities leads to significant losses, both technological and financial. As a result, the consequence of errors in choosing an indicator organism is the loss of the effectiveness of online biomonitoring: loss of sensitivity, reliability and automaticity, complication of the process, increase in labor intensity and share of manual labor, a large number of failures, interference, false alarms, as well as premature death of animals, which leads to financial losses and a strong decrease in the quality of systems to the point of their unsuitability.

A known bioindication method according to the RF patent for the invention No. 2213350 C2 dated 12.24.2001, including the selection of a group of indicators, the formation of a reference medium with a breakdown by quality classes, the determination of the types of indicators that can exist in the range of quality classes of a reference medium, extraction of all possible views from the group of indicators. For each class of environmental quality, the types of indicators are established and the class of environmental quality is determined by the maximum value of the total class significance of the indicators. They assess the possibility of self-cleaning the environment by additionally extracting indicators from the environment and determining the total class significance of the indicators until the maximum value of the total class significance and the highest value after it are located in neighboring areas of the environmental quality class. The method can be used as an express method for a one-time examination and for monitoring media, has the short duration of the collection of bio-indicators, low cost, the ability to compare information about the ecological usefulness and economic significance of the examined media. This method is not applicable for operational bioindication systems.

Closest to the claimed method is a method of forming reference groups of test organisms described in the article "Bioelectronic monitoring of surface water" (S. V. Kholodkevich, A. V. Ivanov, E. L. Kornienko, A. S. Kurakin, V. A. Lyubimtsev Magazine "World of Measurements", No. 1. - 2011). To form such groups, a choice is made for each type of test organism of physiological, biochemical, or behavioral indicators (biomarkers) that would be necessary and sufficient to assess their physiological state (PS) and adaptive ability. The following selection criteria for test organisms for bioindication and biomonitoring are proposed:

- the presence in the ecosystem under study (if possible in large numbers) of potential bioindication objects with homogeneous properties;

- wide geographical distribution of the bioindicator organism and its representation in various habitats;

- ease of identification of bioindicator and accessibility in obtaining material;

- lack of seasonal differences in the availability and use of bio-indicators;

- the relative stability of the bioindicator to the effects / accumulation of the stressor;

- the presence of a correlation or functional relationship between the response of the bio-indicator organism and the level of stressor impact on the ecosystem. In order to select animals, namely crayfish, as bioindicators, it is proposed to apply an additional series of stress test effects to them, since the differences in crayfish taken from the same natural population and selected according to the above indicators can reach 40 ... 50% in terms of cardiac activity (HR) and total hemolymph protein. Based on their reactions to specially developed standardized loads (test effects), a reference group of test organisms is formed, the functional state of which is uniform in physiological parameters. The disadvantage of this method of selection of bioindicators is its significant complexity, cost and narrow focus. First, there are not two, but many characteristics of heterogeneity in addition to cardioactivity and hemolymph protein; secondly, indicators that are uniform during the test can vary greatly afterwards; thirdly, it is the homogeneity of the reference individuals that reduces the objectivity of the bioindication of natural waters, since the heterogeneity (genetic, physiological, biochemical) of organisms of one species is an integral property of nature. The variety of reactions (in terms of strength and speed) reflects the objective reaction of the entire population, and not of one group selected for a number of characteristics, whose individuals may be a minority in the population. Consequently, the creation of artificial groups, convenient for work, makes the indication biased in relation to natural objects. However, such an artificial group is suitable for testing such artificial objects as sources of tap and industrial water, aquariums; or to conduct biotests in experiments. The main drawback is the focus on the selection of a group of healthy individuals within one species (crayfish), and not on the selection of the best biosensor species among several potential species.

The inventive method, as well as known, includes the selection of bioindicator organisms by determining the distribution and abundance of the species (s) and assessing their availability, applying a number of test effects to the selected organisms.

The problem solved by the invention is to improve the quality of on-line bio-monitoring by developing the basics of the proper selection of bio-indicators, as well as reducing resource losses, including financial, by ensuring the reliable operation of bio-indicators-biosensors without replacing them for a certain period.

The technical result consists in creating a method for identifying and selecting biosensor organisms for operational bioindication and biomonitoring in order to ensure their effective and reliable operation in conditions of monitoring and reducing the number of failures, interference, false alarms.

