HEAVY METALS CONTENT IN PANICUM VIRGATUM CULTIVATED ON CONTAMINATED SOIL UNDER DIFFERENT FERTILIZATION

Heavy metal contamination of soils is a major problem worldwide. As a result, arable land polluted with heavy metals is unsuitable for food production. Utility of energy crops which allow the commercial exploitation of these soils by establishing biofuel feedstock production systems can offer a solution. Additionally, plant cultivation offers opportunities for site remediation. Field experiments have been performed on heavy metal contaminated arable soil located in southern Poland, in the vicinity of a former smelting factory. Although heavy metal concentration exceeded standards, the area has been used for agricultural purposes. Experiments involved testing Switchgrass (Panicum virgatum) cultivated with standard NPK fertilizers and commercially available microbial inoculum. Biomass water, macronutrients (N, P, K, Mg, Ca) and heavy metal (Cd, Pb, Zn) content in aboveground plant organs were assessed at the end of two growing seasons. Switchgrass biomass water content was higher after the second year for nearly 40%. Additionally, after the first as well as the second year fertilizers increased it. Magnesium content, essential in chlorophyll biosynthesis, was higher in the first year and additionally more evident in fertilized variants after every year. Heavy metals accumulation in aboveground organs was lower after the second year compare with the first year. Similar trend was observed for Ca and N plant accumulation. However P and K accumulations were higher after the second year of experiment. In conclusion, due to acclimatization, switchgrass reduce heavy metal uptake, what could result in increase of two biogenic elements (P, K) essential in plant growth.


INTRODUCTION
Heavy industry (i.e., smelters, coal mine) is main emitter of heavy metals (HMs) to the environment.The most dangerous directly for human health is spreading HMs to vicinity agricultural areas where it can be introduce to food chain by accumulation in consumed plant organs (Järup, 2003).Such contaminated arable lands should be excluded from agricultural production.The alternative for such production can be establishing biofuel feedstock production systems on those areas.Except biomass production for energy purposes there is additional benefit in those land management what protect soil from erosion (Meerset al., 2010).Mineral macro-and micronutrients are essential in plant development and proper growth of plants is undeniable depends on its content in soil.Mineral macronutrients are need for plants in higher amount in comparison to micronutrients.Mineral macronutrients can be divided in to two groups: primary mineral macronutrients (N, P, K) and secondary mineral macronutrients (Ca, Mg, Fe, S) (Tripathi et al., 2014).There are reports indicates negative impact of HM on macronutrients accumulation in above ground organs in agricultural plant as well as in plant cultivated for medical purposes (Bello et al., 2004, Siedlecka, 2014).There is a dearth of papers concerning on macronutrients status in energy crop cultivated HMs contaminated soils.Panicum virgatum (Switchgrass) is C4 photosynthesis perennial grass belongs to second generation energy crops, however it can be also cultivated for feed purposes.It is originated from prairie of Northern America where it is dominant species.Switchgrass due to those features is plant very resistant to drought conditions (Parrish and Fike, 2005).
The aim of the study was to describe relationships in HMs and mineral macronutrients accumulation in fertilized Switchgrass during two growing season (2014 and 2015) after plantation establishment.Plants were treated with standard NPK fertilizer and commercial available microbial inoculum (EmFarma, Probiotics Magdalena Górska, Poland).

Site description
The trial was established in Bytom (Upper Silesia Region, 50°20'43.0"N18°57'19.6"E,Poland) on arable land contaminated with HMs.Soil was contaminated over the last century due to dust fall with HMs deposition (Zn, Cd and Pb) emitted by already non-existent Zn/Pb smelter.The climate conditions at the site are moderate, with monthly average temperature and precipitation presented in Figure 1 (based on Institute of Meteorology and Water Management data).
Experiment design Switchgrass plots were established at the beginning of May 2014 from the seedlings obtained from seeds, pre-cultivated in growing room.On each of the three plots 49 plants were planted with density of 3plants per 1 m 2 .Due to high soil homogeneity (Tab. 1) and apprehension of uncontrolled fertilizer application pseudo-replication were performed.On each plot four section were distinguish (Fig. 2): edge plants excluded from further analysis(i) and three section (pseudoreplication) from which samples were taken(ii).Each plot was treated in a different way and 4 meter buffer zone between each experimental plot was set: •P -C -control (no treatment); •P -NPK -NPK standard fertilization, applied directly to the soil once before planting (nitrogen 70 kg ha -1 , phosphorus 30 kg ha -1 as P 2 O 5 and potassium 45 kg ha -1 as K 2 O); •P -INC -commercial microbial inoculum (EmFarma, ProBioticsPolska Magdalena Górska, Poland) applied on seedlings roots before plantation and on the leaves as aerosol in the middle of every month during the growing seasons (from May to September 2014 and 2015).Initial soil samples were collected before planting, one from each plot sections.The plant samples for HMs and mineral macronutrients analysis were collected in the middle of October each year: one sample were collected from the middle plot section and two samples were collected from upper and down plot section.Soil organic matter (OM) was measured by loss on ignition as follows: air dry soil was sifted to pass a 2 mm sieve, dried at 105°C for 24 h and then (5 g) treated with 550°C for 4 h.Soil organic carbon (C-org) was assessed according to PN-ISO 14235:2003.Figure 2. Section on plot distinguished for sample collection.Red -section excluded from sampling, greensection where two samples were collected, yellowsection where one sample was collected

Statistical analysis
Data were analyzed using a one-way ANOVA, followed by a post hoc comparison using the Fisher LSD test (P < 0.05).Statistical analyses were performed using Statistica 10 (Statsoft, USA).Spider charts were constructed using Excel MS Office (Microsoft, USA) and standardization of data for charts construction were performed using Statistica 10 Software (Statsoft, USA).

