Metals Phytotoxicity Assessment and Classification

. In this paper, the influence of trace metals (Cd, Pb, Cu, Co, Ni, Zn) on plants of spring barley ( Hordeum vulgare L.) was investigated in polluted sod podzolic sandy loam on layered glacial sands and calcareous deep chernozem on loamy loess soils. We propose to estimate the phytotoxicity with help of phytotoxicological classification. The phytotoxicological classification of trace metals gives the possibility to assess their hazard for plants. On the base of indicators such as plant up-taking index (UI), phytoletal dose (PhLD 50 ), Dipole moment (µ), Phyto Maximum Allowable Concentration (PMAC) a phytotoxicological classification of hazardous trace metals was suggested. The four classes of danger in phytotoxicological classification of hazardous trace metals were offered. According to phytotoxicological classification, Cd, Co, Ni belong to the first class of hazard, Cu – to the second class of hazard, Zn – to the third class of hazard, Pb – to the fourth class of hazard. Phytotoxicological classification of hazardous trace metals gives the possibility to comprehensively estimate the danger of trace metals for plants as a biological object that plays a very important role in the life of ecosystem. This approach may be applied for another trace metals risk assessment for other plants.


Introduction
One of the main harmful and widespread in the territorial and nomenclature aspects of ecosystem contaminants is trace metals, the assessment of which hazards among different groups of toxicants is ambiguous [1,4,29]. After all, trace metals-trace elements, on the one hand, provide normal livelihoods of organisms, and on the other hand, in the high concentration they are toxic to biota [4,15,28]. Anthropogenic trace metals contamination of ecosystems as a result of the application of industrial, transport, agrarian and other technologies causes a damage of the functioning of plants as an important component in ecosystem [2,3,9,11]. Often plants are the main accumulator of trace metals in polluted ecosystem. In the same time, plants play an important role in ecosystem as biomass producers and as biodiversity creators [11,12,17,19,21]. That's why it would be reasonable to get the tools of objective assessment of trace metals influence on plants in polluted ecosystem. Complex approach to trace metals hazard assessment for plants was suggested. It relies on using the phytotoxicologic classification which includes several criteria: PhLD 50 value, PhLD 5 value, Phyto Maximum Allowable Concentration, polarity of metal compounds, Bioaccumulation. Obtaining the PhLD 50 value, PhLD 5 value, Polarity (µ) of metal compounds and assessment of trace metals by these values was represented in previous papers [20,23].

Materials and Methods
Spring barley (Hordeum vulgare L.) was selected as a model plant. Spring barley (Hordeum vulgare L.) is one of the important cereals crop in Ukraine. 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.
The soils of experimental pots were: sod podzolic sandy loam on layered glacial sands (sod podzolic) and calcareous deep chernozem on loamy loess (chernozem). Sod podzolic soil has the following physic chemical characteristics: pHsalt 5 Studied trace elements: Cd, Pb, Zn, Cu, Co, Ni were applied separately in amount equal to the following concentration in the soils: 1. Cu 2+ : 100 mg kg -1 of the soils, 150 mg kg -1 of the soils, 200 mg kg -1 of the soils, 300 mg kg -1 of the soils.

Cd 2+
: 15 mg kg -1 of the soils, 30 mg kg -1 of the soils, 60 mg kg -1 of the soils, 90 mg kg -1 of the soils, 150 mg kg -1 of the soils, 300 mg kg -1 of the soils.
The metals were added to the soil in such quantities before sowing the spring barley. That amount corresponds with adopted in Ukraine Maximum Allowed Concentration (MAC) in soil [27]. The investigation was conducted in green house conditions. Plants grew in plastic Mitcherlikh's pots. Soil preparation, pots filling, and trials were carried out in accordance with standard methodic [5,27]. The trace metals were added to soil during soil preparation before filling the pots. Then, spring barley germinated seeds were planted into the pots and, in the stage of 3 leaves, the recommended population was established.
The studied elements were extracted by 1 M HCl from the soils. The method of trace metals determination was thin layer chromatography (TLC). The method is based on the extraction of metal ions from solutions by diphenyldiithiocarbazone (ditizon). Complex compounds of metals (dithizonates) are formed in a certain range of pH. Further, the colored dithizonates of metals are identified by chromatography in a thin layer of the adsorbent with qualitative and quantitative determination. Method officially recognized in Ukraine [13].
Trace element determination in the plants was carried out after wet digestion by mixture of H 2 SO 4 and HClO 4 [8]. The method of the trace metals determination was the thin layer chromatography (TLC). The plant up-take index (UI) was calculated in equation 1 [21]: where Cplant -metal's concentration in plant (or a part of plant), mg kg -1 ; Csoil -metal's concentration (available form) in 0-20 cm soil layer, mg kg -1 . Now a methodology that would determine the safe concentration of trace metals directly for plants in the soil is absent. After all, the existing standards for the content of trace metals in environmental objects are sanitary-hygienic and focused just on human health [14,16,25]. Determination of the trace metals safe level in the soil for plants can help to objectively assess state of the ecosystem and prevent the trace metals dangerous influence on plant [24]. In this studies, the algorithm of calculation of Phyto Maximum Allowable Concentration (PMAC) was proposed similar to the existing approach of calculation of Maximum Allowable Toxic Concentration (MATC) (equation 2) [18]. In the toxicology practice, the scheme to substance toxicity assessment using the LOEC and NOEC is quite effective and widely used [6,16,25]. These indicators are used also to

