(AIDED)-PHYTOSTABILISATION OF Cu-RICH MINE TAILINGS USING WOODY CROPS AND GRASS SPECIES

Petra Kidd<fnref n="1" />, Vanessa Álvarez-López<fnref n="1" />, Beatriz Rodríguez-Garrido<fnref n="1" />, Carmen Trasar-Cepeda<fnref n="1" />, María Touceda-González<fnref n="1" />, Michel Mench<fnref n="2" />, Markus Puschenreiter<fnref n="2" />, Felipe Macías-García<fnref n="3" />, Ángeles Prieto-Fernández<fnref n="1" />

Corresponding author email: pkidd@iiag.csic.es

ABSTRACT

Mine-spoils and tailings present hostile environments for plant growth (low nutrients, low organic matter content, high acidity, and elevated trace metal content). Phytostabilisation techniques aim to establish a vegetation cover and progressively promote in situ inactivation of trace metals by combining the application of soil amendments with the cultivation of metal-tolerant populations of plant species.

This study was carried out in an abandoned copper mine in Touro, Galicia (NW Spain). The mine operated from 1974 until 1988 but is now confined to the extraction of materials for road construction. Mine tailings cover an area of approximately 550 ha. The objectives of the study were to reduce Cu mobility and availability in the mine-soils and establish either a short rotation coppicing system or a grass cover. The geological substrate is amphibolite, with significant quantities of metal sulphides (pyrite, pyrrhotite, and chalcopyrite). The mine-soils (Spolic Technosols (Episkeletic)) are characterised by their extreme acidity (pH 2.8-3.5), low C, N and P, and high concentrations of Cu (319-774 mg/kg). Field plots (10m x 45m) were established in Spring 2011 and treated with one of three amendments. Amendments consisted of composted municipal solid wastes (compost) and two technosols. Technosols were mixtures of organic (anaerobic and aerobic sewage sludge) and inorganic wastes (aluminium oxides, iron oxides, fly ash from wood bark combustion, and foundry sand). Application rates of all amendments were equivalent to 250 kg Mg⁻<fnref n="1" /> dry tailings. Compost-amended plots were planted with different metal-tolerant clones of *Salix* (*S. caprea* and *S. viminalis*) and *Populus nigra*. The experimental plot was sub-divided into 5m x 5m sub-plots, and three replicate sub-plots were used for each plant species/clone. *Agrostis capillaris* cv. Highland was planted in three replicate sub-plots in both the compost- and technosol-amended plots. Soil samples were collected to determine the baseline trace metal content and general physico- and bio-chemical properties before amendment addition/planting. Plant growth/survival and soil properties (improvements in pH, nutrient availability and soil enzyme activities, and reductions in Cu bioavailability) were determined one and two years after planting. Shifts in the soil microbial community with time were evaluated using the denaturing gradient gel electrophoresis (DGGE) technique.

Soil Cu concentrations at time zero showed a high heterogeneity across the experimental field plots. Nonetheless, the addition of amendments led to an increase in soil pH, C/N content, cation exchange capacity and nutrient availability. At the same time amending soils led to a significant decrease in the NH₄NO₃- and NaNO₃-extractable concentrations of Cu (<0.2 mg kg⁻<fnref n="1" /> extractable [Cu]) and shoot Cu concentrations remained consistently below 25 mg kg⁻<fnref n="1" />. Few differences were observed in soil physicochemical properties between the three amendments after 2 years of treatment. However, plant growth, soil enzyme activities and microbial community structure were significantly influenced by both the amendment type and plant species.

Acknowledgements: The financial support of the FP-7 project GREENLAND (contract No. 266124) by the European Commission, and from the Spanish Ministerio de Economía e Competitividad and FEDER (project CTM2012-39904-C02-01), is greatly appreciated.

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<footnote n="1">¹ Instituto de Investigacións Agrobiolóxicas de Galicia (IIAG), Consejo Superior de Investigaciones Científicas (CSIC), Santiago de Compostela, Spain</footnote>

<footnote n="2">² University of Natural Resources and Life Sciences (BOKU), Department of Forest and Soil Sciences, Tulln, Austria</footnote>

<footnote n="3">³ Tratamientos Ecológicos del Noroeste s.l. (TEN s.l.), Touro, Spain</footnote>
EDTA AIDED PHYTOREMEDIATION OF CADMIUM BY BRASSICA NAPUS L.

Mujahid Farid, Shafaqat Ali, Muhammad Bilal Shakoor

Department of Environmental Sciences, Government College University, Allama Iqbal Road, Faisalabad, 38000, Pakistan

Corresponding author email: shafaqataligill@yahoo.com Ph.: +92 345 6281213

ABSTRACT

Heavy metals pollution of soil is a prevalent global problem and oilseed rape (Brassica napus L.) are considered useful for the restoration of metal contaminated soils. Phytoextraction is an in-situ environment friendly technique for the clean-up of contaminated soils. Response to cadmium (Cd) toxicity in combination with a chelator, Ethylenediaminetetraacetic acid (EDTA) was studied in oilseed rape grown hydroponically in greenhouse conditions under three levels of Cd (0, 10, and 50 µM) and two levels of EDTA (0 and 2.5 mM). Cd decreased plant growth, biomass and chlorophyll concentrations while the application of EDTA enhanced plant growth by reducing Cd induced effects in Cd-stressed plants. Significant decrease in photosynthetic parameters was found by the Cd alone. Addition of EDTA improved the net photosynthetic and gas exchange capacity of plants under Cd stress. Cd at 10 and 50 µM significantly increased electrolyte leakage, the production of hydrogen peroxidase (H₂O₂) and malondialdehyde (MDA) and a significant reduction was observed in the activities of catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase under Cd stress plants. Application of EDTA at the rate of 2.5 mM alone and with combination of Cd increased the antioxidant enzymes activities and reduced the electrolyte leakage and production of H₂O₂ and MDA. Oilseed rape (Brassica napus L.) actively accumulated Cd in roots, stems and leaves and the addition of EDTA boosted the uptake and accumulation of Cd in oil seed rape by dissociating Cd in culture media. The present results suggest that under 8 weeks Cd-induced stress, application of EDTA significantly improved the plant growth, chlorophyll content, photosynthetic, gas exchange capacity, improving enzymes activities and increased the metal uptake in roots, stems and leaves of oilseed rape (Brassica napus L.) respectively which shows that this technique is very useful for heavy metal pollution management.

