Bio-Test Procedures 

Procedures for biological testing with representatives of the family Lemnaceae

• at the cellular level ( method on inhibition of phototaxis , A.G.Lomagin, L.V.Ul'yanova, 1993.),
• at the level of organs ( changes in morphology or pigmentation of duckweed plants from their normal appearance in reaction to a contaminant: chlorosis, yellowing, or bleaching of leaves, and other specific reactions),
• at the level of the organism and or population ( method of calculating the realization of reproductive potential, the method of vital staining ). 

Equipment for conducting the testing:

1. Crystalizer of 2-5 L capacity, used as an incubator for the duckweed cultures.
2. Light meter
3. Petri dishes (4 pcs for the control and for each concentration of the studied material).
4. Pipettes of 3 - 5 mL
5. Micro-spatula.
6. Binocular microscope.
7. Centrifuge.
8. Bagorov's camera, a glass box of 30 x 40 cm. 

Inhibition of phototaxis.
This method is based on a reaction that leads to inhibition of phototaxis. As an index for change in the functioning of the cell the number of chloroplasts in the epistrofnom position is used. For this fronds of duckweed from the stock culture were placed into the clinical laboratory centrifuge, and spun for a period of 10-15 minutes at the maximum speed - 3000 rpm. After this, the fronds were removed and were placed into a drop of water on a microscope slide.

Because of positive phototaxis, chloroplasts in the cells of parenchyma on the upper side of the frond and part of the thallus were located in the upper wall of cells in the epistoform position, i.e., perpendicular to the light beam. Without moving the preparation, a strong illumination was directed on the subject in the microscope, as strong as possible without damaging the cells.

After 10 minutes the illumination of the subject was decreased to a level convenient for observation, and under 400-power magnification the number of chloroplasts in the epistoform position were counted. In the control (distilled water) the number of epistoform chloroplasts is minimal, since the majority of the chloroplasts will move away because of the negative phototaxis to the parastoform position, being moved against the side walls and being turned with their thin edge to the light beam.

Fixation of morphological changes.
Fixation of morphological changes in duckweed plants from the control under the action of contaminants: chlorosis, yellowing, or the bleaching of leaves, and other specific reactions. The historically precise morphological reactions of organisms to industrial chemicals is routinely practiced for the evaluation of water quality. The biological parameters are change in pigmentation of fronds: yellowing, browning, the loss of the intensity of pigmentation. This method became the basis for the methods of the machine analysis of the Lemna-Tec firm, where the calculation is conducted with the aid of video image analysis, which identifies fronds on a defined color range: dark green, green, yellow, brown, white, tissue chlorosis.

Method of calculating the realization of reproductive potential. 
For the evaluation of the action of contaminants, the time of the doubling of number (t of udv) is used. This index is calculated from the coefficient of the instantaneous increase in the population (r), whose change reflects the resistance of the medium, i.e., is characterized the sum of all the limiting factors of the medium which impede the realization of the reproductive potential (r swing), which is calculated from the growth of the control culture.

During the time of exposure of the fronds in the control and in each concentration, the total number of fronds are counted, including maternal individuals and fronds which have separated from the maternal individual. 

In the control and in each concentration the coefficient of an instantaneous increase in the population (r) is calculated on the basis of theresults obtained:
 

r = ln(Nt) - ln(N0) / t,

where N0 - initial number of fronds; Nt - final number of fronds; t - duration of experiment (twenty-four hours).

Further for each t is calculated the frond number doubling time (t _{of udv}):
 

t of udv = ln(2)/r = 0,6931/r.

The index of change of the frond number doubling time for the duckweed in the solutions being investigated with respect to the control, is expressed in percentages (Dt):
 

Dt =((tdoub.K. - tdoub.exm..) / tdoub.K.) * 100%

Dt indicates the quality of the tested aqueous solution. A deviation of more than 20% from the doubling time of the control is considered statistically significant. Deviation to the side of decrease during the toxicological analysis shows the degree of the action of toxicant, with biomonitoring - indicating a decrease of the eutrophication of water or the presence of toxicants. In exceptional cases in biomonitoring it is possible to observe an increase in growth rate as a response to increasing concentrations of the test solution, as in the case of an increase in eutrophication.

