RU2519070C1 - Method of evaluation of toxicity of environmental components of azov and black seas - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: method comprises placing the fluorescent test-objects in the control and analyzed samples, irradiation with the excitation light, definition of fluorescence characteristics, by which change the toxicity of the controlled environment is assessed. Microalgae of species Scenedesmus apiculatus are used as test-objects, which are previously isolated from environmentally safe areas of the test water reservoirs.

EFFECT: use of the claimed method enables to assess quickly and accurately the toxicity of water and bottom sediments of the Azov and Black Seas.

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Description

The invention relates to ecology and toxicology and can be used to assess the toxicity of water and bottom sediments of the Azov and Black Seas both in the conditions of economic activity and in emergency situations.

It is known that under the influence of various environmental factors and anthropogenic pollution on aquatic ecosystems, the photosynthetic activity of the cells of photosynthetic organisms changes in the first place. These changes will subsequently lead to changes in all other parts of the ecosystem.

To assess the toxicity of the waters of the Azov-Black Sea basin, a strain of luminescent bacteria Ph. phosphoreum (Cohn) Ford (I.Yu. Malygina, A.M. Katsev. Luminous bacteria of the Black and Azov seas. Ecology of the sea. 2003. Issue 64) (1), the bacterial test "Ekolyum", developed in Russia (TU 6 -09-20-236-93, Moscow State University, Moscow) (2), strains of the bacteria Vibrio fischeri VKPM B-9579 (Patent for the invention of the Russian Federation No. 2346035. MKI C12N 1/20 2007) (3) and Vibrio fischeri VKPM B-9580 (RF patent No. 2342434 MKI C12N 1/20) (4). These strains are isolated from the Black Sea. Biotesting is based on the sensitivity of bioluminescent bacteria to the action of toxicants present in water and bottom sediments of marine water bodies. In known methods, the assessment of toxicity is based on determining the change in the intensity of the bioluminescence of bacteria when exposed to toxic substances present in the analyzed sample, compared with the control. However, the content of bacterial cultures is demanding on the conditions of cultivation, their frequent reseeding is necessary, which leads to high costs of labor and funds. In addition, there are known cases of the loss of a strain of bioluminescent activity (properties) during storage.

In addition to bacterial cells, other organisms, such as algae cells, can be used to control the sea waters of the Azov-Black Sea basin. Algae, like all autotrophs, play a vital role in the ecosystem’s food web. Violation of their physiological activity by toxins, as well as the structure of algocenosis itself, has serious consequences for ecosystems. Methods for the study of phytoplankton, based on the measurement of fluorescence, are now widely used both in laboratory conditions in experimental cultures of algae, as well as in water bodies, in the field. By measuring the fluorescence of phytoplankton, it is possible to calculate the concentration of chlorophyll in microalgae (Gold V.M., Gaevsky N.A., Shatrov I.Yu., Popelnitsky V.A., Rubtsov S.A. Experience in using fluorescence for differential estimation of chlorophyll a content in planktonic algae // Gidrobiol. Zh. 1986. T. 22, No. 3) (5).

A known method for determining water toxicity (USSR AS No. 1405745 MKI A01K 61/00, G01N 33/18) (6), in which the change in the intensity of pigment release into the environment under the influence of toxic compounds is controlled, where seaweed is used as a pigment-containing test object. genus Callithamnion. The standard international methods for biotesting sea water developed under the auspices of ISO (International Standard Organization) are test systems using microalgae Phaeodactylum tricornutum and Skeletonema costatum (Water quality - Algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum. Draft International Standard ISO / DIS 10253.2. 1994. 12 p.) (7).

Microalgae analyzes give a statistical advantage over many test objects, since it is easy to use a larger number of cells, much smaller sample volumes and testing time are required, the maintenance of reserve cultures, due to their rare reseeding and low demands on cultivation conditions, does not require large labor costs and funds. The testing process is easy to automate.