Keywords: antioxidant enzymes, cadmium, chelator, EDTA, growth, oilseed rape

<figure><img src="image_160.png" /></figure>
# PHYSIOLOGICAL STRESS FACTORS ASSOCIATED WITH DIFFERENT MINE TAILINGS ON THE CHLOROPHYLL FLUORESCENCE OF PLANTS

Jacques Berner<fnref n="1" />, Pieter W. van Deventer<fnref n="2" />, Cindy Faul<fnref n="2" />

Corresponding author email: 22869794@nwu.ac.za

## ABSTRACT

Phytoremediation is an emerging technology for the remediation of mine tailings. Phytostabilization is a technique within this field which involves the establishment of vegetation in mine tailings in order to immobilize potentially toxic trace elements, and which can also refer to the physical stabilization of the surface of tailings materials. Physical stabilization is required to avoid or minimise erosion and reduce surface runoff, thereby decreasing the distribution of trace elements, e.g. arsenic, copper, nickel, cobalt, cadmium, into the surrounding environment and/or groundwater system.

The aim of this investigation was to evaluate the ability of selected plant species to adapt to the physiological stress factors associated with mine tailings, thereby facilitating a successful phytostabilization methodology. The vitality of the plants subjected to various soil stress conditions was measured as a function of photosynthetic ability. It is expressed as $PI_{ABS}$, and takes into consideration the ability of the plant to absorb and convert sunlight into chemical energy.

Chlorophyll fluorescence (JIP-test) was used to measure $PI_{ABS}$ in six plant species subjected to various stress factors, i.e. water stress, acidity, salination and low nutrient status. The stress factors associated with the different mine tailings negatively affected plant vitality, thereby limiting the ability to convert sunlight into chemical energy. Comparisons between the reactions of the different plant species to the stress factors revealed significantly different behaviours. Very low water retention levels are characteristic of coarse tailings materials, i.e. kimberlites and coal discard, and this was reflected in the chlorophyll fluorescence results. Gold and coal tailings negatively impacted the plants by causing a decrease in the vitality index. Plants that were grown in more favourable conditions and plants adapted to survive in mine tailings showed a higher vitality index.

To conclude: Chlorophyll fluorescence assessments proved to be a very useful indicator in the identification of physiological stress in plants established on mine tailings.

**Keywords:** Chlorophyll fluorescence, Physiological stress factors, Phytostabilization, Plant vitality ($PI_{ABS}$), Potentially toxic trace elements, Remediation, Mine tailings

**Acknowledgements:** Financial support from the Technology and Human Resources for Industry Programme (THRIP) of the National Research Foundation (NRF) [South Africa] as well as from Frazer Alexander Tailings is greatly appreciated.

<figure><img src="image_161.png" /></figure>

<footnote n="1">¹School for Biological Sciences, North-West University, Potchefstroom, South Africa</footnote>

<footnote n="2">²School of Geo- and Spatial Sciences, North-West University, Potchefstroom, South Africa</footnote>
# HEAVY METAL TOLERANCE AND ACCUMULATION OF SWEET SORGHUM- POT OUTDOORS EXPERIMENT

Wang Xu, Wang Jian-long, Li Shi-zhong, Fan Gui-fang, Chen Can

Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, China

Corresponding author email: chencan@tsinghua.edu.cn

## ABSTRACT

High biomass producing species are considered as tools for remediation of contaminated soils by heavy metals, radionuclides or organic pollutants. Sweet sorghum (Sorghum bicolor), with high biomass, is a typical model plant for bioenergy crops. In this study, the characteristics of Zn, Cu, Cd, As, Hg or Cs tolerance and accumulation of sweet sorghum grown for three months were studied in pot outdoors, Hainan Province, P.R. China. Each treatment had three replications. No visual toxic symptoms on shoots of S. bicolor were observed at Cd level ≤3 mg/kg or Cs level ≤100 mg/kg or Hg level ≤10 mg/kg, with the height and dry biomass of the plants were similar to that of the control. Obvious toxic effects of S. bicolor growth occurred at Cu level of 200 or 400 mg/kg, at As level of 25 or 50 mg/kg, at Zn level of 500 mg/kg, at Cd level of 15 mg/kg, or at Cs level of 400 mg/kg, with height and dry biomass of the plant decreased much. The shoot Zn concentration and root of the treatment with 250 mg Zn/kg was 1657.6 mg/kg and 1370.0 mg/kg, respectively when the dry biomass of the plant decreased by 45%. The shoot and root Cs concentration at Cs level of 100-400 mg Cs/kg reached 687.5-2716.3 mg/kg and 336.9-1147.5 mg/kg, respectively. The TF value and BCF value of the plant under Cs pressure (100 or 400 mg/kg) reached 2.0 and over 6.0, respectively. It' estimated that 10.2 kg Cs/ha at Cs level of 100 mg/kg as well as 6.9 kg Cs/ha at Cs level of 400 mg/kg could be removed from the soil with one time of harvest (every three-month growth). Hg concentration in the plant was lower than detection limit. The above results showed that sweet sorghum was promising for phytoremediation on low-level Cs or Zn polluted soil.

**Acknowledgements:** The financial support by the National Natural Science Foundation of China (Grant No. 51378284;50830302; 50808111).

<figure><img src="image_162.png" /></figure>
# NATURAL PLANTS ON BORON (B) RESERVE AREAS IN TURKEY AS PHYTOREMEDIATION AGENTS OF B CONTAMINATED ENVIRONMENTS

Harun Böcük, Cengiz Türe

Biology Department, Science Faculty, Anadolu University, Eskişehir, Turkey

Corresponding author email: hbocuk@anadolu.edu.tr

## ABSTRACT

Boron (B) is one of the necessary micro nutrients for plant metabolism. However, the window between the amount which is necessary for growth and the amount which causes toxicity is very narrow and this spectrum varies among plant species. 4 (5) mg kg⁻¹ available B in soil for plants is generally accepted as toxicity border for most plants.

B excess is a common problem in the world and Turkey differs from all other countries with securing almost 70% of total B in the world. This situation is resulted in forming a special habitat in terms of soil-plant relationships due to high B concentrations at or near environments of the reserve areas.

In this research, therefore, we aimed to determine both (1) the most tolerant natural plant species to plant available B in soil and (2) their B accumulation potentials. To reach these aims, we studied in and around seven natural B reserve areas (tertiary deposits) located in four different provinces in Turkey. Soil and its corresponding plant samples were taken every 200 m from the centres of all of the B reserve areas to their environments (up to 2 km) along the transects (Belt Transect Method) on the main directions. "Minimum area method" was used in determining the size of sampling area. Plants species were identified and soil and plant samples were analysed chemically.