The technical result is achieved by the fact that the criteria for the selection of biosensor organisms as operational bioindicators are the determination of their survival, manufacturability, reactivity and assessment of the adequacy of reactions, while first determining the survival of species of organisms - potential biosensors by conducting experiments with the placement of these species in specific monitoring conditions (surface water, thickness or bottom of the reservoir), evaluate the technological effectiveness of the use of organisms - potential biosensors - put m testing their ability to tolerate manipulations in experiments, transportation, drying, temperature changes and the possibility of using it in a specific technical monitoring system and operational bioindication, then they test the selected organisms of potential biosensors for reactivity by determining the speed and stability of physiological and behavioral reactions to various limiting and non-toxic environmental factors with the establishment of response potential for each type of test timed exposure; select the monitoring parameters of biosensors based on the frequency of certain reactions under the same environmental influences, evaluate the adequacy of the selected body reactions to the environmental pollution factors of interest by testing them, then determine the range and thresholds of the responses of the selected biosensors to changes in various types of pollution; Based on the data obtained, the final selection of biosensor organisms is carried out.

Experiments on the survival of biosensor organisms are carried out for 1-2 winter (critical) months under extreme conditions similar to natural ones, or field experiments are conducted under specific monitoring conditions.

When selecting biosensor organisms from other ecosystems, a reserve group of biosensor organisms is created by placing them under monitoring conditions in suspended cages.

Determining the survival of organisms - potential biosensors by conducting experiments with the placement of these species in specific monitoring conditions (water surface, thickness or bottom of the reservoir), provides the possibility of operation of operational bioindication and biomonitoring systems in these conditions, increasing their efficiency and reliability, as well as reducing resource losses including financial.

An assessment of the technological use of an organism - a potential biosensor - by testing its ability to tolerate manipulations in experiments, transportation, drying, temperature changes and the possibility of using it in a specific technical monitoring system or operational bioindication ensures that the biosensor organism is adequate for the technology used, including the correspondence of the type of animal used monitoring installation and recording equipment, without which holding a biosensor monitor Inga is technically impossible.

Testing the body - a potential biosensor - for reactivity by determining the speed and stability of physiological and behavioral reactions to various limiting and non-toxic environmental factors with the establishment of the response potential for each type of test exposure provides the desired level of monitoring sensitivity to external influences, reducing the number of failures, interference, false alarms.

The choice of reactions and monitoring parameters of the biosensor organism based on the technical capabilities of registration, as well as studies of the repeatability of the selected reactions under identical environmental influences; assessment of the adequacy of the selected reactions of the body to the environmental pollution factors of interest by testing them, determining the range and thresholds of the responses of the selected biosensors to changes in various types of pollution, ensures the reliability and accuracy of biosensor monitoring and bioindication.

The combination of the above distinguishing features, combined, provides the claimed technical results.

The invention is as follows.

After determining the monitoring object (sea water, or fresh, or effluent, or tap water) and its tasks, the selection of the types of potential biosensors from among the known bio-indicator species begins. To do this, in the beginning, according to available data, a study of the biology and ecology of potential biosensor species is carried out: determining their survival under monitoring conditions, their tolerance ranges, accessibility both for selecting and conducting experiments, and subsequent application in monitoring. On the basis of criteria such as survival, manufacturability, reactivity, contact, availability, adequacy of reactions and others, a list of potential biosensor species is selected.

To do this, first conduct field and laboratory experiments to study the reactions and survival of these species under monitoring conditions (surface of the water, thickness or bottom of the reservoir). In subsequent experiments, the selected species are kept for a long time under constant aquarium conditions with special experiments for:

- determining the variability of the biosensor parameters in constant conditions (the presence of stable rhythms, trends);

- establishing the speed and stability of reactions (physiological and behavioral) to various effects, that is, the range and speed of reactions to non-toxic effects (stressors) - light, vibration, water movement, oxygen, etc .;

- determination of survival under conditions of starvation and the influence of limiting factors (temperature, salinity, suspension, etc.);

- testing of reactions and survival in several pollution groups (suspension, heavy metals, petroleum products, model toxicants).

The selected biosensor organisms should, as a rule, be available in sufficient quantity or distributed throughout the study (monitoring) area and be available for collection in all seasons. They should provide the possibility of gradation of the reaction to stress depending on its duration and intensity and, if possible, demonstrate the specificity of the reactions so that it is possible to determine the type of impact. The biosensor contact is important, i.e. the degree of its physiological perception of anthropogenic influences through a constant connection with the local environment.