RESULTS AND DISCUSSION
Results of soil analyses indicate that among experimental plots there were no significant differences between measured parameter, except Zn (Tab.1).Soil Zn content was slightly higher on P -INC plot when compare to control.There were no significant differences between P -NPK plot and P -INC plot.Soil texture on the experimental field was classified as silty loam.According to obtained results and regulation of Polish law (D.2002. r.165 poz.1369) soil HMs content exceed the limits defined by government regulation.In this case experimental area should be classified as marginal, moreover whole agriculture production should be abandoned (Gopalakrishnan et al., 2011).Spider charts (Fig. 3) were used as a tool for assess values pattern changes of measured parameters in above ground organs of Switchgrass among different treatments, as well as annual changes for two growing seasons (2014 and 2015).Spider charts can be divided in to three representative sections: water with secondary macronutrient content (Ca, Mg) (1), primary macronutrients content (N, P, K) (2) and HMs content (Zn, Cd, Pb) (3).Overall, the highest HMs accumulation in above ground organs were found for control plants and it was conditioned by Cd accumulation in the first growing season.The lowest values of Mg and Ca for control plants could be associated with that highest HMs uptake.Hermans et al. (2011) reported that in Arabidopsis thaliana old leaves the content of Cd was lower forplants treated with Cd and Mg, when compare to plant treated only with Cd.It can be assumed with agreement to presented results that higher Mg content can reduce Cd accumulation in elder plants.It is well known that magnesium plays significant role in chlorophyll biosynthesis due to this is essential for photosynthesis process (Cakmak, 2014).Other measured parameters were equal among each treatment in the first growing season.In the second growing season it was found, that there is similar trend among treatments for Mg and Ca accumulation, however it was found that those parameter values are lower when compare to the second growing season.pH (KCl) EC (µS cm -3 ) OM (%) C-org (%) P -C 6.49 ± 0.03a 5.97 ± 0.05a 89.92 ± 1.41a 5.39 ± 0.02a 2.18 ± 0.03a P -NPK 6.57 ± 0.04a 6.06 ± 0.07a 82.28 ± 2.80a 5.52 ± 0.06a 2.11 ± 0.03a P -INC 6.57 ± 0.03a 6.12 ± 0.02a 89.33 ± 2.89a 5.44 ± 0.06a 2.10 ± 0.01a Soil heavy metals content Pb (mg kg -1 ) Cd (mg kg -1 ) Zn (mg kg   It also has been found that there is overall decrease in accumulation of HMs and N for plants from each plot, except Cd accumulation in NPK treated plants when compare to the first growing season.Increased K concentration in plant samples was found for each variant and P was found to be increased on each plot treated with fertilizer, when compare to the first growing season.Gonçalves et al. (2009) reported, that higher Cd concentration in hydroponic solution can decrease K uptake to above ground organs.This paper is with agreement with obtained results, however the tendency is only contrary to NPK treated plants.Water content in plant stems was 40% higher for plant in the growing season 2015 when compare to the growing season 2014 and treatments slightly increased it.This phenomena can be associated with ability to water capture, which is correlated to root distribution.It is known that perennial grasses root system become fully developed after 4 th growing season (Ferchaud et al., 2015).In this case water content could be increased due to annual increase of roots density.Spider charts with description corresponds to overall annual and treatment changes in biomass HMs, water and mineral macronutrient content.Its allows to track changes in tendencies while detailed statistical analysis for measured parameters is presented in Table 2.
Table 2. Matrix of statistical significant differences among analyzed parameters presented on spider charts (Fig. 2 A lower case letters (a, b, c, d, e, f -where "a" corresponds to the highest value and "f" to the lowest) denotes significant differences among plants elements and water content taken from different plots at P ≤ 0.05 according to Fisher LSD test.Each measurement was performed in 5 replicate (n = 5)

CONCLUSION
It could be concluded that HMs in soil affect mineral macronutrients status in Panicum virgatum, especially Mg and K. Additionally there are differences mostly in each measured parameters when compare growing seasons.It can be associated with acclimatization of plant to contaminated site.There is a dearth of papers concerning mineral nutrients as well as HMs status in the first two years after establishment, because most of them are focused on fully establishment trials, where perennial energy crops achieves its maturity after 3 rd -4 th year after establishment.

ACKNOWLEDGEMENT
Research financed by the PHYTO2ENERGY project -7FP EU, Grant Agreement No. 610797 and IETU statutory funds by Polish Ministry of Science and Higher Education.

Figure 3 .
Figure 3. Spider charts constructed on macronutrients, heavy metals and water content show patterns indicated changes of those parameters; among treatment (Ccontrol, NPK -NPK fertilizer treated plant, INCmicrobial inoculum treated plant) and growing season (a-2014 and b-2015) for Panicum virgatum(P).Mg, Ca, P, K, N, Pb, Cd, Znelements accumulation in above ground plants organs.For better data visualization all presented values were standardized.Each measurement was performed in 5 replicate (n = 5) Panicum virgatum, Ccontrol; NPK -NPK fertilized plants, INCmicrobial inoculated plants.

Table 1 .
Soil physico-chemical characteristic, heavy metal content and macronutrients content in initial soil samples Soil physico-chemical characteristic pH (H 2 O)