RU2499256C2 - Method of biotesting of water contaminated with crude oil according to length of test plant roots - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: test plant is chosen, uniform seeds of test plant placement is carried out on the filter paper in the control and test Petri dish of 10 cm in diameter. Then 5.0 ml of water is poured in each control and the test Petri dish with 4-8 replicates of watering, at that the level of liquid in the dishes must be below the surface of the seeds. At that in the control Petri dish the water sample with a zero concentration of crude oil is taken, and in the test dishes before watering the water emulsions of test crude oil with different concentrations are prepared. Then the seeds of test plant in Petri dishes are watered with prepared water emulsions with the specified concentration of crude oil for 72 hours, and the concentration of crude oil in different water emulsions is increased until the moment of measurement of root length when the germination of round red radish is not less than 50 %.

EFFECT: assessment of dangerous levels of contamination of water objects with crude oil, by means of improving the accuracy of indices of effects of crude oil of different volume concentration in the aqueous solution on the root growth of plants in a particular area.

3 cl, 12 dwg, 3 tbl, 1 ex

Description

The invention relates to environmental engineering and environmental protection. It can be used in environmental regulation of the effect of the concentration of oil and oil products on the natural environment in the form of vegetation. At the same time, environmental rationing is carried out by biotesting on the growth of the roots of various types of test plants, for example, in environmental monitoring of the quality of water in rivers and reservoirs contaminated with oil discharges.

There is a method of testing water pollution by the growth of plant roots in accordance with the method of biotesting for seed germination (Appendix 10. Sanitary rules and norms SanPiN 2.1.7.573-96 "Hygienic requirements for the use of wastewater and their rainfall for irrigation and fertilizer" (approved by resolution State Committee for Sanitary and Epidemiological Supervision of the Russian Federation dated October 31, 1996 No. 46), including uniform seed placement on filter paper in a Petri dish with a diameter of 10 cm, with 5 ml of test water being poured into each Petri dish at 4-8-fold repetition, with ur the liquid level in the cups should be lower than the surface of the seeds, then the cups are covered and placed in a thermostat at a temperature of 20 ° C, and in the absence of a thermostat the experiment is possible in room conditions, but then, due to temperature fluctuations, it is difficult to compare the results carried out at different times, experiment ends after 72 hours, after which the length of the roots is measured.

The disadvantage is that the concentration of oil is not taken into account in wastewater. Therefore, the method is not applicable for environmental regulation. However, the advantage is that in specific places during biotesting of wastewater for irrigation it is recommended to use precisely those cultivated plants that grow and are used in agriculture on this land plot as test plants.

There is also a known method of testing water pollution by the time of plant root growth according to patent No. 2402765, which includes selecting a test plant, for example, in the form of red round radish seeds, uniformly placing seeds of the test plant on filter paper in a control and test Petri dish with a diameter of 10 cm, moreover, 5 ml of water is poured into each Petri dish at 4-8-fold watering repetitions, while the liquid level in the cups should be lower than the surface of the seeds, the experiment is completed after 72 hours, after which the length of all roots in each cup is measured Peter.

The disadvantage is that the concentration of oil in the water sample of rivers and reservoirs is not measured prior to biotesting, and therefore, the change in the root length of the test plant is unknown. However, there are no methods of environmental regulation before testing samples of river or other water

The technical result is the extension of the functionality of biotesting for environmental regulation of water pollution by oil and oil products, increasing the accuracy of indicators of the influence of oil of various volume concentrations in aqueous solution on the growth of plant roots in a particular area, and in general on the growth of the roots of the generally accepted test plant - red round radish with white tip.

