RU2315006C1 - Method of biotesting of the water on the heavy metals pollution - Google Patents

Abstract

FIELD: environmental protection; methods of biotesting of the water on heavy metals pollution.

SUBSTANCE: the invention is pertaining to the method of the analytical biotesting of water. For the biotesting -cultivate the plants of the duckweed small Lemna minor L. at the permanent daylight and the temperature mode and the composition of the growth mineral medium. The biotesting duration makes 7 days. The toxicity degree is estimated through the mean day gain of the growth share of the duckweed small and compute it by the formula: D =(B-A)/A·t, where A is the initial number of the small leaves of duckweeds in the probe; B - the final number of the small leaves; t - growth time (in days). The number of the small leaves is counted in the probes on 2-d, 5-th and 7-th day of the test. Availability of the pollutants determine by the complex of the morphological changes of the duckweed. In the capacity of the morphological changes of the duckweed use the specific changes of the color of the young and mature plants with the characteristic localization on the small leaves, conservation of the groups of the duckweeds small leaves or their decay - partial or complete, conservation or dropping of the roots. The method allows to increase the information value of the water biotesting system.

EFFECT: the invention ensures the increased information value of the water biotesting system.

1 tbl

Description

The invention relates to a method for analytical bioassay of water for contamination with some heavy metals using small duckweed plants, Lemna minor L., as a sensitive indicator organism (test organism).

A known method for assessing the total toxicity of the soil by testing aqueous extracts of soil samples on duckweed plants is small and based on an estimate of the number of dead cells of plant leaves when stained with safranin (Patent No. 2135884 - Method for biotesting water and soil for pollutant contamination. Tsatsenko L.V., Malyuga N.G., 1997). However, this method can only be used to assess the acute toxicity of samples with a relatively high pollutant content. In real conditions, it is often necessary to detect the effect of low concentrations of pollutants, which manifests itself only with prolonged exposure to the test organism. In addition, this method does not allow us to judge the presence of specific pollutants in the samples, which seems very important in a detailed analysis of toxic samples.

Closest to the proposed one is a method for biotesting water for pollution with heavy metals, including using duckweed plants, Lemna minor L., as a test organism and assessing water pollution by the level of growth of plant leaves and changing their morphology (Patent No. 2096781 - Method for assessing pollution soils of agrolandscape with pollutants (Malyuga N.G., Tsatsenko L.V., Avetyants L.Kh., 1996).

The disadvantages of this method is the following.

1) He maintains completely standard conditions for the cultivation of an indicator organism, which are necessary for an unambiguous interpretation of the results.

2) The adopted system for assessing the degree of toxicity of water - the growth rate of plants for the entire period of the experiment - does not fully reflect the nature of changes in the growth of duckweed minor throughout the duration of the biotest.

3) Of the whole complex of specific reactions of the indicator organism, mainly the discoloration of the leaves is recorded - in some cases this is insufficient to judge the presence of a toxic metal in the test sample.

The technical solution to the problem is to increase the information content of the biotesting system.

The problem is achieved in that in the method of biotesting water for pollution with heavy metals, including the use of small duckweed plants, Lemna minor L., as a test organism and assessing water pollution by the level of growth of plant leaves and changing their morphology, according to the invention, small duckweeds are cultivated under constant light and temperature conditions, biotests are carried out under the same conditions for 7 days and assess the degree of toxicity of the samples through the average daily fraction of the growth of duckweed small, calculated by the forms le:

D = (B-A) / A t

where A is the initial number of duckweed leaves in the sample; In - a finite number of leaves; t is the growth time in days. The presence of pollutants - copper, nickel and zinc ions is determined by the complex of morphological changes in the duckweed plants, which are used as a specific color change of young and mature plants with a characteristic localization on the leaves; keeping groups of duckweed leaf small or their decay, partial or complete; conservation or loss of roots in duckweed plants small.

Determination of the toxicity of the studied samples and the presence of copper, nickel or zinc in them is carried out as follows.

1. Small duckweed plants are cultivated at a temperature of 25 ± 2 ° С and light intensity of 3000 lux fluorescent lamps, the period of plant illumination is 10 hours per day. For cultivation using a mineral medium containing the following components, dissolved in distilled water (mg / l):

potassium nitrate (KNO ₃ ) - 175;

monosubstituted potassium phosphate (KH ₂ PO ₄ ) - 30;

potassium phosphate disubstituted three-water (K ₂ HPO ₄ × 3H ₂ O) - 60;

tetrahydrous calcium nitrate (Ca (NO ₃ ) ₂ × 4H ₂ O) - 147;

magnesium sulphate heptahydrate (MgSO ₄ × 7H ₂ O) - 50;

boric acid (H ₃ BO ₃ ) - 1.2;

molasses - 4.

Once every 4-5 days, the plants are transplanted into a container with freshly prepared medium, choosing young plants of similar size with bright green color of leaves. For biotesting use a culture after 2-3 days of growth in fresh medium.

2. The analyzed water samples are poured into clean glass beakers of 100 ml each; small duckweed plants in the amount of 29-31 leaves are placed in each of them. The growth medium serves as a control. The glasses are covered with a transparent celluloid film to prevent evaporation of water and left for the entire testing period under constant temperature and light conditions.