The closest solution is the method of evaluating fluid toxicity selected as a prototype (ed. Certificate of the USSR No. 1515105 MKI G01N 33/18) (8), which provides for the cultivation of a photosynthetic test object, excitation of the glow of the test object, and determination of fluorescence characteristics, by changing which judge the toxicity of the controlled fluid.

When using algae to assess the toxicity of sea waters in different seas, the adaptation of the test object to specific waters is not always achieved, which reduces the reliability of the results. In particular, when assessing the toxicity of natural waters that do not correspond to the natural hydrochemical composition of the water in which the culture of the test object was grown, the reaction of this test object can be regarded as the toxic effect of the test water. Therefore, the search for test objects in the Azov and Black Seas was aimed at selecting microalgae that could serve as a test object for water pollutants in the Azov and Black Seas.

The problem solved by the invention is the expansion of the number of test objects for assessing the toxicity of sea waters of the Azov-Black Sea basin, as well as increasing the reliability of information in assessing the toxicity of the environment. The problem is achieved in that in the known method, which includes placing fluorescent test objects in control and analyzed samples, irradiation with exciting light, determination of fluorescence characteristics, the changes of which are used to judge the toxicity of the controlled environment, according to the invention, microgenuses of the form Scenedesmus are used as test objects apiculatus, which are previously isolated from the ecologically clean area of the studied reservoir.

Using the microalgae Scenedesmus apiculatus as a test object expands the number of test objects for assessing the toxicity of the sea waters of the Azov-Black Sea basin and reduces the cost of testing.

Moreover, the use of native algae Scenedesmus apiculatus, isolated from ecologically clean areas of the Azov and Black Seas as the most adapted to the environment of the studied water bodies, as a test object, will reduce errors and significantly increase the sensitivity of fluorescence biotesting.

The set of distinctive features of the described method ensures the achievement of the task.

Comparison of the prototype with the claimed solution showed that the above signs are distinctive, and therefore, the claimed method meets the criterion of "novelty."

The method is as follows.

From ecologically clean areas of the Azov and Black Seas, water samples are taken. Using multiple dilutions and reseeding methods, green algae of the species Scenedesmus apiculatu is isolated from the selected samples, which is used as a test object. Suspensions of microalgae are placed in control (no toxicants) samples and analyzed samples. They act on the samples with exciting light pulses to excite the fluorescence of the test objects. The fluorescence characteristics of the test objects are determined, by the change of which the toxicity of the analyzed samples is judged.

Example 1. From ecologically clean areas of the Azov and Black Seas in May-June 2008, i.e. during the period of active vegetation of the main types of microalgae, water samples were taken. During this period, algae of 7 sections vegetate in the Sea of Azov and the Black Sea: Cyanophyta, Chrysophyta, Bacillariophyta, Dinophyta, Cryptophyta, Chlorophyta, Euglenophyta. Five algologically and bacteriologically pure cultures of certain types of green and blue-green algae were isolated by repeated dilutions and reseeding from the selected samples. According to their taxonomic affiliation, 3 isolated strains belonged to blue-green algae (Cyanophyta), and 2 strains belonged to green algae (Chlorophyta). Blue-green algae are identified as Oscillatoria laetevirens (Crouan) Gom. (= Phormidium laetevirens (Crouan et Gom.) Anagn. Et. Kom.), Oscillatoria Agardhii Gom. (= Planktothrix Agardhii (Gom.) Anagn. Et. Kom.) And Snowella rosea (Snow) Elenkin; green - like Oocystis borgei Snow and Scenedesmus apiculatus (W. et W.) Chod. The isolated algae strains were prepared for spectral analysis. Suspensions of microalgae were placed in control (without toxicants) water samples. Fluorescence spectra of solutions containing microalgae were recorded on an RF-5301PC spectrofluorophotometer (Shimadzu, Japan). Using the Panorama fluorescence 1.1 program in the scanning mode (2D synchro measurement), the excitation and luminescence spectra were analyzed and wavelengths with characteristic excitation maxima were selected for each strain according to the results of their synchronization.