At the end of the study, it was determined that 11 natural plants distributed on the areas with high plant-available B concentrations over 10 mg kg⁻¹. Two of them were aquatic (*Typha latifolia* L. (Typhaceae), *Phragmites australis* (Cav.) Trin. ex Steudel (Poaceae)) and the others were terrestrial species (*Isatis glauca* Aucher ex Boiss. subsp. *glauca* (Brassicaceae), *Elymus elongatus* (Host) Runemark subsp. *turcicus* (McGuire) Melderis (Poaceae), *Glaucium corniculatum* (L.) Rud. subsp. *corniculatum* (Papaveraceae), *Alyssum sibiricum* Willd. (Brassicaceae), *Polygonum equisetiforme* Sibth. & Sm. (Polygonaceae), *Chenopodium album* L. subsp. *album* var. *album* (Chenopodiaceae), *Tamarix tetrandra* Pallas ex Bieb. emend. Willd. (Tamaricaceae), *Gypsophila perfoliata* L. var. *perfoliata*, *Puccinella distans* (Jacq.) Parl. subsp. *distans*. Moreover, it was also revealed that these natural species also accumulated B in quite high amounts. The highest B accumulation amount was determined in *P. distans* subsp. *distans* (270.24 ppm in leaf, 85.74 ppm in stem and 124.30 ppm in root).

As a result, most of the mentioned plant species are reported for the first time as B tolerant and accumulator with this research. It is thought that these species could serve as phytoremediation agents for both aquatic and terrestrial environments affected with B. So that, this phyto-technological application could help sustainable management of plant-available B concentration in natural environments.

**Acknowledgements:** The financial support by Anadolu University (BAP, No: 061010). ETİ Maden Works General Management and their personnel in four different provinces are in greatly appreciated.

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# STUDY OF ORGANIC AMENDMENTS USED IN PHYTOREMEDIATION AND ITS INTERACTIONS WITH METALS BY ¹³C CPMAS NMR SPECTROSCOPY

**Consuelo Escolástico**<fnref n="1" />, **Javier Pérez-Esteban**, **Ángeles García**<fnref n="1" />, **Ana Moliner**<fnref n="2" />, **Alberto Masaguer**<fnref n="2" />

*Corresponding author email: cescolastico@ccia.uned.es*

## ABSTRACT

The application of organic amendments in soils of urban areas (gardens, parks, etc.) and in other sectors such as agriculture is a common practice since they provide organic matter and nutrients and therefore help to improve fertility. However, it is important to analyze its chemical composition in order to determine effect produced on the mobility and bioavailability of contaminants in soils, because depending on the amendment can have contradictory effects. The objective of this work is to study the components of different organic amendments (commercial humic acids and sheep and horse manure compost) by NMR ¹³C CPMAS, since in both cases, it has been found that when are used in combination with different plant species such as *Vetiveria zizanioides* and *Atriplex halimus* for phytoremediation of soils contaminated with heavy metals, immobilization of the same occurs. Furthermore it has been determined by spectroscopic analysis of mixtures of amendments with different concentrations of metals that functional groups of these amendments are involved in the retention of metals.

*Acknowledgements:* This work was financed by the Spanish Ministry of Science and Innovation (Project CTM2009-13140-C02-01).

<figure><img src="image_164.png" /></figure>

<footnote n="1">¹Departamento de Química Orgánica y Bio-Orgánica. Facultad de Ciencias. Universidad Nacional de Educación a Distancia (UNED), Madrid, Spain</footnote>

<footnote n="2">²Departamento de Edafología. Escuela Técnica Superior de Ingenieros Agrónomos. Universidad Politécnica de Madrid (UPM), Madrid, Spain</footnote>
# BORON TOLERANCE IN *PUCCINELLIA FRIGIDA*: AN EXTREMELY BORON TOLERANT SPECIES

Consuelo Rámila<fnref n="1" />, Samuel Contreras<fnref n="2" />, Camila Di Domenico<fnref n="2" />, Gonzalo Pizarro<fnref n="1" />

Corresponding author email: cdramila@uc.cl

## ABSTRACT

Boron (B) is an essential element for plant growth, but is required only at trace concentrations due to its high toxicity. Many arid and semi arid regions suffer from B toxicity, limiting their agricultural activities. Current B removal technologies are either impractical or too expensive for arid regions; hence, much attention has been paid to using B tolerant plants for phytotechnologies during the last decades. Different kinds of phytotechnologies for B-polluted soils and water have been studied: evapotranspiration, phytoextraction, phytorestoration, and constructed wetlands. For any of these, new B-tolerant plant species must be studied in order to find suitable candidates for the different applications and sites.

We recently discovered a new B-tolerant species - *Puccinellia frigida* - that spontaneously colonizes and acts a B hyperaccumulator in an extremely B-contaminated site in Northern Chile (Rámila and Pizarro, 2013). The aim of this work was to start evaluating the potential of *P. frigida* for B phytoremediation purposes.

To investigate this, we studied the tolerance of *P. frigida* to B in hydroponic culture in a controlled greenhouse environment. Seeds were collected from the Colpitas Basin, Northern Chile, and germinated in filter papers. Seedlings were transferred to soil and, after one month, plants were transplanted to hydroponic culture and acclimated during several weeks. Plants were then treated with 10 different B concentrations for 5 weeks. Each treatment was done in triplicate, with four plants per pot per replicate. Finally, plants were harvested. Shoot and root biomass were determined, and chlorosis (visual symptom of B-toxicity) was visually estimated.

<figure><img src="image_165.png" /></figure>

We found that *P. frigida* tolerated more than 750 mg B/L without showing major symptoms of toxicity (32 % chlorosis). This concentration is extremely high, taking into account that concentrations over 3 mg B/L may be considered toxic for plants. Moreover, shoot growth was inhibited by 50% only at ~1500 mg/L, which exceeds the concentrations reported for other species with extreme B tolerance (Stiles et al., 2010). Plant biomass production was estimated in the range of 2 - 5 ton ha⁻<fnref n="1" /> year⁻<fnref n="1" />. As a conclusion, *P. frigida* is the (or at least one of the) most B-tolerant species reported so far. Hence, this plant could be used to phytorestore extremely B-polluted sites or evapotranspirate high B-waters to reduce B-leaching to adjacent areas. Analysis of the plant's tissue B concentration will reveal its applicability for phytoextraction purposes.

## References:

1. Stiles, A., Bautista, D., Atalay, E., Babaoğlu, M., and Terry, N. (2010) Mechanisms of boron tolerance and accumulation in plants: a physiological comparison of the extremely boron-tolerant plant species, *Puccinellia distans*, with the moderately boron-tolerant *Gypsophila arrostil*. *Environ. Sci. Technol.* 44: 7089-7095.

