Probit analysis for Cd, Pb, Cu, Zn phytotoxicity assessment

The aim of investigation was to develop a new approach in Cd, Zn, Cu, Pb phytotoxicity assessing. This approach provided the probit analysis using. Methods included probit analysis, thin layer chromatography, statistical methods (calculation of the least significant difference, correlation analysis). We applied «doze-effect» model to obtain the ranking of the metals according to their phytotoxicity in spring barley field. We offered to estimate the phytotoxicity by PhLD 50 index. Research results were: graphic formalization of "dose-effect" dependence and calculation of phytotoxic doses (PhLD 50 and PhLD 95 ) for Cd, Cu, Pb, Zn in polluted soil. According to PhLD50 value we conducted a comparative assessment of Cd, Cu, Pb, Zn phytotoxicity relatively to spring barley. According to PhLD 50 value metals could be ranked: Cd> Cu> Pb> Zn. The most toxic metal was Cd. PhLD values, on which the estimation of metals phytotoxicity, were: Cd – 50, Cu –129, Pb –537, Zn –603 mg / kg mobile forms in turf-podzol sandy loam soil. Our findings are relevant to estimating the metal hazard and controlling the condition of the crop growth.

Investigation of damaging effect factors (for ex.pollutants toxic effects) on biological objects usually requires probit analysis.The idea of probit analysis was first published by Bliss in 1934 in an article devoted to the impact of pesticides on the dead pests percentage [1].Bliss suggested to calculate the percent of dead pests using block probability-probability unit (or probit), which got the complete definition by Finney [2].It is known that measuring of the lethal dose for an exact individual is almost impossible, because the death of the plant organism often occur not immediately.If the dose is not sufficient to cause the death of the plant organism, it also turns out over time.Moreover, the accurately determination of the dose at which 100% of individuals die is not possible and not acquitted.That's why usually there is the determination of the dose at which 50% and 95% of individuals are dying (or biomass decreasing).These doses are taken as averages characteristics of lethal effect of damaging factors and indicate respectively LD 50 and LD 95 [3].Methods of probit analysis using, and calculation of plant or animal organisms death resulted by the toxic factor in agrocenoses in field are described in [3].
Transformation of dead plants percent to probit, and "dose-effect" curve are shown in Table 1 and in Figure 1.
Probit analysis is widely used in Toxicology because LD 50 and LD 95 are among the most important indexes in assessing the toxicity of the substance.The more LD 50 and LD 95 , the less toxic to certain populations.Usually LD 50 index is used in toxicology as "dozeeffect" correlation for assessment of toxicity substances for human's life with following methodology of extrapolating data [4,5].However, there are not ecotoxicological studies which should to determine the LD 50 and LD 95 for living components of the ecosystem.Although, the biological productivity of ecosystems is an important parameter which characterized the condition of ecosystems and comfortable human existence in it as the last link in the food chain [6,7].
Modern ecotoxicologic assessment of pollutants danger includes several indexes bypassing attention of calculation lethal doses for biological objects in ecosystem [8][9][10][11][12].In multimetallic chronic pollution, which are typical for natural conditions, it is impossible to establish a clear "doze-effect" dependence for each metal.But, in practice, single heavy metals pollution occurs rarely.Therefore, the simulation conditions of soil contamination impact allow to define the "dose-effect" with various concentrations of metal in the soil and to establish lethal dose in plant (LD 50 and LD 95 ).Such an experiment will be assessed phytotoxicity each of the pollutants that can be used in calculating the risk of contamination of toxic metals in the environment.The need for such research is of particular relevance in terms of pollutant contamination of agro-ecosystems, quantity and quality crop production which is very important for humans as consumers of it [6][7][8][9][10][11][12].In addition, the establishment of indicators lethal doses will enable comparative evaluation of the toxicity of pollutants in relation to biological objects.
We offer to define the lethal indexes for plant (phytotoxicity) using known probit analysis analogously to LD 50 and LD 95 .
Phytotoxic lethal dose (PhLD 50 and PhLD 95 ) is the amount of pollutants in the soil (or plant) (mg kg -1 ) which results 50% (or 95%) death of plants.
Our research has been devoted to defining indexes PhLD 50 and PhLD 95 of Cd, Zn, Cu, Pb for spring barley in polluted turf-podzol sandy loam soil.
Impact pollution is a one-time (or nonsystematic) contamination of soil pollutants leading to disruption of biotic component of ecosystems, i.e. it is formed the artificially created biogeochemical endemic [13][14][15].
Impact and chronic pollution of toxic metals in agro-ecosystems generally occurs near proximity anthropogenic (industrial) landscapes.In order to predict the harmful effect of metals behavior in polluted conditions in such landscapes, it is necessary to have ecotoxic preliminary data about these pollutants, for example, their kinetics, soil profile migration, effects on micro biota, plant up-taking and others properties.For this purpose, the model experiment included the artificial adding of different dozes of Cd 2+ , Pb 2+ , Cu 2+ , Zn 2+ salts in sod podzolic sandy loam soil.