At the next stage of the work, the sensitivity of the isolated microalgae cultures to the action of standard toxicants was determined. Sensitivity was assessed by the relative difference in the intensity of bioluminescence of the control and experimental samples. A suspension of microalgae was introduced into samples with potassium dichromate K ₂ Cr ₂ O ₇ (solution concentration from 0.001 to 100 mg / l), copper sulfate CuSO ₄ (concentration from 0.0001 to 100 mg / l) and phenol (5-1500 mg / l) . The exposure time was from 10 to 30 minutes. The response of microalgae to the effects of selected toxicants was investigated by exciting a glow in the region of the maxima established for each strain of algae. In this case, fluorescence spectra were recorded, recording changes in the luminous intensity at established emission maxima.

Based on the results of studies of the sensitivity of all selected microalgae cultures to the action of standard toxicants - potassium dichromate (K ₂ Cr ₂ O ₇ ), copper sulfate (CuSO ₄ ) and phenol, the most sensitive (prospective for biotesting) microalgae species Scenedesmus apiculatus and Snowella rosea were selected.

The change in the sensitivity of the isolated microalgae species under the influence of standard toxicants and the fluorescence intensity (in arbitrary luminescence units, ECE) of the microalgae Scenedesmus apiculatus and Snowella rosea in the experiment are given in Tables 1, 2, 3.

Table 1 The fluorescence intensity of microalgae Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) and Snowella rosea (λ _exc. 340 nm, 440 nm) in the experiment with potassium dichromate (UES) The control The concentration of the toxicant K ₂ Cr ₂ O ₇ (MPC 0.05 mg / l) mg / l 0.001 0.01 0.1 0.5 1,0 5,0 10.0 100.0 Fluorescence Intensity of Scenedesmus apiculatus, UES 24.5 20.9 20.3 19,4 19.5 19.5 17,2 14,4 0.7 Fluorescence intensity of Snowella rosea, UES 11.3 11,4 11.5 12.5 15.3 18,4 16.5 15.0 2,4

table 2 The fluorescence intensity of microalgae Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) and Snowella rosea (λ _exc. 220 nm and λ _emission. 370 nm) in the experiment with copper sulfate (UES) The control CuSO toxicant concentration ₄ mg / l 0.0001 0.001 0.01 0.1 1,0 10.0 100.0 Fluorescence Intensity of Scenedesmus apiculatus, UES 24.5 21.8 20.5 19.3 17.5 15.9 12,2 1,2 Bioluminescence intensity of Snowella rosea, UES 11.3 11,4 11.9 12.8 13,4 14.1 8.5 7.9

Table 3 The fluorescence intensity of microalgae Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) and Snowella rosea (λ _exc. 340 nm, λ _emission. 440 nm) in the experiment with phenol (UES) The control Toxicant concentration phenol mg / l 5,0 50,0 100.0 300 700 1000 1200 1500 The fluorescence intensity of Scenedesmus apiculatus, UES 24.5 16.7 0.5 0.1 0 0 0 0 0 Fluorescence intensity of Snowella rosea, UES 11.3 12.9 14.3 15.1 11.0 9.2 7.5 6.7 6.0

From table 1 it is seen that under the influence of K ₂ Cr ₂ O ₇ (0.01-10 mg / l) (MPC 0.05 mg / l), the fluorescence intensity (λ _exc. 220 nm, λ _emission. 366 nm) of the Scenedesmus apiculatus culture decreased to the maximum 45-50% of the control level (depending on the exposure time and fluorimetric conditions).

For a culture of Snowella rosea in the presence of the same concentrations of K ₂ Cr ₂ O _7, a maximum increase in the fluorescence intensity was found to be 60% (λ _exc. 340 nm, λ _emission. 440 nm).

Higher concentrations of K ₂ Cr ₂ O ₇ (at the level of 50-100 mg / L) suppressed the fluorescence of both algal cultures by 45-99%.