This technical result is achieved in that a method for biotesting the length of the roots of a test plant with oil-contaminated water, including selecting a test plant, for example, in the form of red round radish seeds, uniformly placing the seeds of the test plant on filter paper in a control and test Petri dish with a diameter of 10 cm, with 5.0 ml of water being poured into each Petri dish at 4-8-fold watering repetitions, while the liquid level in the cups should be lower than the surface of the seeds, the experiment is completed after 72 hours, after which the length of all the roots in each Petri dish is determined, characterized in that the total number of tested Petri dishes is taken according to the number of volumetric oil concentrations in the aqueous solution, while a water sample with a zero oil concentration is taken in the Petri dish, the following seed irrigation in each Petri dish within 4-8 times the repetition is performed with an increase in the period between them, before the next filling, an aqueous solution of the test oil is prepared, for this oil of a given volume is collected and poured into a volumetric flask, last why water is collected and poured into the same volumetric flask, then the contents of the flask are mixed, and the seeds of the test plant in the Petri dish are poured with the prepared solution with the given oil concentration for 72 hours, and the seeds are watered in the first test Petri dish at 4-8 -fold repetition with the same low concentration of a new aqueous oil solution, and in each subsequent Petri dish, watering is performed with an aqueous oil solution in order to increase the volume concentration until, after measuring the root length, n radish red round does not exceed 50%, then the statistical simulation reveal biotechnical length change pattern of red radish root round on the volume of oil concentration on the totality of the petri dishes and each of them individually.

Before filling, an aqueous solution of the test oil of a given concentration for one Petri dish is prepared, for this a sample of oil of a given volume in milliliters is taken with a graduated pipette using a rubber bulb and poured into a volumetric flask, another graduated pipette is filled with natural water from a nearby oil pipeline or refinery and poured in the same volumetric flask, and the total volume of the prepared oil solution, for example, is 5.0 ml according to the table of the concentration of oil in the aqueous solution:

The volume of oil, ml 0 0.1 0.2 0.3 0.4 0.6 0.8 1,0 Volume concentration 100 C / 5,% 0 2 four 6 8 12 16 twenty

The following watering of seeds in each Petri dish within four repetitions under room conditions is performed with an increase in the period between them for 72 hours in the following sequence:

Watering Number one 2 3 four Root length measurement Time from the beginning of watering, h 0 12 36 60 72

According to the results of measurements after watering the seeds in each Petri dish with an aqueous solution of different oil concentrations in ascending order of magnitude, taking into account the control Petri dish with zero oil concentration, the seed germination rate is simulated for 72 hours using the formula:

$$n_{PR}=a_{\mathrm{one}}\exp (-a_2{\mathrm{FROM}}^{a_3})cos(\pi \mathrm{FROM}/(a_{\mathrm{four}}+a_5{\mathrm{FROM}}^{a_6})+a_7)$$

where n _pr - the number of sprouted seeds of red round radish, pcs;

C is the volumetric concentration of oil,%;

and ₁ ... a ₇ are the parameters of the individual components of the finished statistical regularity, taking specific values for the specific conditions of seed germination.

According to the maximum root length of seedling leaders in each Petri dish with different volumetric oil concentrations, statistical modeling reveals a biotechnical pattern:

$$L_{\max }=a_{\mathrm{one}}\exp (-a_2{\mathrm{FROM}}^{a_3})$$

where L _max - the maximum root length from the best quality seed in each Petri dish, mm;

C is the volumetric concentration of oil,%;

and ₁ ... and ₃ are the parameters of the statistical model.

Statistical modeling of the general biotechnological regularity of the change in the length of the root of the red radish round from the volumetric oil concentration over the entire set of Petri dishes and for each individually is performed in the form of a generalized law

, $$L=L_0\exp (-a_{\mathrm{one}}{\mathrm{FROM}}^{a_2})$$

,

where L ₀ is the length of the root when watering with an aqueous solution with zero concentration

oil, mm;

C is the volumetric concentration of oil,%;

and ₁ - the activity of the death of a natural pattern;

and ₂ - the intensity of the decrease in volumetric oil concentration.

The essence of the technical solution lies in the fact that environmental regulation establishes the allowable effect of the concentration of oil and oil products on the natural environment, including in the event of emergency spills. The volume concentration of oil and oil products is normalized by the test method for the growth of the roots of various types of test plants from the global list, for example, red round radish.

The essence of the technical solution also lies in the fact that the number of tested Petri dishes is taken equal to seven, that is, by the number of values (we hope that they will be standardized) of the volume concentration of oil in an aqueous solution. The total number of Petri dishes in the test is eight, taking into account the control Petri dish, which is taken as a water sample with a zero oil concentration.