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calculate the Maximum Allowable Toxic Concentration (MATC) for assessing the toxicity of substances in the aquatic environment. MATC is calculated by the formula [18]: We propose to determine the Phyto Maximum Allowable Concentration by the formula: where Ccontr -background concentration (on the control variant of experiment -without additional metal input); The PhLD5 is phytotoxic dose 5% (PhLD5) caused reduction of 5% of initial weight (height, length of root etc.).
In our opinion, 5% reduction of initial weight (height, length of root etc.) is the minimal effect, which is similar to the LOEC shows the preliminary changes in the productivity of the plant population. Moreover, the level of significance of deviations, which are considered sufficient for ecological and biological research at the level of 5% (p <0.05) was chosen.
Bioaccumulation capacity and the metal toxic effects are characterized by the plant up-take index [7]. In our investigation, the plant up-take indexes at 10% and 50% reduction of initial weight of plants were proposed for the phytotoxicologic classification.
For each indicator (PhLD5, PMAC, PhLD50, μ, UI10, UI50), the number of classes in phytotoxicological classification is determined by the Sturges (1926) equation [26]: where n -the number of options. The number of options is 12 because in two soils we investigated 6 trace metals. That's why the number of classes was calculated by equation (4): k=1+3,32 lg(12) ≈ 4.
The ranges of classes of each indicator (UI10, UI50, PhLD50, µ, Ccontr, PMAC) were obtained by following equations [10]: 1. The lower limit of first class was calculated by formula: where dx = Lim / k, 2. The upper limit of first class was calculated by formula: Upper limit 1 class = xmin + dx/2-ʊ, where ʊ is measurement uncertainty.
3. The upper and lower limits of second and other classes were calculated by formula 9 and 10: Lower limit 2 class = Upper limit 1 class+ʊ, Upper limit 2 class = Lower limit 2 class + dx-ʊ.
International Letters of Natural Sciences Vol. 73  [22]. The PMAC could be used as an environmental standard that regulate the safe level of pollutants in the soil for plant. Whereas the Phyto Maximum Allowable Concentration is the harmless level of trace metals in the soil for plant, this value was suggested for developing of phytotoxical classification. The plants up-taking (bioaccumulation) is one of the most important indices in the study of the harmful influence of the pollutants on plant. In the case of trace metals, plant up-taking availability is especially important, because trace metals, on the one hand, are nutrient elements, and on the other hand, in high concentrations, are toxic to plants. Besides, the plant up-taking index (UI) gives an opportunity to compare the different bioavailability of the trace metals for plant. In addition, investigation of trace metals up-take by plant in polluted soil is important because pollutants concentration in crops determines the quality of agricultural products. Thereby, index of plant uptaking helps to assess the danger of trace metals and to predict the harmful effects both for human and plants. The plant up-taking indexes of trace metals were obtained in the toxic diapason of concentration in the soil. Reduction of phytomass depending on cobalt's concentration in plant and sod podsolic soil is shown in Fig. 1 as an example. Reduction of phytomass depending on other trace metals in plant and both soils was obtained similar to cobalt.

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ILNS Volume 73 Results of plant up-taking indexes are shown in Table 2. Plant up-taking indexes were found at 5, 10, 50, 80 % reduction of biomass of spring barley. Cadmium plant up-taking indexes were the highest among all investigated trace metals. Lead and cobalt had lowest plant up-taking indexes.
There were obtained the coefficients of variation (CV) and coefficients of oscillation (Kr) of plant up-taking indexes in both soils ( Table 2). The coefficients of variation allowed revealing insignificant variability of plant up-taking in contaminated soil by trace metals. This made it possible to conclude that in conditions of contamination, each metal has a rather narrow range of bioaccumulation ability.  (1 N HCl), mg kg-1

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It has been proved that under the conditions of ecosystem pollution the most intense bioaccumulation is characterized by Cd, Cu, Ni, moderate -Zn, Co, and the smallest -Pb. There were obtained the plant up-taking indexes (UI) at 5, 10, 50, 80% reduction of phytomass of Hordeum vulgare L. The highest plant up-taking indexes were recorded for Cd (0.533-0.645) in polluted ecosystem. Plant up-taking availability allows predicting the amount of metal present in plant and helps to determine the quality of agricultural products. There was obtained the following ranking of the trace metals Cd> Ni> Cu> Zn> Co> Pb according the value of their up-taking index in polluted ecosystem.
The ranges of each value in phytotoxicological classification of hazardous trace metals are shown in the Table 3. According to the obtained ranges of each of the studied values, all trace metals were classified into one of the four classes of danger (Fig. 2). The phytotoxicological classification of hazardous trace metals is shown in Table 4. According to phytotoxicological classification, Cd, Co, Ni belong to the first class of hazard, Cu -to second class of hazard, Zn-to third class of hazard, Pb -to fourth class of hazard.

Conclusions
As a result of this investigation, it was proposed the phytotoxicological classification of trace metals hazard. The phytotoxicological classification by the following indicators: plant up-taking index (UI10, UI50), phytolethal dose (PhLD50), dipole moment of trace metals ditizonate (µ), Phyto Maximum Allowable Concentration (PMAC). We offer to use the four classes of danger in phytotoxicological classification of hazardous trace metals.
According to phytotoxicological classification, Cd, Co, Ni belong to the first class of hazard, Cu -to second class of hazard, Zn-to third class of hazard, Pb -to fourth class of hazard. Phytotoxicological classification of hazardous trace metals gives the possibility to estimate the danger of trace metals directly for plants as a biological object that playing a very important role in the life of ecosystem. We