Table 2 shows that in CuSO ₄ solutions (0.01-10 mg / l) Scenedesmus apiculatus showed similar sensitivity, as evidenced by a decrease in fluorescence intensity (λ _exc. 220 nm, λ _emission. 366 nm) by a maximum of 50% of the control level . In a solution with a CuSO ₄ concentration _of 100 mg / L, almost complete quenching of the fluorescence of a suspension of Scenedesmus apiculatus was observed.

Under the influence of low concentrations of CuSO ₄ (0.0001-1.0 mg / L), the fluorescence spectra of Snowella rosea did not noticeably change. The highest luminescence induction (up to 25% of the control, λ _exc. 340 nm, λ _emission. 440 nm) was recorded in a CuSO ₄ solution with a concentration of 1.0 mg / L. Exposure of algae in a culture medium with CuSO ₄ at a concentration of 10 and 100 mg / l causes quenching of the glow by 25 and 30%. Therefore, the sensitivity of the Snowella rosea culture to CuSO ₄ was approximately 2 times lower than Scenedesmus apiculatus.

Table 3 shows that phenol at a concentration of 5 mg / L inhibited the fluorescence of Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) by 10-55%, and in a solution with a phenol content of 50 mg / L, fluorescence quenching reached almost one hundred%. At the same time, Snowella rosea showed low sensitivity to phenol: the fluorescence intensity was 1.5 times inhibited only by a phenol concentration of 1000 mg / L.

Example 2. For a comparative assessment of sensitivity, we used the characteristic EC ₅₀ (effective concentration) - the concentration of a substance that causes a 50% decrease in the bioluminescence of a suspension of microalgae.

The use of the EC ₅₀ indicator (an effective concentration of a toxicant that reduces luminescence by 50%) made it possible to compare the sensitivity of the isolated algae Scenedesmus apiculatus and Snowella rosea with the sensitivity of bioluminescent bacteria E. coli PT-5, Ph. phosphoreum (Cohn) Ford and native bacteria strains Vibrio fischeri VKPM B-9579 and Vibrio fischeri VKPM B-9580 isolated from the water of the Azov and Black Seas (table 4).

Table 4 The sensitivity of the isolated algae and known strains of luminous bacteria to the action of various toxicants (inhibition of luminescence), EC ₅₀ , mg / l Algal cultures and bacterial strains used as test objects EC ₅₀ mg / l CuSO ₄ K ₂ Cr ₂ O ₇ Phenol Scenedesmus apiculatus 10-40 10-50 5-30 Snowella rosea 20-50 20-50 - Vibrio fischeri B 9579 ¹ 1-1.5 10-50 125-150 Vibrio fischeri B 9580 ² 2 10-50 125-150 E.soISbOO (pPLS-5) ³ 5-7.5 150 > 300 P. phosphoreum (Cohn) Ford ⁴ 250 170 MPC _pkh (mg / l) 0.005 - in terms of C ²⁺ 0.05 - by substance 0.001 ¹ patent of the Russian Federation No. 2346035, 2007; ² RF patent No. 2342434, 2007; ³ RF patent No. 79581,2001; ⁴ Malygina, Katsev, 2003.

Comparison of the sensitivity of indigenous microalgae to the studied toxic substances (Table 4) showed a 2 times higher sensitivity of the microalgae Scenedesmus apiculatus in comparison with the sensitivity of Snowella rosea. Therefore, for testing, the microalgae Scenedesmus apiculatus was chosen as a test object.

Comparison of the sensitivity of the native microalgae Scenedesmus apiculatus in terms of average EC ₅₀ to the studied toxic substances showed that Scenedesmus apiculatus is more sensitive than the Ph. phosphoreum (Cohn) Ford. In particular, on average, it is approximately an order of magnitude more sensitive to K ₂ Cr ₂ O ₇ . Thus, the EC ₅₀ value for the microalgae Scenedesmus apiculatus in potassium dichromate is in the concentration range from 10 to 50 mg / l, and for Ph. phosphoreum is 250 mg / l.