The essence of the technical solution also lies in the fact that during the test a single irrigation regime is established regardless of specific territories. Watering seeds in each Petri dish within four times (for the middle strip of the European part of Russia, on the lands of the south of the country five or even more than multiple) repetitions are performed with increasing period between them. In this case, the first watering time is conditionally (for the watering time scale) 0 hours, the next watering after 12 hours, the third and fourth watering after 36 and 60 hours, respectively, from the beginning of the watering process, and after 72 hours (here we took the standardized time according to SanPiN, not to be confused with our previous invention) the germination of red round radish seeds measures the length of the root of the seedlings.

The essence of the technical solution also lies in the fact that the method indirectly determines the effect of a given concentration of oil in water in 72 hours. Thus, it is possible to find out the environmental damage from the loss of vegetation during an accidental spill of oil and oil products on the territory of a particular territory, if you quickly measure the volumetric water concentration of the spill.

The positive effect is achieved by the fact that an indirect assessment of the effect of water samples with a given content of oil or oil products is carried out, which allows us to determine the degree of possible pollution by oil and oil products of non-flowing water bodies located near agricultural lands and settlements.

The novelty of the technical solution lies in the fact that the experiment on seed germination is performed by changing the root length of the test plant when water of various concentrations and properties is added to the sample before irrigation, and then a table of the influence of the volume concentration of oil or oil products on the parameters of the test plant is compiled - maximum root length and seed germination.

The proposed technical solution has significant features, novelty and a significant positive effect. We have not found any materials discrediting the novelty of the technical solution.

Figure 1 shows 12 seeds of red round radish in a Petri dish when irrigated with an aqueous solution with a volumetric oil concentration of 2%; figure 2 is a graph of the distribution of the length of the root of the radish red round from the volumetric concentration of oil throughout the totality of Petri dishes; figure 3 is a graph of the ranking distribution of 12 seeds in the first Petri dish with a volumetric oil concentration of 0%; in Fig.4 - the same in Fig.3 in the second Petri dish with a volumetric oil concentration of 2%; in Fig.5 - the same in Fig.3 in the third Petri dish with a volumetric oil concentration of 4%; in Fig.6 - the same in Fig.3 in the fourth Petri dish with a volumetric oil concentration of 6%; in Fig.7 - the same in Fig.3 in the fifth Petri dish with a volumetric oil concentration of 8%; in Fig.8 - the same in Fig.3 in the sixth Petri dish with a volumetric oil concentration of 12%; in Fig.9 - the same in Fig.3 in the seventh Petri dish with a volumetric oil concentration of 16%; figure 10 is the same in figure 3 in the eighth Petri dish with a volumetric oil concentration of 20%; 11 is a graph of the distribution of the maximum root length in each Petri dish versus volumetric oil concentration; on Fig is a graph of the change in the number of germinated seeds of the plant in each Petri dish depending on the volumetric concentration of oil.

The method of biotesting along the length of the roots of a test plant artificially contaminated with oil for environmental normalization with oil includes the following main steps.

A sampling of natural water is carried out in the given territory, its conservation and preparation. In laboratory conditions, a biotesting test is carried out along the length of the roots of the test plant with the addition of oil and oil products in part of the water sample.

The seeds of the test plants are uniformly placed on filter paper in a Petri dish 10 cm in diameter. 5.0 ml of water sample is poured into each Petri dish at 4-8-fold repetition, while the liquid level in the cups should be lower than the surface of the seeds, the experiment is completed after 72 hours, after which the length of all the roots in each Petri dish is measured.

The total number of tested Petri dishes is taken according to the number of values of the volumetric concentration of oil in an aqueous solution, while a water sample with a zero oil concentration is taken in a control Petri dish. The following watering of seeds in each Petri dish within 4-8-fold repetition is performed with increasing period between them.

Before the next pouring, an aqueous solution of the test oil of a given concentration for one Petri dish is prepared, for this a sample of oil of a given volume in milliliters is taken with a graduated pipette using a rubber bulb and poured into a volumetric flask, a water sample is collected with another graduated pipette and poured into the same volumetric flask, then the contents of the flask are mixed.