The selection of biosensor organisms from the list of bioindicator species is carried out according to the following parameters (indicators), passing sequential stepwise filtering.

1. Survival. First of all, the choice of a biosensor organism is determined by its ability to survive under monitoring conditions. For example, few organisms survive in brackish and estuary zones or discharge sites. Biosensors can be either local animals (preferable), or introduced from other ecosystems that have proven themselves to be reliable biosensors. To determine the ability of animals - potential biosensors (local or introduced) - to survive for a long time in the conditions of the place (point) of monitoring, experimental and field trials are conducted. The biosensor organism must remain active under test conditions, i.e. an assessment of its actual tolerance is made.

Moreover, it is preferable if the body is able to feed itself; only in the absence of such species are biosensor species (for example, crustaceans) that need to be fed regularly.

2. Manufacturability. The biosensor organism should not only be sensitive to changes in environmental factors of interest (which depends on the tasks chosen for monitoring), but also manifest its reactions in a noticeable way, which can be detected and recorded by the sensor-sensor automatically, without disturbing normal physiological processes .

According to its morphology and environmental abilities, the organism itself must be suitable for use in a specific technical (recording) installation: in particular, it must be resistant to manipulations, including experiments, meet the size and strength requirements of the attachment of recording devices (sensors) to it, transfer drying, heating and temperature changes or other influences during technological operations without involving special complex and costly processes and observing a set of strict rules containing tions of animals.

First, at the first stage, manufacturability is assessed by external signs, then, in the course of experiments - by the ability to maintain normal life activity in the installation: contact, behavior stability and reliability of attachment.

3. Reactivity. The ability of an organism, a potential biosensor, to respond to both gradual and abrupt (acute) changes in environmental factors that are selected for monitoring is determined. Physiological or behavioral reactions are checked in a series of experiments with the creation of artificial gradients of factors (temperature, salinity, pollution, etc.) and with individual acute influences. The response potential for each type of test animal is established.

4. The choice of reaction and biomonitoring parameters. The choice of the process itself is important (physiology - cardiac activity, respiration, nutrition or behavior): on the one hand, the technical feasibility of continuous monitoring of this type of indicator organism reactions under the required conditions is established, on the other hand, the suitability of this reaction for specific biosensor monitoring. For this, in addition to the results of paragraph 2, a qualitative assessment is carried out - testing the selected reaction to the factor of interest, usually to various types of pollution - biosensor monitoring objects.

5. Adequacy. When the reaction and parameters are determined, it is important to establish its adequacy to the effect. That is, the parameter must necessarily change quantitatively depending on the strength of the effect and specifically (preferably) so that the reactions to different types of exposure (especially pollution) differ.

The selection criterion is the repeatability of the reaction under the same impact (for example, with the same type of contamination).

Ranges and thresholds of reactions to changes in natural factors and pollution gradients of different types are determined. The difference in the characteristics of the response to pollution will ensure the generation of an alarm in case of real (rather than false) danger. As in paragraphs 2 and 4, the adequacy of reactions is assessed using certain instruments and devices (in particular, actographs, tensometers, valvometers for behavior, cardiographs, plethysmographs for cardiac activity). To measure the characteristics of environmental conditions, oximeters (oxygen meters), particle counters and fluorimeters (for determining chlorophyll), mini-probes (for measuring fluctuations of several environmental factors) can be used. In turn, the set of devices is checked by the conditions and goals of monitoring and also depends on the type of animal, its biology and ecology.

6. Contact. The presence of a permanent connection with the local habitat. With regard to pollution monitoring, the body’s availability for the perception of anthropogenic influences. The view should preferably be sedentary, attached or sedentary. Firstly, because it must reflect the state of a given local environment; secondly, modern technologies have not yet been developed for monitoring the activity of mobile species (crabs, crayfish, fish) in natural conditions; moreover, when choosing mobile species, one has to artificially limit their activity and movements, and at the same time carry out their regular feeding, which violates the body’s natural lifestyle, the process and adequacy of monitoring, increases the cost and complexity of monitoring.

It is necessary to establish initially the degree of contact of the biosensor organism with its habitat, and then with the monitoring environment: sea, fresh, drinking or wastewater. It is important to determine the range and conditions of use of the bio-indicator-biosensor. For example, in the Netherlands it is necessary to expel chlorine from tap water so that the mussel-zebra mussel can monitor its quality.