Materials and Methods
The soil of experiment was sod podzolic sandy loam on layered glacial sands (sod podzolic).Sod podzolic soil has the following physic chemical characteristics: pH salt 5.5; organic matter by Turin 0.87%, CEC 6.3 mg eqv/100 g.Crop was spring barley.Research conducted at the Institute of Chernigov APP UNAAS.Experimental studies conducted over the years 1999-2012.That amount corresponds with those adopted in Ukraine Maximum Allowed Concentration (MAC) in soil.
The following metals salts: CdSO 4 , were used for the trace elements application.Spring barley (Hordeum vulgare L.) was selected as a model plant.Soil preparation, pots filling, and trials were carried out in accordance with standard methods [3].The studied elements were extracted by 1 M HCl from the soils.The method of HM determination was thin layer chromatography (TLC).Method widely used in our previous investigation and officially recognized in Ukraine (№ 50-97, 19.06.1997) [16].
For studying the total biomass we used the average value of biomass within agrodempopulation, which, in our case, consists of a set of spring barley individuals in per unit area with a certain concentration of mobile and potentially mobile forms of metals in the soil.Mean standard deviations, variance, and minimum, maximum, standard errors were calculated from at least three replicates.The experimental results were interpreted using standard statistical methods.Probit analysis was applied according to Dospekhov V. [3].This method applies to the generalized system modifications probit.It allows only estimating of PhLD 50 and PhLD 95 approximately, because it cannot allow calculating the confidence intervals of these values.Thus it may be possible some errors in values of "dozaeffect", and as a result in PhLD 50 and PhLD 95 .However, on the accuracy of PhLD 50 it affects very slightly.To avoid these shortcomings and draw the line that best fits the experimental set points, it is necessary to use more complex system modifications probit.However, the uses of sophisticated methods are not always beneficial, because in most cases this accuracy is not required.

Results and Discussion
Experimental data are shown in Table 2. Except experimental data Table 2 includes the values of lg D (where D is a 1 M HCl extracted forms in soil, mg kg -1 ) and probit values.
Relationship between LgD of Cd 2+ , Pb 2+ , Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ and probit on the studied soils are shown in figures 2, 3, 4. Lg of dose marked on the ox, and values of probit marked on the oy.

The correlation between between LgD and probit is presented in table 3. Values of PhLD 50
According to PhLD 95 the metals can be ranked by descending phytotoxic order as follow: Cd>Cu>Zn>Pb.This is because the large doses of lead and other metals causes the defense mechanisms of plant organisms that are responsible for the normal up-taking.Often, in such cases, the break downing of defense mechanisms results to freely uptaking toxicants instead nutrients.Such phenomenon describes in many environment axioms, in particular in Shelford's regularity.Diapason of resistance (tolerance) is between organism limit factors (pessimums).That's why commonly known toxicological practice of lethal dose establishing operates 50% changing of population.Furthermore, it is known that plants root up-taking of lead has less intensity than other ways of plant up-taking.
Some studies indicate that low doses of lead are often not only inhibit the production of biomass crops, but also cause a stimulating effect which is observed when feeding micronutrient [15][16][17][18][19][20].It is established that small amounts of lead needed to plant organisms.Although lead is present in all living organisms, it is proven its vital necessity.But on the other hand, toxicity or biological role or mechanisms of lead's action are studied very poorly [19][20][21].Therefore, setting up of experiments for studying the "dose-effect" dependence should consider not only the effect of depression, but stimulating effect of metals in small quantities.Indeed, all metals in small quantities play role as ultratrace elements [15][16][17][18][19][20][21].
We offer to use the probit analysis for assessing the phytotoxicity metals.
Graphic formalization of "dose-effect" dependence for Cd, Cu, Pb, Zn relative to spring barley was built.The PhlD 50 and PhLD 95 for each metal were calculated.
We suggested to estimate the heavy metals phytotoxicity by means of PhLD 50 value.The results helps to compare phytotoxicity of studied metals Cd, Cu, Zn, Pb for plants of Spring barley (Hordeum vulgare L.) on sod podzolic sandy loam on layered glacial sands (sod podzolic).The PhLD 50 value is indicates not only 50% reduction of biomass for spring

Fig. 2 . 3 ) 4 )
Fig. 2. Correlation between LgD of Cd and probit in the condition of sod podzolic sandy loam on layered glacial sands

Fig. 3 .Fig. 4 .
Fig. 3. Correlation between LgD of Pb and probit in the condition of sod podzolic sandy loam on layered glacial sands