2. Rámila C. and Pizarro G. (2013) *Puccinellia frigida*, a native species from Andean wetlands, as an alternative for boron phytoremediation. In Proceedings of the 5th International Symposium on Wetland Pollutant Dynamics and Control, WETPOL 2013, Nantes, France.

**Acknowledgements:** The financial support by CONICYT-PCHA/ Doctorado Nacional/ 2010-21100787 scholarship is greatly appreciated.

<figure><img src="image_166.png" /></figure>

<footnote n="1">¹Dept of Hydraulic and Environmental Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile</footnote>

<footnote n="2">²Faculty of Agronomy and Forest Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile</footnote>
# MYCORRHIZATION OF BETULA CELTIBERICA INCREASES THE TOLERANCE TO GROW IN A HEAVY-METAL POLLUTED SOIL

Daniel Fernández-Fuego<fnref n="1" />,<fnref n="2" />, Elena Fernández-Miranda<fnref n="1" />,<fnref n="2" />, Abelardo Casares<fnref n="1" />,<fnref n="2" />, Ana Bertrand<fnref n="1" />,<fnref n="2" />, Aida González<fnref n="1" />,<fnref n="2" />

Corresponding author email: fernandezfuedaniel@uniovi.es

## ABSTRACT

In natural conditions, roots of many plant species, especially in the case of trees are associated with mycorrhizal fungi. The presence of mycorrhizae, increases root absorption area and water and important nutrients bioavailability. That symbiotic association seems to gain importance when plants grow in poor or metal-polluted soils, where mycorrhizal fungi can improve the survival and development rates of trees under stress conditions, although the effect of the mycorrhization on metal uptake seems to be fungus and plant specific.

The aim of our research was to evaluate the effect of mycorrhization of *Betula celtiberica* in order to consider its use in phytoremediation programs, studying its physiological responses when growing in a heavy-metal polluted soil and verifying whether or not plant-fungus association persists along culture time. Plants of a selected clone of *B. celtiberica*, whose Cd accumulation capacity exceeded the hyperaccumulation threshold in a previous *in vitro* screening (Fernández et al., 2008), were cultured in greenhouse in a peat-perlite mixture (3:1, v/v) (control plants) or in a polluted soil coming from a deserted nitrate factory located in Asturias, Spain, with a wide range of heavy metals including Zn, Hg, Cd, Pb, As and Fe. In addition, some of these plants were inoculated with an isolation of the ectomycorrhizal fungus *Paxillus ammoniavirescens* collected also in a metal-polluted soil. After 60 days, plant growth, photosynthetic efficiency, pigment content and metal accumulation were measured.

In a controlled *in vitro* mycorrhization, the fungus present is known. However, when it pass to field or greenhouse, it is possible that the fungus inoculated disappears or is displaced by other mycorrhizal fungi. In order to assess whether plant-fungus interaction persisted along culture time and to verify that the observed ectomycorrhizae were formed by *P. ammoniavirescens*, the species of fungus was determined analysing the sequence of Internal Transcribed Spacer (ITS) region of the ribosomal DNA amplified with the primers ITS1 and ITS4. The obtained sequence was compared with the one previously obtained from an isolation of *P. ammoniavirescens* confirming that plant-fungus interaction persisted along culture time.

Results showed that mycorrhized plants had a higher fresh weight and shoot length than non-mycorrhized ones. No differences in root length or number of leaves were found among treatments. Regarding photosynthetic pigments content, chlorophyll, carotenoids and anthocyanin levels were significantly higher in mycorrhized plants than in the non-mycorrhized ones when plants were cultured in polluted soil. However, no differences in photosynthetic efficiency were found among treatments. Mycorrhized plants accumulated fewer metals than non-mycorrhized both in roots and in shoots. This result may indicate that *P. ammoniavirescens* presence in rhizosphere could reduce metal bioavailability in soil or immobilize the metals in its mycelium preventing therefore its transport to host plants. This behavior leads to an increasing metal tolerance of *B. celtiberica* plants as it could be observed in the results obtained from the analysis of biomass and photosynthetic pigments content. In any case, in both treatments, *B. celtiberica* plants were able to bioaccumulate several times the amount of Zn, Cd and Mn available in soil.

**References:** Fernández et al. (2008). *Env. Poll.*, 152 (3), 522-529.

**Acknowledgements:** Project funding by CTM2011-29972. Fellowships: FICYT foundation.

<figure><img src="image_167.png" /></figure>

<footnote n="1">¹ Área de Fisiología Vegetal, Departamento de Biología de Organismos y Sistemas, University of Oviedo, Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain</footnote>

<footnote n="2">² Instituto Universitario de Biotecnología de Asturias</footnote>
# SEED GERMINATION OF PLANT SPECIES USEFUL TO RECLAMATION OF WASTES AFTER Zn-Pb ORES FLOTATION USING *IN VITRO* AND *IN VIVO* ASSAYS

Zbigniew Gajewski<fnref n="1" />, Ewa Muszyńska<fnref n="1" />, Ewa Hanus-Fajerska<fnref n="1" />, Tomasz Czech<fnref n="2" />

Corresponding author email: zbychor@poczta.fm; emuszynska@ogr.ur.krakow.pl

## ABSTRACT

The reconstruction of habitats strongly contaminated with heavy metals require taking up significant measures. Waste material obtained after zinc and lead ore flotation are ranked among particularly hard to introduce biological life. Application of native local floristic biodiversity is the fastest way to create compact vegetation cover, what sometimes offer the possibility of restraining the mobility of toxic ions in relatively short time.

The Olkusz district Zn-Pb ore-mining region located in southern Poland is known to be a habitat of plant species adapted to grow in such waste areas. Among others *Alyssum montanum* L. (Brassicaceae), *Biscutella laevigata* L. (Brassicaceae), *Dianthus carthusianorum* L. (Caryophyllaceae), *Gypsophila fastigiata* L. (Caryophyllaceae), *Scabiosa ochroleuca* L. (Dipsacaceae) spontaneously appear in areas contaminated with heavy metals. For above mentioned reasons fruit samples were collected from specimens representing calamine flora of the Olkusz Ore District in the autumn 2012. Seed samples were preserved at room temperature (22° C) and they were not subjected to any additional treatment. Representative samples of respective species were used to test the energy and ability of germination in the experiment which were set up in 2013. First of all, the energy and ability of germination were evaluated in the field condition on experimental plot established on the post-flotation waste heap (50°17"N, 19°30"E). Seeds were sown during the autumn on three types of substrates: (1) post-flotation material, (2) post-flotation material enriched with NPK fertilizers (3) post-flotation material mixed with municipal sewage sludge. Photoblastic seeds of *G. fastigiata* and *D. carthusianorum* were not covered by layer of substratum on the contrary to the others species seeds. At the field conditions the number of seedlings was started to be summed up after 28 days period. As additional control treatments, the seed germination in *vitro* using paper test (4) at room temperature (22° C), and (5) the outdoor temperature (28 - 0,3° C day/night) was carried out. In those two control treatments seedlings were counted 3, 7, 18 and 28 days after sowing.