7. Availability. The view must be available for use at the monitoring site at any time. Studies are being conducted on the distribution and abundance of local species, and an assessment of their availability. If it is impossible to use local species, you can apply proven species of biosensors taken from another ecosystem.

As a result, we get the “Proficiency” of the selected species as bioindicators and / or biosensors.

In accordance with ISO TS 16649-3, mollusks are included in the list of biosensor organisms and test reactions (ISO // Official ISO website: http://www.iso.org/iso/home/store/catalogue_ics/catalogue).

Examples of the practical implementation of the method.

Example 1. To conduct operational biomonitoring of water quality, mussels were selected near the enclosures for seals in the Kola Bay.

Initially, bivalve mollusks were selected among all groups of bottom invertebrates, since both the technology and the continuous biomonitoring equipment in the sea were developed and tested in the world for this particular group of animals.

Since, when selecting mollusk biosensors, they rely on the already known data on the biology, ecology, physiology, and behavior of these animals obtained earlier, a preliminary study of the malacofauna of this biotope was carried out according to sampling data and literature. The massive mollusk sestophages available for collection in the area were mussels, scallops, hiatella, and modioli. These species-bioindicators were selected as potential biosensors according to the results of previous studies and experiments, which showed a high potential for the use of precisely mollusk-sestonophages (filtrators) for operational biomonitoring.

It turned out that in the surface water layer where the enclosures are located, seasonal variations in water salinity are significant - desalination caused by spring coastal runoff reduces salinity from 34 to 20 ‰ and lower.

It is known that under the conditions of desalination, many species of marine bivalves do not survive. Of the species living in the area, only mussels and, to a lesser extent, hiatella are able to live with low salinity.

Therefore, further selection was carried out among these two species. However, unlike the mussel, which massively populated the pontoons of enclosures with seals, the hiatella was rare and in smaller numbers. This was an indication of her relatively worse survival under the given conditions. At the same time, the hiatella was small (up to 1-2.5 cm), a fragile shell with many surface curvatures. All this made it unsuitable for technological reasons.

Thus, edible mussel Mytilus edulis, which has a high population density under these conditions, remained the only candidate for mollusk biosensors, which indicated its excellent survival in enclosures, with significant fluctuations in salinity, temperature, and other environmental factors. In addition, previous studies have shown good manufacturability and reactivity of mussels.

The survival of mussels was determined by a two-week exposure in mesh cages under extreme conditions - low temperature and low salinity of water in spring. Survival was higher than 95%.

The experimental determination of reactivity was carried out in the range of natural variability of environmental factors in natural conditions. It was found that mussels are able to respond even to minor fluctuations in environmental factors: for example, temperature - by 0.1 ° C (in winter), salinity - by 0.2 ‰ (in spring) - depending on the season.

When choosing a parameter for biosensor monitoring, we studied both physiological (respiration rate, filtration rate, and cardiac activity) and behavioral parameters of mollusks. Since the measurement of physiological parameters in the field is technically problematic, the main indicator of the activity of the mollusk-biosensor was chosen for its behavioral reactions (Fig. 1), the registration technology of which is the most developed and available. In addition, it was found that physiological indicators are too variable, and it is difficult or not always adequate to judge changes in the environment (Fig. 2).

Changes in the behavior of mussels were evaluated by elementary behavioral acts and three parameters of the movements of the mollusk valves: the level and amplitude of the opening of the valves (URS and AMP,% of the maximum) and the frequency of shell collapse - adduction (ADD).

After choosing the monitoring parameters, the range and adequacy of the behavioral responses of mussels under various conditions was determined by determining the reactions to both individual effects and gradients of change in natural factors and various types of pollution (Fig. 3-7).

Based on a set of tests and the data obtained, the final selection of mussel Mytilus edulis was made as a biosensor organism for operational biomonitoring under these conditions.

Subsequently, for the operational biomonitoring of water in enclosures with seals, the behavioral reactions of 30 specimens of adult mussels (from 30 to 65 mm) collected from the natural fouling of the ropes of attachment of enclosures in the immediate vicinity of the enclosures and at the same depth were recorded.