According to phenol, the EC ₅₀ value for the microalgae Scenedesmus apiculatus was 5-30 mg / l, which is also an order of magnitude lower than the EC ₅₀ for Ph. phosphoreum.

Comparison of the sensitivity of the microalgae Scenedesmus apiculatus to toxic substances with the sensitivity of the lux strain of E. coli PT-5 showed similar results, with the exception of CuSO ₄ . The microalgae Scenedesmus apiculatus is more sensitive to K ₂ Cr ₂ O ₇ , (~ 5 times), as well as to phenol (about an order of magnitude), but inferior in sensitivity to CuSO ₄ (~ 4 times).

The data obtained indicate the promise of using the microalgae Scenedesmus apiculatus to determine the toxicity of aquatic environments.

Example 3. We tested the sensitivity of the algae of the species Scenedesmus apiculatus used as a test object, isolated from the water of the Black and Azov Seas. Water samples were taken from ecologically clean regions of the Azov and Black Seas. The microalgae Scenedesmus apiculatus, selected from different seas, were placed in them.

As studies have shown, cultures of Scenedesmus apiculatus from different seas, placed in “native” and “non-native” environments, differed in fluorescence intensity (λ _exc. 220 nm, λ _emission. 366 nm) within 12-17% depending on the exposure conditions ( table 5).

Table 5 Deviation from the control of the fluorescence intensity of Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) in the water of the Black and Azov Seas (exposure time - 10 minutes),% No. Water option Scenedesmus apiculatus (from the water of the Sea of Azov) Scenedesmus apiculatus (from the Black Sea) one Black Sea Water Sample -17.15 4.45 2 Water sample of the Sea of Azov 3.30 -12.45

Thus, native algae placed in a “non-native” medium showed 2-3 times greater deviations of the fluorescence level from that of microalgae placed in a “native” medium. Such a deviation can distort the results of measuring toxicity of samples in a “non-native” environment, which indicates the need to use native microalgae (as the most adapted to the conditions of a given reservoir) in the practice of biotesting water and aqueous extracts of bottom sediments.

Example 4. Experiments were conducted to determine the toxicity of the components of the environment (water, bottom sediments) of the Black Sea using the native microalgae Scenedesmus apiculatus as a test object. We studied 3 water samples and 3 samples of bottom sediments taken in the area with high anthropogenic pollution (the water area of the Black Sea port). To prepare the extracts of bottom sediments, pure Black Sea water, pre-filtered through a bacterial filter, in a weight ratio of 10: 1 with soil dried at room temperature was used as a solvent. Samples of bottom sediments were shaken on an orbital shaker at 65 rpm at room temperature for one hour and then sedimented until the suspension settled. The settled extracts were filtered through a siphon (kapron network No. 76) and used in further toxicity studies.

The toxicity of water and aqueous extracts of bottom sediments was determined by the change in the fluorescence intensity of Scenedesmus apiculatus (λ _exc. 220 nm, λ _emission. 366 nm) relative to the control (clear sea water of the Black Sea). Exposure time - 10 minutes. Moreover, the deviation of the luminous intensity in the test sample relative to the control (both downward and upward) of less than 20% indicates the absence of toxicity, from 20 to 30% - low toxicity, from 30 to 50% - moderate toxicity, over 50% - about acute toxicity.

The research results are shown in table 6.

Table 6 The results of biotesting of water samples and bottom sediments from the polluted waters of the Black Sea on the native algae culture Scenedesmus apiculatus (deviations from the control of fluorescence intensity,%; in the denominator - toxicity index, points) Test samples λ _exc. 220 _nm , λ _emission. 366 nm Exposure time, min 10 thirty Water 1