At room conditions germination began in three days (except *S. ochroleuca*), and 18 days after sowing the germination level of every tested species was proved to be similar (above 80% of seeds germinated). The highest germination ability was ascertained for both *D. carthusianorum* (94.6 %) and *A. montanum* (98.66 %). Whereas in the outside conditions seeds started to germinate seven days after sowing. *G. fastigiata*, *S. ochroleuca* germinated as last two species. Consequently the number of *G. fastigiata* and *S. ochroleuca* seedlings were the lowest (36 % and 49.3%, respectively). On the contrary *Alyssum* and *Dianthus* quickly began the process of germination, so in a week period over 90% of seeds germinated, and 18 days after sowing this result increased up to 97.3 % for *Alyssum*, 98.6 % for *Dianthus*. Due to the high germination ability of, *Alyssum* and *Dianthus* calamine ecotypes, the most promising in vegetation cover creating are proposed exactly these two species. Whereas during this experiment the germination of *B. laevigata* seeds were poor, except *in vitro* test at room temperature.

The results of seed germination ability tested on the field plot with poor post-flotation material were similar to germination ability tested *in vitro* in case of *G. fastigiata* (41.3%) or significantly lower for *D. carthusianorum* (32 %). However, those results may be disturbed by intense flurry of wind. Uncovered seeds could be blown by the wind on neighboring plots or outside the experimental plots. Therefore, it should be underlined that for calculations only the seedlings growing in the respective plot were taken into account. In every treatment, germination of *A. montanum* was proved to be very high, and comparable to the paper test (88 %). Generally on "NPK plots" it was observed for most examined species significantly lower seed germination ability in comparison to all control treatments. It may be the effect of higher salinity of the substrate (additionally, some seedlings have been yellowish). The greater ability of seed germination was noticed on the plots with sewage sludge, probably due to better properties of the ground. Undoubtedly, the lowest germination ability was ascertained in the case of *Scabiosa ochroleuca*, and it amounted to 2.6% and 5.3% on the plots with NPK and sewage sludge, respectively.

Recapitulating, in terms of seed germination ability the most difficult plant material was *B.laevigata* while the best – *A. montanum*. Additionally easily germinated *D. carthusianorum* seeds can be helpful in the reconstruction of habitats degraded by centuries-old industrial activity.

<figure><img src="image_168.png" /></figure>

<footnote n="1">¹ Unit of Botany and Plant Physiology, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture, Krakow, Poland</footnote>

<footnote n="2">² Department of Agricultural and Environmental Chemistry, Faculty of Agriculture and Economics, University of Agriculture, Krakow, Poland</footnote>
# REMOVAL EFFICIENCY OF COPPER AND CHROMIUM BY AQUATIC MACROPHYTES *EICHHORNIA CRASSIPES* AND *PISTIA STRATIOTES*

**A.B. Tabinda**, R. Irfan, A. Yasar, G. Riaz

Sustainable Development Study Centre, GC University, Lahore, Pakistan

*Corresponding author email: amtulbaritabinda64@yahoo.com; Yasar.abdullah@gmail.com*

## ABSTRACT

Heavy metal contamination in water has become a serious issue in Pakistan and this problem requires environmentally safe technology like phytoremediation. Present study focussed on comparative phytoremediation potential of two aquatic plants *Eichhornia crassipes* and *Pistia stratiotes* for the removal of chromium (Cr) and copper (Cu) at their different concentrations (mg/l) i.e., 2 (T₁), 4(T₂), 6(T₃) and 8(T₄) and Control (T₀) for a period of 30 days. It was observed that during 30 days experiment, percentage removal of Cr by *Eichhornia crassipes* and *Pistia stratiotes* was 95 and 86% while the percentage removal of Cu was 99 and 97% respectively. The percentage removal of Cr by different parts (roots, shoots and leaves) of *Eichhornia crassipes* was 57, 25, 18% and 56, 28 and 16%. Whereas percentage removal of Cr by different parts (roots, shoots and leaves) of *Pistia stratiotes* was 51, 32, 17% and for Cu was 52, 32 and 16% respectively. Results showed significant removal (p<0.05) of chromium and copper by *Eichhornia crassipes* and *Pistia stratiotes*. Both the species are efficient for the removal of Cu as compared to Cr but roots are significantly (p<0.05) more efficient for removal of Cu and Cr as compared to shoots and leaves for both the species. Significant increase (p<0.05) in the plant biomass was observed for all the treatments after 30 days of experimentation but increase in biomass decreased with increase in both the metal concentrations for both the plants.

*Acknowledgements:* Department of Chemistry GC University Lahore Pakistan is greatly appreciated for providing equipment facilities.

<figure><img src="image_169.png" /></figure>
# EFFECT OF Pb AND Cu TOXICITY ON ANTIOXIDANT ACTIVITY AND PHOTOSYNTHESIS IN SEEDLINGS OF *CITRUS AURANTIUM* L.

Giannakoula A.<fnref n="1" />, Sarafi E.<fnref n="1" />, Therios I.<fnref n="1" />, Chatzissavvidis C.<fnref n="2" />

Corresponding author emails: agianna@cp.teithe.gr

## ABSTRACT

Heavy metal stress is a major environmental factor which adversely affects plant growth and development. However, low concentrations may ameliorate the toxic effects of metals and help to sustain development under stress. Rootstocks of fruit trees, commonly used to regulate growth, precocity, fertility, and yield, can also be used to solve problems associated with soils pollution such as copper and lead excess. Despite their potential use for phytoremediation, information on the physiological and biochemical aspects of heavy metal toxicity stress on fruit tree rootstocks remains limited. This study was conducted to determine the effects of varying concentrations of PbNO₃ and CuSO₄ on physiological operation, antioxidant activity and flavonoids content of *Citrus aurantium* L. subjected to heavy metal stress at different levels. The results showed that PbNO₃ and CuSO₄ addition significantly affected growth in a concentration-dependent manner; generally, low concentrations enhanced while high concentrations limited growth. The results also showed that, generally, low PbNO₃ and CuSO₄ concentrations significantly increased photosynthetic rate and water potential while it decreased flavonoids content and antioxidant activity. These results suggest that PbNO₃ and CuSO₄ influence many physiological parameters and especially for CuSO₄ the function of the photosynthetic apparatus and this is dependent on the metals concentration.