Field experiments on operational biosensor monitoring showed that this was the only bioindicator species that retained the sensitivity of reactions (tests for background reactions and pollution) under conditions of severe desalination and could live under these conditions for almost unlimited time (survival tests).

Example 2. For biosensor monitoring in the Dalnaya Zelenetskaya Bay under the project of joint Russian-French research in 2012-2014. Chlamys islandica scallops, caught at a distance several miles from the lip, were successfully used as biosensor organisms, along with mussels. When choosing this species, all the above stages of selection of the biosensor organism were used.

Operational biomonitoring in the lip was carried out at a depth of 15 m, under conditions of relatively stable and high salinity of the water. From literature and previous studies it was known that scallops do not tolerate significant fluctuations in salinity and especially desalination, but in conditions of high (marine) salinity they are highly sensitive to changes in environmental factors. Earlier scallops lived in the lip, but even then they were found in small quantities. Other potential biosensor mollusks at these depths were absent.

The survival of scallops (50 specimens) in Dalnaya Zelenetskaya Bay was checked during their annual exposure in cages at a depth of 12-15 m, it amounted to 98% on average.

Many signs indicated that Ch. islandica may be suitable as a biosensor organism. This mollusk-sestonophage is widespread and abundant in the waters of the Murmansk coast. The scallop shell is large, strong, flattened; in manual operations, it tolerates handling and short-term (up to 30 min) drying, as well as gluing sensors with special glue, mechanical stresses. All this makes it a suitable object according to the criterion of manufacturability. Testing the reactivity and adequacy of the scallop reactions was carried out similarly with that of mussels both under the influence of changes in natural factors and for individual types of pollution (Fig. 7-11).

The scallop behavioral reactions were studied using the same basic parameters as mussels (URS, AMP, ADD). The results showed that scallops are sensitive to many influences and environmental changes, their responses to pollution are adequate to the strength of the effect (level of toxicity and concentration).

A sharp increase in the frequency of closure of the valves (adduction) with increasing temperature and vice versa reflects a linear relationship between adduction and temperature (Fig. 8). The dependence is most pronounced in the temperature range in the natural habitats of mollusks.

Testing of scallops showed their full compliance with biosensor organisms and their suitability for operational biomonitoring of coastal waters at depths of 12-15 m and more.

In the subsequent 16 copies. scallops were used for operational (on-line) biomonitoring of the aquatic environment in the Dalnaya Zelenetskaya Bay in 2012-2015. Data on the behavior of mollusks were automatically transmitted via a mobile communication channel via the Internet in real time.

The inventive method provides efficient operation and increasing the reliability of operational bio-indication and biomonitoring systems, reducing the number of failures, interference, false alarms.

Claims (3)

1. A method for identifying and selecting biosensor organisms for operational bioindication and biomonitoring of marine and fresh waters, including drinking and wastewater, including the selection of bioindicator organisms by determining the distribution and abundance of the species (s) and assessing their availability, applying a number of tests to selected organisms -exposures, characterized in that the selection criteria for biosensor organisms as operational bioindicators are the determination of their survival, manufacturability, reactivity and assessment of the adequacy of reactions, At the same time, the survival of species of organisms — potential biosensors — is first determined by conducting experiments with the placement of these species under specific monitoring conditions (water surface, thickness or bottom of the reservoir), the technological effectiveness of the use of organisms — potential biosensors — is assessed by checking their ability to tolerate manipulations in experiments, transportation, drying, temperature differences and the possibility of using it in a specific technical monitoring system and operational bioindication; then they check the selected organisms of potential biosensors for reactivity by determining the speed and stability of physiological and behavioral reactions to various limiting and non-toxic environmental factors with the establishment of the response potential for each type of test exposure; select the monitoring parameters of biosensors based on the frequency of certain reactions under the same environmental influences, evaluate the adequacy of the selected body reactions to the environmental pollution factors of interest by testing them, then determine the range and thresholds of the responses of the selected biosensors to changes in various types of pollution; Based on the data obtained, the final selection of biosensor organisms is carried out. 2. The method according to p. 1, characterized in that experiments on the survival of biosensor organisms are carried out for 1-2 winter months in extreme conditions similar to natural ones, or field experiments are carried out in specific monitoring conditions. 3. The method according to p. 1 or 2, characterized in that after the final selection of biosensor organisms from other ecosystems, a reserve group of biosensor organisms is created by placing them under monitoring conditions in suspended cages.

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