Method of vital staining.
This method is based on the staining of dead cells - by the dye safranin. Living cells strongly limit permeability inside the cell protoplasm, and those placed into the dye solution are practically are not colored. However, the stain penetrates freely into the dead cells, thanks to which it is possible to immediately identify them (V.D. Pashkovich, B.S. Yudin, 1978). Safranin is used as the stain, since it possesses the ability to pass through cell walls very well. Dissolve 0,25 grams safranin in 100 mL of 10% alcohol (Z.P.Pausheva, 1980; The operating instructions in the cytological and cytoembryological technology, 1991). The number of stained cells in the percent ratio to the total frond area is accepted as the index of the toxicity of pollutants (L.V.Tsatsenko, N.G.Malyuga, 1998).

Studies have been conducted in the prolonged multifactor stationary experiment since 1992, placed in the experimental field KGAU.

Soil of the test area:  light, super-power, low-humus black soil.

The stationary multifactor experiment examines the following factors: Factor A - fertility of the soil; Factor B - system of fertilizers; Factor C - the system of plant protection, and factor D - the system of the basic cultivation of the soil. In the experiment four models of the levels of the fertility of soil are created on the basis of the existing normative indices due to the introduction under the first culture of crop rotation (the sugar beet) of organo-mineral fertilizers. For creating the soil background A1 - 200 kg/ha P₂O₅ and 200 T/ha of bedding manure were applied. For creating the background A2 - the doses of fertilizers were doubled, and for background A3 - they were tripled. A0 was the natural background (Table 1).

Variant	Fertility Level	Adapted to fertilizers on 1 ha of cultivated land		Adapted to plant protection treatments on 1 ha of cultivated land
		NPK, kg/ha	Bedding manure T/ha	Pesticides	Biological controls
1	2	3		4
000	Natural background fertility	No added fertilizer		No plant protection applied
		0	0	0	0
111	Average/mean background of fertility (200 kg/ha P2O5 +200 T/ha of manure)	Minimum application of fertilizers		Biological controls only
		91	4,5	0	5,2
002	Natural background fertility	No added fertilizer		Integrated system for protection from weeds
		0	0	5,1	0
020	Natural background fertility	Average application of fertilizers		No plant protection applied
		181	9,0	0	0
200	Increased background of fertility (400 kg/ha P2O5 +400 T/ha of manure)	No added fertilizer		No plant protection applied
		0	0	0	0
222	- // - // -	Average application of fertilizers		Integrated system for protection from weeds
		181	9,0	5,1	0
333	High level of fertility (600 kg/ha P2O5 +600 T/ha of manure)	High application of fertilizers		Integrated system for protection from weeds
		361	18,0	12,1	0

The second factor studied in this stationary experiment was the system of fertilizers (Table 1.). The ranges of the fertilizer applications were determined on the basis of balance method, taking into account the planned productivity for the required quality of production, the assigned rates of soil fertility, and favorable state of environment.

The system for protection of plants from pests, disease and weeds was the third factor studied in the experiment. In all experimental variants seeds were disinfected using a insecticide-fungicide mix.

The system of the basic cultivation of the soil was the fourth factor studied in the experiment. In the case of this experiment, the cultivation recommended for this region was adopted.

During the development of present program the basis was a multifactor experiment design (4*4*4)*3. The objective was to study the fertility of the soil, the system of fertilizers and the system of plant protection at doses 0,1,2,3 using the recommended soil cultivation.

The area of the experimental plot: 4,2 m * 25,0 m = 10⁵ m², stock-taking 2,0*17,0=34 m². Repetition of the experiment - triple.  Each field of crop rotation had 144 plots (72 plots*2 blocks) and occupied an area of 1,75 ha (160 m*109,2 m).