**Keywords:** phytoremediation, *Citrus aurantium* L., heavy metal toxicity, stress

**Introduction:** Copper (Cu) is considered as a micronutrient for plants and plays important role in CO₂ assimilation and ATP synthesis. Copper is also an essential component of various proteins like plastocyanin of photosynthetic system. Lead (Pb) is one of the most abundant toxic elements in the soil. High level of Pb also causes inhibition of enzyme activities, water imbalance and oxidative stress by increasing the production of ROS in plants [3]. It is evident that heavy metals as Cu and Pb modify flavonoid content in plants. Accumulated heavy metals in plants may interact with the photosynthetic machinery and resulted in a wide variety of toxic effects, including photooxidative damage. In particular, they alter the chloroplast membrane functions and components of the photosynthetic electron transport chain (ETC), thus impairing the light phase of photosynthesis [4]. It is known that heavy metals decrease photosynthetic rate, stomatal conductance, transpiration rate, plant growth and agricultural productivity [6]. The aim of this work was to determine the effects of the abiotic stress caused by lead and copper on photosynthetic apparatus and on the flavonoid levels in young citrus plants.

**Materials and Methods:** *Plant Material:* One year old *C. aurantium* plants were grown with different concentrations of Cu and Pb in a greenhouse in Thessaloniki, Greece, (40°34′ 35′′ N 22°57′19′′E), 2013.

*Determination of total antioxidant:* The ability of the leaves to act as hydrogen or electron donors in

the transformation of DPPH into its reduced form DPPH-H was investigated. Total antioxidant activity was determined following the method of Su *et al.* [2]. *Photosynthesis:* Photosynthetic parameters were monitored with a portable photosynthesis system (Li-Cor 6400, USA). The measurements were made on the third mature leaf in control and heavy metals treatments. Photosynthetic assimilation rate (A), stomatal conductance (gs),

<figure><img src="image_170.png" /><figcaption>Figure 1. Assimilation rate (A) and Transpiration rate (E) photosynthetic parameters as affected by heavy metals concentrations [µM] in the nutrient solution.</figcaption></figure>

<figure><img src="image_171.png" /></figure>

<footnote n="1">¹ Laboratory of Pomology, School of Agriculture, Aristotle University, 54124 Thessaloniki, Greece</footnote>

<footnote n="2">² Department of Agricultural Development Democritus University of Thrace, 68200 Orestiada, Greece</footnote>
intercellular CO₂ concentration (Ci) and transpiration rate (E) of leaves were measured using the youngest fully expanded leaves on a fine morning. *Determination of flavonoids:* The total flavonoid content was measured using a modified colorimetric method [7]. The flavonoid content was expressed as g rutin equivalents (CE) per 100 g of dry weight (dw).

**Results and Discussion:** *Effect of copper stress on photosynthesis:* In our study, at 800µM and 1000µM copper treatment, the photosynthetic assimilation rate (A), stomatal conductance (gs), intercellular CO₂ concentration (Ci) and transpiration rate (E) of leaves were significantly decreased compared to control. While at low concentration (200µM), copper promoted the increase of photosynthesis (A). Changes in CO₂ assimilation may be attributed to either stomatal or non-stomatal factors, or both. Photosynthetic assimilation rate (A) was significantly decreased at both 600µM and lead, accompanied by the decrease of Ci and gs suggested the stomatal factor was the main factor influencing CO₂ assimilation. However, when A and stomatal closure (E and gs decreased) significantly declined in 600 µM Pb compared to that at 150 µM and 300 µM Pb, Ci even showed increased, which implied that non-stomatal limitations to photosynthesis also occurred [1].

In conclusion, excess copper and lead inhibit photosynthesis and flavonoid content the inhibition of copper on the photosynthesis is not only by stomatal but also by non-stomatal factors. At low concentrations copper promoted photosynthesis. Pb treatment had also a stimulating effect on photosynthetic machinery, flavonoid content, antioxidant activity at low concentration and an inhibitory effect at higher concentration.

References:

1. Mysliwa-Kurdziel B, Strzałka K (2002) Influence of metals on biosynthesis of photosynthetic pigments. In: Prasad MNV. pp 201-228

2. Su M, Silva, J.L. (2006) *Food Chem.* **97**, 447–451.

3. Sytar O, Kumar A, Latowski D, Kuczynska P, Strzałka K, Prasad MNV (2013) *Acta Physiol Plant* **35**:985-999.

4. Ventrella A, Catucci L, Piletska E, Piletsky S, Agostiano A (2011) *Bioelectrochemistry* **77**:19–25.

5. Vesely T, Neuberg M, Trakal L, Szakova J, Tlustoa P (2011) *Wat. Air Soil Pollut* **223**:1847–1859.

6. Yusuf M, Fariduddin Q, Varshney P, Ahmad A (2012). *Environ Sci Pollut Res* **19**:8–18.

7. Zhishen J, Mengchen T, Jiamming W., 1999. *Food Chem*, **215**:655-660.
# PRELIMINARY STUDY FOR THE PHYTOREMEDIATION OF HIGHLY ACIDIC MINE TAILINGS: SELECTION OF AMENDMENTS

Tania Pardo, Clémence Bes, Mª Pilar Bernal, Rafael Clemente

Department of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, Murcia, Spain

Corresponding author email: rclemente@cebas.csic.es

## ABSTRACT

Tailings formed from mine spoils accumulation and storage are considered one of the principal sources of contamination associated to mining activities. They generally contain high amounts of trace elements (TEs) whose availability and dispersion (by wind and water erosion and lixiviation) suppose a serious threat to surrounding populations and ecosystems. This is the case of the mining district of La Unión-Cartagena (Murcia, SE Spain), where 89 abandoned tailings have been identified and which remediation is needed [1]. Phytostabilisation using native halophyte shrubs such as *Atriplex halimus* seems to be an efficient remediation technique [2], because of their tolerance to TEs, high canopy and deep root system, and their adaptation to the Mediterranean semi-arid climate. However, given the extreme conditions of mine tailings, the application of an adequate combination of amendments is required to reduce soil toxicity and improve soil fertility [2]. The aim of this work was to assess the viability of two inorganic materials (calcium carbonate and ViroBindTM reagent), alone or in combination with organic amendments, for their use in the phytoremediation of highly acidic and TEs-contaminated mine tailings from La Unión-Cartagena.