A prepared solution with a given oil concentration is poured over the seeds of the test plant in a Petri dish for 72 hours, and the seeds are watered in the first test Petri dish with 4-8-fold repetition with the same low concentration of a new aqueous oil solution.

In each subsequent Petri dish, watering is performed with an aqueous solution of oil to increase the volume concentration until, after measuring the root length, the seed germination of the test plant does not exceed 50%. Then, statistical modeling reveals the biotechnological regularity of the change in the length of the root of the plant from the volume concentration of oil in the entire set of Petri dishes and each of them individually.

The method of biotesting the length of the roots of a round red radish contaminated with oil, for example, water samples with a predetermined volumetric oil concentration, includes the following steps.

A water sample is taken, for example, river water from stagnant places (elders and other bodies of water, floodplain meadows, etc.), its conservation and preparation.

In laboratory conditions, a biotesting test is carried out along the length of the roots of the test plant - red round radish.

Red round radish seeds are uniformly placed on filter paper in a Petri dish 10 cm in diameter. 5.0 ml of a sample of river water are poured into each Petri dish at 4-8-fold repetition, while the liquid level in the cups should be lower than the surface of the seeds, experiment complete after 72 hours, after which the length of all the roots in each Petri dish is measured.

The total number of tested Petri dishes is taken according to the number of values of the volumetric concentration of oil in the aqueous solution, while in the control Petri dish a sample of river water with a zero oil concentration is taken.

Before the next pouring, an aqueous solution of the test oil of a given concentration for one Petri dish is prepared, for this a sample of oil of a given volume in milliliters is taken with a graduated pipette using a rubber bulb and poured into a volumetric flask, river water is added with another graduated pipette and poured into the same volumetric flask, and the total volume of the prepared oil solution, for example, is 5.0 ml according to the table of the concentration of oil in the aqueous solution:

The volume of oil, ml 0 0.1 0.2 0.3 0.4 0.6 0.8 1,0 Volume concentration 100C / 5,% 0 2 four 6 8 12 16 twenty

A prepared solution with a given oil concentration is watered with red round radish seeds in a Petri dish for 72 hours, and seeds are irrigated in the first tested Petri dish with 4-8-fold repetition with the same low concentration of a new aqueous oil solution. The following watering of seeds in each Petri dish within four repetitions under room conditions is performed with an increase in the period between them for 72 hours in the following sequence:

Watering Number one 2 3 four Root length measurement Time from the beginning of watering, h 0 12 36 60 72

In each subsequent Petri dish, watering is performed with an aqueous solution of oil to increase the volume concentration until, after measuring the length of the root, the germination rate of red round radish seeds does not exceed 50%.

According to the results of measurements of the length of the root of the red round radish after watering the seeds in each Petri dish with an aqueous solution of different oil concentrations, they simulate seed germination over 72 hours using the formula:

$$n_{PR}=a_{\mathrm{one}}\exp (-a_2{\mathrm{FROM}}^{a_3})cos(\pi \mathrm{FROM}/(a_{\mathrm{four}}+a_5{\mathrm{FROM}}^{a_6})+a_7)$$

where n _pr - the number of sprouted seeds of red round radish, pcs;

C is the volumetric concentration of oil,%;

and ₁ ... a ₇ are the parameters of the individual components of the finished statistical regularity, taking specific values for the specific conditions of seed germination.

According to the maximum root length of seedling leaders in each Petri dish with different volumetric oil concentrations, statistical modeling reveals a biotechnical pattern:

$$L_{\max }=a_{\mathrm{one}}\exp (-a_2{\mathrm{FROM}}^{a_3})$$

where L _max - the maximum root length from the best quality seed in each Petri dish, mm;

C is the volumetric concentration of oil,%;

and ₁ ... and ₃ are the parameters of the statistical model.

Statistical modeling of the general biotechnological regularity of the change in the length of the root of the red radish round from the volumetric oil concentration over the entire set of Petri dishes and for each individually is performed in the form of a generalized law

, $$L=L_0\exp (-a_{\mathrm{one}}{\mathrm{FROM}}^{a_2})$$

,

where L ₀ is the length of the root when watering with an aqueous solution with zero oil concentration, mm;

C is the volumetric concentration of oil,%;

and ₁ - the activity of the death of a natural pattern;

and ₂ - the intensity of the decrease in volumetric oil concentration.