3. On the 2nd, 5th and 7th day of testing, the number of leaves for a comparative assessment of growth is calculated and the following morphological signs of indicator plants in the experimental and control samples are noted:

preservation or change of color of leaves: yellowing or pallor (chlorosis), complete discoloration (necrosis), as well as the localization of areas with a changed color and the nature of the color change in leaves of different ages, young or mature;

preservation of small duckweed plant groups, consisting of 4-8 leaves, connected by hyaline threads, or the decomposition of groups, partial or complete, to separate leaves;

conservation or loss of roots in duckweed plants small.

The proposed method makes it possible to detect the presence in the analyzed sample of zinc, copper or nickel ions in concentrations corresponding to the maximum permissible concentrations (MPC) for these metals according to the standards adopted in the Russian Federation for drinking water (Kutseva N.K. et al. / / Journal of Analytical Chemistry, 2005, 60 (8), 886-893), according to the characteristic set of morphological changes in duckweed minor. In the experiments, solutions of salts of CuSO ₄ , NiCl ₂ , ZnSO ₄ , CdSO ₄ were analyzed; for the convenience of calculations, the concentrations of metal salts in solutions were expressed in micromol / L (μM), correlating these values with the concentrations in mg / L adopted for the dimension of the MPC of pollutants. In all cases, a decrease in the growth of duckweed was observed in samples containing the studied heavy metals in concentrations at the MPC level or lower, especially during long-term cultivation (table 1).

Table 1 Heavy metal ions, their concentration and The average daily percentage of small duckweed growth share of the maximum permissible concentration (MPC) for drinking water After 2 days 2 to 5 days 5 to 7 days Control, 0 0.267 0.341 0.338 Cu ²⁺ , 6.00 μM (0.38 mg / L, 0.38 MAC) 0.217 0 0 Ni ²⁺ , 0.60 μM (0.035 mg / L, 1.75 MAC) 0.150 0.299 0 Zn ²⁺ , 7.00 μM (0.455 mg / L, 0.09 MAC) 0.167 0.125 0.045 Cd ²⁺ , 0.01 μM (0.001 mg / L, 1.00 MAC) 0.200 0.238 0.264

The assessment of the percentage of growth of duckweed plants in small time intervals (0-2 days, 2-5 days and 5-7 days) during the entire biotesting period seems more informative than the estimate of the growth from the beginning to the end of the test (0-7 days), so as reflects the development of toxic effects, uneven for different pollutants.

The reaction of plants to copper at a concentration of Cu ^{2+ ions of} 6.00 μM (0.38 g / L) was observed with this biotesting method on the second day and manifested itself as a pale or almost complete discoloration (necrotization) of adult leaflets over the entire surface, whereas in young leaflets of the region growth points (proximal areas) retained a bright green color, and only their distal areas were discolored. After 5 days, the groups of plants were disconnected, the adult leaves discolored and acquired a bluish tint, and in young the growth points remained bright green. The roots fell off all the leaves. On the 7th day, all plants almost died, single adult foliage was completely discolored, young growth retained bright green growth points.

On the second day, the duckweed also reacted to zinc at a concentration of Zn ^{2+ ions of} 7.00 μM (0.455 g / l): young leaves began to turn yellow in the region of growth points (in proximal areas). After 5 days, adult leaflets partially remained green, and partially were discolored in the proximal areas on 1/2 or 3/4 of their surface. Young leaflets were discolored completely or on most of their surface. The groups of plants did not disunite, the roots of the plants did not fall off. The same picture was observed on the 7th day of the test.

In samples containing nickel in the concentration of Ni ^{2+ ions of} 0.6 μM (0.035 mg / L), the duckweed reaction was not observed on the second day of the test. After 5 days, adult leaflets remained green, while young foliage turned pale green, sometimes with discoloration (necrotization) of the area of growth points (proximal sites). After 7 days, discoloration of the proximal areas was well pronounced in small young leaves, some of the leaves completely discolored. Partial separation of plant groups was observed; roots did not fall off.

In samples containing cadmium ions at a concentration of 0.01 μM Cd ²⁺ (0.001 mg / L), duckweed leaves retained a bright green color throughout the test. Only a decrease in the level of plant growth was noted.

Thus, in the proposed version of water biotesting, one can judge the degree of toxicity of the tested water samples by the growth of duckweed plants as the main criterion, while the characteristic set of changes in leaf morphology suggests the presence of specific pollutants in the samples under study - copper, nickel or zinc ions. The reaction of indicator plants, manifested at a level below the MPC of copper and zinc, indicates the high sensitivity of the proposed biotesting method against these pollutants.

Claims (1)

A method for bioassaying water for heavy metal pollution, including the use of small duckweed plants, Lemna minor L., as a test organism and the assessment of water pollution by the level of growth of plant leaves and their morphology, characterized in that the small duckweed is cultivated at constant light and temperature mode, conduct biotests under the same conditions for 7 days. and assess the degree of toxicity of the samples through the average daily fraction of the growth of duckweed small, determined in the intervals 0-2 days, 2-5 days, 5-7 days, and calculated by the formula D = (BA) / A · t, where A is the initial number duckweed leaves in the sample; In - a finite number of leaves; t is the growth time in days, and the presence of pollutants - copper, nickel and zinc ions - is determined by the complex of morphological changes in small duckweed plants, which are used as a specific color change of young and mature plants with a characteristic localization on the leaves, preservation of the groups of duckweed leaves small or their decay, partial or complete, conservation or falling roots in small duckweed plants.