Two pot experiments were carried out using two spoil soils (D1 and D2) from mine tailings situated in an area known as "El Descargador", within the mining district of La Unión-Cartagena. Both soils are extremely acid (pH 3.3), have high concentrations of TEs (≈1100; 4700-6900; 5000-20000 mg kg⁻¹ of As, Pb and Zn) and elevated EC (2.9 and 9.0 dS m⁻¹). As amendments, four different formulations of a commercial reagent (ViroBindTM, mainly composed by Fe and Al oxides and Ca and Mg minerals) and its combination with a mature compost were applied in soil D1, while calcium carbonate and its mixtures with a digestate from an anaerobic digestion plant or with the previous compost were selected for soil D2. Four weeks after amendments addition, 0.3 g of A. *halimus* seeds were sown in each soil and pots were kept in a climate chamber during 5 and 3 months for soil D1 and D2, respectively. The evolution of the treatment effect on soil physicochemical properties and TEs availability in soil and soil solution (periodically extracted in *situ*), as well as plant growth and foliar TEs accumulation were studied.

The application of the amendments led to a significant pH increase in both soils, reaching values of 6-7.8 at the end of the experiments. Soluble and extractable concentrations of TEs were very high, especially in soil D2, but treatments successfully decreased their solubility as a consequence of the pH rise (>99% decrease of TEs dissolved in pore water). A. *halimus* was not able to grow in untreated soils, but all the treatments allowed its growth, reflecting a clear improvement of soil conditions. The supply of essential nutrients by the organic amendments (principally compost) was crucial for the increase observed in biomass production and nutritional status of the plants, and for reducing foliar TEs accumulation (Figure 1). Therefore, the use of A. *halimus* with the combined application of calcium carbonate or ViroBindTM and compost can be a good phytostabilisation strategy to remediate highly acidic TEs-contaminated mine tailings.

<figure><img src="image_172.png" /><figcaption>Figure 1. Principal components analysis resulting from the relation of soil and plant properties (soil D2).</figcaption></figure>

## References:

1. Robles-Arenas, V.M., Rodríguez, R., García, C., Manteca, J.I., Candela, L. (2006) *Environ Geol* 51, 47-64.

2. Pardo, T., Clemente, R., Epelde, L., Garbisu, C., Bernal, M.P. (2014) *J Hazard Mat* 268, 68-76.

**Acknowledgements:** This work was funded by the Fundación Séneca (Murcia, Spain) and the Spanish Ministerio de Ciencia e Innovación (MICINN) through the projects ref. 11785/PI/09 and CTM2010-21922-C02-01, respectively.

<figure><img src="image_173.png" /></figure>
# THE EFFECT OF ORGANIC AND MINERAL SUBSTANCES ON THE MOBILITY OF CADMIUM IN THE SOIL - PLANT SYSTEM

**Alžbeta Hegedúsová<fnref n="1" />, Alena Andrejiová<fnref n="1" />, Miroslav Šlosár<fnref n="1" />, Alena Vollmannová<fnref n="2" />, Janette Musilová<fnref n="2" />, Ondrej Hegedűs<fnref n="3" />, Tímea Pernyeszi<fnref n="4" />**

Corresponding author email: alzbeta.hegedusova@uniag.sk

## ABSTRACT

Current findings highlight the need for a comprehensive approach to address the contamination of soil and corp growth. The position of the Slovak Republic within Europe stipulates that this land is contaminated by long-term global transmission of large industrial and energy complexes. These impacts have a negative effect on soil with an increased to extremely high content of risk elements.

Ecological risks of accumulation of hazardous substances in the soil are reflected in the ability of soils to provide hygienic, harmlessly food. The danger lies mainly in the change of their mobility influenced by various factors. This is particularly true for heavy metals with highest biotoxicity.

South Slovakia is the most intense producer of vegetables where Cd is shown to be the most dangerous heavy metal. Its accumulation in vegetables is affected mainly by different soil conditions. Soil remediation is very expensive, but its impact to some extent can be reduced by applying sorbents based on organic and natural resources. The aim of our study was to analyze the influence of selected sorbents on the accumulation of cadmium in carrots.

Two sorbents were used in the experiment: the Humavit (organic humus fertilizer) and the Zeolite (sorbent of mineral origin). The study showed that increased Cd concentration in soil resulted in a non-linear Cd accumulation in carrot roots. The study found that application of the Humavit (5%) into soil contaminated by Cd (2 mg and 5 mg Cd.kg⁻<fnref n="1" /> soil, respectively) significantly decreased Cd content in carrot roots (approx. from 25% to 35%). In comparison, application of the Zeolite (5%) into soil contaminated with 5 mg Cd.kg⁻<fnref n="1" /> soil significantly decreased Cd content in carrot roots (about 28%). Application of lower rates of the Humavit (5%) and the Zeolite (5%) into soil contaminated by Cd did not significantly affect Cd content in carrot roots. The study confirmed that application of both sorbents into soil contaminated with 2 mg Cd.kg⁻<fnref n="1" /> soil significantly decreased Cd accumulation in edible parts of carrot (the values below hygienic limits – 0,1 mg.kg⁻<fnref n="1" />). Under conditions of higher concentration of Cd in soil (5 mg Cd.kg⁻<fnref n="1" /> soil), only application of the Zeolite (5%) into soil significantly reduced Cd content in carrot (below the hygienic limits). The study showed that carrot cultivation in highly Cd-contaminated soils is not recommended. However, application of the Humavit (5%) into soil lowly polluted with Cd establishes hygienic and safe conditions for cultivation of carrot.

Intensive and systematic research focused on specific ion-exchange properties of the Zeolite sources opens wide possibilities of the sorbents use in land protection.

**Acknowledgements:** The financial support by VEGA project No. 1/0456/12 is greatly appreciated.

<figure><img src="image_174.png" /></figure>

<footnote n="1">¹ Dept of Vegetables-Production, Faculty of Horticulture and Landscape Engineering, Slovak University of Agriculture in Nitra, Slovak Republic</footnote>

<footnote n="2">² Dept of Chemistry, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic</footnote>

<footnote n="3">³ Dept of Public Health University of SS Cyril and Methodius in Trnava, Slovak Republic</footnote>

<footnote n="4">⁴ Dept of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Hungary</footnote>
# TISSUE KINETICS OF METAL ACCUMULATION IN THE RECOVERY OF INDUSTRIAL WATER SLUDGE BY PLANTS

Juan Navarro-Aviñó<fnref n="1" />, J.J Fernández Navarro<fnref n="1" />, C. Cioranu<fnref n="2" />, Leonor Lapeña<fnref n="2" />, Pilar García-Agustín<fnref n="2" />

Corresponding author email: *jpnaupv@gmail.com*

## ABSTRACT

The overall pollution in the planet is reaching unbearable limits for humanity. Never before mankind had come to adopt measures agreed between countries in the magnitude of the current ones. Among the measures taken to alleviate the problem are increasing green alternatives. In this paper, it is shown how GMOs operate which have proven over the last decade to be by far, the best overall tool for combating pollution. A kinetic study of the accumulation of heavy metals (Ni, Cu, Zn, Cd, B, Pb, and Hg) in sludge from a treatment plant of industrial water is performed. The results confirm again with another specific case, the capacity and usefulness of this tool as a solution to the problems of global pollution.