$$\frac{-4.27}{0}$$

$$\frac{-8.85}{0}$$

Water 2 $$\frac{-21.77}{\mathrm{one}}$$

$$\frac{-10.67}{0}$$

Water 3 $$\frac{-8.52}{0}$$

$$\frac{-\mathrm{13,42}}{0}$$

Bottom sediments 1 $$\frac{-34.49}{2}$$

$$\frac{-34.86}{2}$$

Bottom sediment 2 $$\frac{-40.95}{2}$$

$$\frac{-41.92}{2}$$

Bottom sediments 3 $$\frac{-52.09}{3}$$

$$\frac{-48.84}{2}$$

According to the test results, the decrease in fluorescence intensity relative to the control in water samples No. 1, 2 and 3, depending on the exposure time, was, respectively, -4.27 - -8.85%; -21.77 - -10.67% and -8.52 - -13.42%, which, in accordance with the above scale, characterizes water samples No. 1 and 3 as non-toxic, sample No. 2, depending on the test conditions, as non-toxic and slightly toxic.

The fluorescence intensity in extracts of bottom sediments No. 1,2 and 3 depending on the exposure time decreased relative to the control by -34.49 - -34.86%, -40.95 - -41.92% and -52.09 - - 48.84%, respectively. According to the results of fluorimetry, bottom sediments No. 1 and 2 are thus assessed as moderately toxic, sample No. 3 - as acutely toxic. The obtained test results indicate moderate toxicity of samples of bottom sediments No. 1 and 2 and acute toxicity - samples No. 3. According to analytical data, in the bottom sediment sample No. 3, the maximum content of oil products (32.6 g / kg of dry soil), ACAS (91 mg / kg), phenol (4.1 mg / kg) and polycyclic aromatic hydrocarbons (1010) mcg / kg).

Testing the method of biotesting water and bottom sediments taken in an area with high anthropogenic pollution (seaport) using Scenedesmus apiculatus as a test object made it possible to determine the toxicity of water and bottom sediments by changing the fluorescence intensity (λ _emission. 366 nm). The toxicity values for water samples were lower than for extracts of bottom sediments and in some cases correlated with the content of surface-active substances, oil products and phenol in the samples.

In general, the high sensitivity of the isolated algae cultures to tested toxicants and the testing of the biotesting method based on Scenedesmus apiculatus in the Black Sea indicate the promise of using the isolated microalgae cultures as test objects for determining the toxicity of the components of marine water bodies under complex anthropogenic pollution.

The developed bioassay method can be used both for rapid assessment of the toxic substances in liquids, for example, when discharging wastewater into the environment, and for continuous monitoring of environmental toxicity, including in emergency cases and adverse environmental situations.

The inventive method for determining toxicity compares favorably with similar systems to a higher sensitivity to toxicants (it is possible to record the toxic effect of a toxicant at its MPC for water of a fishery reservoir), an express reaction (analysis results are recorded within an hour), inertia (light that excites fluorescence, a little changes the physiological state of the test object), low analysis cost.

Literature

1. I.Yu. Malygina, A.M. Katsev. Luminous bacteria of the Black and Azov Seas. Ecology of the sea. 2003. Issue. 64.

2. TU 6-09-20-236-93

3. Patent for the invention of the Russian Federation No. 2346035. MKI C12N 1/20.

4. Patent for the invention of the Russian Federation No. 2342434 MKI C12N 1/20.

5. Gold V.M., Gaevsky N.A., Shatrov I.Yu., Popelnitsky V.A., Rubtsov S.A. The experience of using fluorescence for differential assessment of chlorophyll content in planktonic algae // Gidrobiol. journal 1986. T. 22, No. 3.

6. USSR author's certificate No. 1405745, IPC A01K 61/00, G01N 33/18.

7. Water quality - Algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum. Draft International Standard ISO / DIS 10253.2. 1994.12p.

8. Copyright certificate of the USSR No. 1515105 MKI G01N 33/18 (prototype).

Claims (1)

A method for assessing the toxicity of components of the environment of the Azov and Black Seas including placing fluorescent test objects in control and analyzed samples, irradiating with exciting light, determining fluorescence characteristics, which change is used to judge the toxicity of the controlled environment, characterized in that microalgae of the type are used as test objects Scenedesmus apiculatus, which are previously isolated from ecologically clean areas of the studied water bodies.