Example. Biotesting along the length of the radish roots of a red round artificially oil-contaminated water was carried out using river water, a sample of which was taken from the Malaya Kokshaga River.

Table 1 shows the germination data at room conditions (seeds were planted at 8 p.m. February 13, 2011) of independent samples of 12 seeds of red round radish with a white tip on eight Petri dishes when irrigated with an aqueous oil solution.

Table 1 The length of the roots of red round radish after irrigation with an aqueous solution of oil of different volume concentrations Volumetric oil concentration C,% Root length 12 seeds L, mm 0 26 0 13 26 16 27 29th 23 16 32 21 fourteen 2 28 26 19 16 10 23 13 twenty 10 24 26 26 four 0 5 17 fifteen 17 8 10 12 24 9 twenty 8 6 0 eleven 25 6 19 eleven twenty four four 0 21 12 8 5 9 0 7 7 fifteen 12 eighteen 26 fourteen 13 10 12 8 fifteen 12 10 23 fourteen 12 5 21 fourteen 8 13 16 13 fourteen 9 9 0 10 10 23 8 10 9 9 twenty 0 0 12 fourteen 5 0 0 0 6 8 four 0

During the experiment on filter paper, red round radish seeds were placed in a Petri dish with a diameter of 10 cm. 5 ml of a sample of river water was added to each Petri dish with the addition of oil of different volume concentrations at four repetitions.

Before the next filling, an aqueous solution of the test oil of the given concentration for one Petri dish was prepared. For this, a sample of oil of a given volume in milliliters was collected with a graduated pipette using a rubber bulb and poured into a volumetric flask, river water was collected with another graduated pipette and poured into the same volumetric flask, the contents of the flask were mixed.

A prepared solution with a given oil concentration was watered with red round radish seeds in a Petri dish for 72 hours, and seeds were irrigated in the first tested Petri dish with 4-fold repetition with the same low concentration of a new aqueous oil solution. The following watering of seeds in each Petri dish within 4-fold repetition in room conditions was performed with an increase in the period between them for 72 hours in the following sequence:

Watering Number one 9 3 four Root length measurement Time from the beginning of watering, h 0 12 36 60 72

In each subsequent Petri dish, watering was performed with an aqueous solution of oil in increasing volume concentration values. For example, Fig. 1 shows a test Petri dish in which 12 seeds of round red radish were irrigated with an aqueous solution with a volumetric oil concentration of 2%.

Measurement of the length of all roots of the red round radish in each test Petri dish was performed after 72 hours of germination.

The regularity of the distribution of the length of the root of the radish red round from the oil concentration in the whole set was obtained in the form of a generalized law of death, i.e.

$$L=\mathrm{20,64957}\exp (-\mathrm{one},09998{\mathrm{FROM}}^{0,7\mathrm{four}558}),(\mathrm{one})$$

where L is the maximum length of the root of the radish red round, mm;

C is the volumetric concentration of oil,%.

Figure 2 presents a graph of the distribution of the length of the root of the radish red round from the volumetric concentration of oil over the entire set of Petri dishes. It can be seen from the graph that the higher the oil concentration, the smaller the values of the length of the red round radish root. With a decrease in concentration, the length of the root increases.

The root length assumes the greatest values at a volume concentration of oil from 0 to 8%. At a volume concentration of 2%, oil has little effect on the growth of the root of the red round radish.

Graphs of the distribution of the length of the root of the radish red round from the volume concentration of oil for each Petri dish are shown in Fig.3-10.

High correlation coefficients of the equations convince of the high adequacy of the obtained statistical models.

