Reactions to heavy metal salts, part 2




Figure 6.  Spirodela polyrhiza. a) control; b) Mn with concentrations from 0,1-0,025 mg/ml; c) Ba - 0,025 mg/ml.

 To  manganese (Mn) in three species, as represented in the tables, a relatively similar reaction was recorded: death and the detachment of roots.(Figures 6-7).





Figure 7.  Lemna minor. a) control; b) Ba with concentrations from  0,001-0,0025 mg/ml; c) Co - 0,1-0,025 mg/ml; d) Mn - 0,125-0,025 mg/ml.

 In Spirodela  the response to the action of heavy metals proved to be expressed most strongly compared with the remaining duckweeds, evidenced by the pigmentaton of the fronds.  The specific character of the action of many heavy metals consists in the fact that they are  biologically active in microcells, necessary for the normal functioning of the organism.  In this connection for many of them there are no barriers of entering into the organism and they possess the ability to be accumulated in the substance of the plant.

The selected test objects - the aquatic plants of the family Lemnaceae - they reflect in our view most fully the spectrum of the action of each metal.  Lemnaceae are characterized by extraordinary sensitivity to change in the composition of water, since they absorb everything of the water of all by their surface.

Practically all concentrations of metals, investigated in the work, caused the retardation of increase/growth, and in a number of cases - death of the plants.

During the analysis of the influence of Co, Mn, Cu, Zn - as the most toxic metals, of the entire group focus attention on themselve considerably.  The most rapid response of duckweeds was to the action of Cu: in several hours with all duckweeds the fronds were disconnected.  It is known that one of the mechanisms of the toxic action of copper can be caused by a change of the conformational characteristics of proteins as a result of the connection/attachment of Cu ions or with a change in the intracellular conditions, as a result of the caused Cu disturbance of the permeability of the cell membrane.  Evidently, the copper ions disrupt the protein structure of the membrane, breaking disulfide bonds, in consequence of which the transparent thread that connects the fronds is torn up and are decomposed the groups of fronds.

Relative to the action of zinc on viability and rate of growth in the aqueous macrophytes there are data, that it can be absorbed as a result of ion-exchange processes, which use metabolic mechanisms, that require expenditure of energy.  The primary object of the damage of zinc, is apparently the breakdown of photosynthetic transport, about which testifies the complete discoloration of duckweeds fronds.

The actions of Сo, Ba, and Mn on the integral indices of duck-weeds - the rate of growth and viability - it is noted to the second day of the experiment:the complete disconnection of fronds, by death starting from the edges and the detachment of roots.  The toxic action of these metals, is connected with action on the protein structures of cells, as a result of which the permeability of the membrane is disrupted.  Ions of Co, Ba, and Mn cause swelling the organelles of cytoplasm, which entails the gap of the cell membrane and the separation of the connection/communication between fronds.

In the work 5 species of duckweeds were used, which made it possible to approach most correctly the selection of appropriate indicator forms.  On the sensitivity of duckweeds to the metals, on the basis of the results of this work the forms were arranged in the following sequence:

Lesser duckweed > Swollen Duck-weed > Greater Duckweed > Ivy-leaf Duckweed  > Wolffia arrhiza (Figure. 8).

 Form very sensitive to the pollution/contamination of soil and water;  reacts to the salt TM with the concentration: Cu (0,000ymg/ml), Zn (0,025 mg/ml), Ba (0.001 mg/ml), Co (0,0001 mg/ml), Mn (0,025 mg/ml).

 Average- sensitive forms to the pollution/contamination.

 Low-sensitivity to the pollution/contamination forms.
Figure 8. Classification of the forms of duck-weeds on the sensitivity to the salts of heavy metals.

In the second stage of work the reaction of duckweeds on pollution/contamination by pollutants was studied on tests 4 and 5 in more detail.

a) Control

 Position of chloroplasts in the epistrofnom (horizontal) position after illumination by a weak beam of light.

 Position of chloroplasts in the parastrofnom (vertical) position after illumination by strong beam of light. 
б) versions with the pollution/contamination

 Position of chloroplasts in the epistrofnom position after illumination by a weak beam of light.

 Position of chloroplasts in the parastrofnom position after illumination by strong beam of light. 
Figure 9. Braking the phototaxis of chloroplasts in fronds of teh duckweed, Lemna minor L. in the control and the treatments with the pollution/contamination of soil by toxicants.

Test 4 - the inhibition of phototaxis.  Chloroplasts with the concentrations being investigated did not disperse to the edges of cell.  On the average, the number of chloroplasts per cell varies from 20 to 23.  In the control after intensive illumination all chloroplasts or 97% of the total number converted to vertical state, i.e., they were localized to the cellular wall.(fig 9).  With the decrease of metal concentration their negative influence on phototaxis is reduced (table 4). 
Table 4.
 Lethal doses of toxic elements for Lemna minor L.
 Metal  Value of toxic concentrations, mg/L*10-6
LD50 LD95
Cu 1800 38700
Co 1600 73500
Zn 2900 76700
Pb 3100 77300
Mn 2800 78400
Ba 2600 76700
Note:  Estimation/evaluation of lethal doses is given in terms of the value of chloroplasts in the horizontal position.

The metals are distributed as follows according to the degree of toxicity for the test object: Co > > Cu > Ba > Mn > Zn > Pb.

Test 5 - Vital staining.  With the aid of the visual estimation/evaluation at low concentrations of metals it not always possible to determine the viability of fronds.  The method of vital staining makes it possible to look over a large number of fronds to immediately see the degree of the damage to the entire plant.  For the analysis of one sample 5-7 min are required.  In the stained standard they proved to be to 10% of the cells from the entire area of fronds.  With a high concentration of metals, old fronds were stained completely, with a decrease in concentration of the metal, for the living fronds, i.e., those not stained, only the points of increase/growth remained unstained.  With some concentrations only parts of fronds were green (fig 10).
 Figure 10.  Vital staining of leaves of duckweed.  Red background - stained, yellow - the green or unstained cells of fronds.  In the upper right corner - metal concentrations are indicated, in the lower right - % stained (i.e. the killed cells).  Control - 0-10% of stained cells.

As can be seen from figure, with the low concentrations of metals (0.001;0,0001 mg/ml) young fronds remain viable, and this allows fronds to continue multiplying.


 This data on the sensitivity of duckweeds to contaminators make it possible to make the following conclusions:
  1. Copper, in comparison with Zn, Co, Ba, Mn, Fe possesses the strongest toxic action and its reaction is manifested in 3 - 5 hours with the concentrations: 0,1; 0,25;0,025; 0.001; 0,0001 mg/ml.
  2. Cu, Co, Ba, Mn - cause the complete disconnection of duckweed fronds; with concentrations 0,1 - 0,25 - 0,025 mg/ml.Mn - death of roots and their detachment from fronds.
  3. The investigated metals possess toxic actions which can stop the growth of duckweeds and affect their viability.
  4. Lesser duckweed, swollen duckweed and greater duckweed - are more sensitive subjects to the action of heavy metals than are ivy-leaf duckweed and Wolffia arrhiza, which is apparently explained by the intensive metabolic processes in the plants themselves.
  5. Lemna species as phytotesters possess high sensitivity to the action of toxicants, since are capable of reacting to the metals at concentrations in the range from 0,1 to 0,0001 mg/mL and thy can be of successfully being used for testing pollution/contamination by the pollutants of the components of the ecosystem.

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Краснодар, 2002