The distribution diagram of the maximum root length (Table 2) in each Petri dish when watering with an aqueous solution of a different oil concentration (Fig. 11) also shows a decrease in root length with increasing concentration and has a pattern in the form of a generalized death law

$$L_{\max }=\mathrm{31,00617}\exp (-0,0\mathrm{four}506{\mathrm{FROM}}^{0,83\mathrm{four}8\mathrm{one}}),(2)$$

where L _max - the maximum length of the root of the radish red round, mm;

C is the volumetric concentration of oil,%.

table 2 The maximum length of the root of the red round radish in each Petri dish at different volume concentrations of oil Volumetric oil concentration C,% Maximum root length L, mm 0 32 2 28 four 24 6 25 8 26 12 23 16 23 twenty fourteen

The maximum number of seeds not sprouted is in a large concentration of oil (Table 3). At a volumetric oil concentration of 20%, the amount of not sprouted red round radish seeds was 50%. Therefore, a further increase in oil concentration was not carried out and the number of tested Petri dishes, taking into account the control during biotesting with red round radishes, was taken to be eight.

Table 3 The concentration of oil in aqueous solution and seed germination Volumetric oil concentration C, ml 0 0.1 0.2 0.3 0.4 0.6 0.8 1,0 Relative concentration 100C / 5,% 0 2 four 6 8 12 16 twenty Absolute germination (of 12 pcs.) N _pr , pcs. eleven 12 eleven 10 eleven 12 eleven 6 Relative germination rate 100 n _pr / 12,% 91.67 one hundred 91.67 83.33 91.67 one hundred 91.67 50.00

By identifying the biotechnical law, a regularity was obtained, presented in the form of the following germination model

$$n_{PR}=\mathrm{10,33736}\exp (0,03\mathrm{one}82\mathrm{FROM})cos(\pi \mathrm{FROM}/5\mathrm{one},7\mathrm{one}6\mathrm{four}8),(3)$$

where n _pr - the number of sprouted seeds of red round radish, pcs;

C is the oil concentration,%.

The resulting model (3) shows the wave perturbation of red round radish seeds to the oil content.

From the graph of the distribution of sprouted seeds of red radish round oil concentration (Fig) also shows that small concentrations of oil do not significantly affect the germination of seeds, but an increase in concentration leads to their death.

The tests carried out indicate a decrease in the growth of the roots of the red round radish with oil pollution of river water. A regular decrease in the length of the root of the red round radish with increasing oil concentration was revealed. The largest number of non-germinated seeds of the plant was noted in a cup with a higher concentration of oil.

The proposed method describes with high accuracy the effect of oil on the distribution of the root length of a seedling during river water pollution.

The application of the proposed method expands the possibilities of environmental monitoring to identify and assess dangerous levels of pollution of water bodies with oil, including as a result of accidental pollution.

Claims (3)

1. A method for assessing dangerous levels of oil pollution of water bodies along the length of the roots of a test plant with oil-contaminated water, including selecting a test plant, uniformly placing the seeds of the test plant on filter paper in a control and test Petri dish with a diameter of 10 cm, and in each control and test 5.0 ml of water is poured into the Petri dish at 4-8-fold watering repetitions, while the liquid level in the cups should be lower than the surface of the seeds, the experiment is completed after 72 hours, after which the length of all roots in each is measured A Petri dish, characterized in that a water sample with a zero oil concentration is taken in a control Petri dish, and water emulsions of the test oil with different concentrations are prepared in the test cups before irrigation, then the seeds of the test plant in the Petri dishes are prepared with prepared water emulsions with a given oil concentration for 72 hours, and the oil concentration in different aqueous emulsions is increased until, at the time of measuring the length of the root, the germination of red round radish seeds is below 50%. 2. The method for assessing dangerous levels of water pollution by oil along the length of the roots of a test plant with oil-contaminated water according to claim 1, characterized in that before the filling an aqueous emulsion of the test oil of a given concentration for one Petri dish is prepared, for this an oil sample of a given volume in milliliters is collected with a graduated pipette using a rubber bulb and poured into a volumetric flask, another graduated pipette draws natural water from a water body adjacent to the pipeline or oil refinery and into Lebanon in the same volumetric flask. 3. The method of assessing dangerous levels of oil pollution of water bodies along the length of the roots of the test plant with oil-contaminated water according to claim 1, characterized in that the following, after the first watering of the seeds in each control and test Petri dishes, are performed under room conditions with an increase in the period between waterings of 72 h in the sequence of 12, 36 and 60 hours

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