Some of the plants used in this work are hyperaccumulators as demonstrated in mining soil containing concentrations of heavy metals close to 20000 ppm [1]. Since the capacity of a hyperaccumulator plant, could be non-existent, in amounts of industrial sludge containing only a few hundred ppm, this study aims to determine whether these plants are able to maintain their usefulness in industrial sludge. Sludge from a treatment plant for industrial water were used to demonstrate the ability to decontamination of species used. The initial data (t0) were taken at 3 weeks of planting, and end at 3 months (tf) From a practical point of view the most important goal was to determine whether GMOs are the best tools available in regard to wild genotype, and above all to know exactly, the ability of the tested genotypes, to decontaminate sludge. In this regard it is noteworthy that GMOs, consistent with previous trials, produced a greater accumulation in virtually all cases. Besides, both wt and GMOs multiplied the overall accumulation in plant at the end of the assay. In the other hand, assuming that a good way to determine the ability to remediate a contaminated environment, is to calculate the bioconcentration factor (BCF). This BCF, is greater than 1 in most cases, (Table 1). Finally, regardless of the initial concentration and specific metal, a greater total accumulation for GMOs is observed, compared to wt. Therefore, it can be concluded that there is no threshold in both. There is no threshold concentration, in the limits of concentrations tested in this assay, for the plant to activate the mechanisms of accumulation. There is also no threshold to activate the differences, between GMOs and wt.

<table><thead><tr><th>Table1</th><th>BFC</th><th>t0</th><th></th><th>tf</th><th></th></tr><tr><th>Metal</th><th>wt</th><th>GMO</th><th>wt</th><th>GMO</th><th></th></tr></thead><tbody><tr><td>Ni</td><td>0,09</td><td>0,09</td><td>0,53</td><td>0,65</td><td></td></tr><tr><td>Cu</td><td>1,03</td><td>0,77</td><td>2,57</td><td>3,22</td><td></td></tr><tr><td>Zn</td><td>2,97</td><td>2,52</td><td>12,90</td><td>14,99</td><td></td></tr><tr><td>Cd</td><td>0,26</td><td>0,34</td><td>0,56</td><td>0,74</td><td></td></tr><tr><td>Pb</td><td>0,03</td><td>0,05</td><td>0,60</td><td>1,13</td><td></td></tr><tr><td>Hg</td><td>0,61</td><td>2,22</td><td>0,31</td><td>1,37</td><td></td></tr></tbody></table>

## Reference:

1. M. Martínez, M.P. Bernal, C. Almela, D. Velez, P. García-Agustín, R. Serrano, J.P. Navarro-Aviñó (2006) An engineered plant that accumulates higher levels of heavy metals than *Thlaspi caerulescens*, with yields of 100 times more biomass in mine soils, Chemosphere, 64 (3), 478-485

<figure><img src="image_175.png" /></figure>

<footnote n="1">¹ ABBA Gaia, ¹Avenida del Sud, 4, 46918. Sueca, Valencia, Spain</footnote>

<footnote n="2">² Department of Experimental Sciences, Area of Vegetable Physiology, ESTCE, University Jaume I Post Code 12071, Castellón, Spain</footnote>
# GERMINATION OF COATED SEED FROM THREE GRASS SPECIES IN DIFFERENT MINE WASTES FOR PHYTOSTABILISATION

Irma Müller<fnref n="1" />, Pieter W. van Deventer<fnref n="2" />, Klaus Kellner<fnref n="1" />

Corresponding author email: 21635617@nwu.ac.za

## ABSTRACT

The adverse characteristics of mine waste materials, together with the loss of ecosystem functionality and stability caused by mining activities, create conditions undesirable for seed germination which can delay the success of phytoremediation and -stabilisation. Attempts to improve germination rates in the harsh environmental conditions associated with mine tailings include the enhancement of seeds. Advance Seed Company enhances seeds by the chemical application of water-soluble, lime-based coatings which are hypothesised to assist in the germination process, especially with regard to the facilitation of water movement and nutrient uptake.

The main objectives of this study were to compare the germination and root development of coated and uncoated seed types in different growth media (including mine tailings), and also to determine the water absorption of seeds relative to the coating. Three grass species native to South Africa (*Eragrostis curvula*, *Digitaria eriantha*, *Cynodon dactylon*) were selected for use in this study. Both seed types (coated and uncoated) were sown and replicated in eight different mine tailings and two control media. Seedlings were removed after six days and fixed for Scanning Electron Microscopy analysis to investigate root development. The hydration percentage of coated and uncoated seeds was also tested during germination by allowing for imbibition to occur; the water content was measured over a time-period of 50 hours.

The preliminary results of the germination experiments showed that the coated seed types had poor germination percentages compared to the uncoated equivalents for all selected species in the majority of the growth media, such as the coal, gold and gypsum mine wastes. The hydration results revealed higher water contents in the uncoated seeds of *Eragrostis curvula* and *Cynodon dactylon* compared to the coated seed types. Seedlings from the uncoated seeds of *Eragrostis curvula* displayed profound lateral root development in several of the growth media compared to seedlings from the coated seeds, while seedlings of *Digitaria eriantha* and *Cynodon dactylon* from both coated and uncoated seeds displayed lateral root development in several of the growth media.

Depending on the components used in the applied seed coating, a negative charge may exist on the coating which could retain water from the enclosed seed and consequently inhibit imbibition. This possibility is reflected in the higher water contents and improved germination results of the uncoated seed types when compared to the coated seed types of the three species; the coating may prevent water from reaching the embryo during germination. The ability of mine tailings to retain water was found to be a determining factor in water availability and consequently, the germination of seeds.

**Keywords:** phytoremediation, phytostabilisation, seed coating, germination, imbibition, mine tailings/waste

**Acknowledgements:** Financial support from Advance Seed (South Africa) is greatly appreciated.

<figure><img src="image_176.png" /></figure>

<footnote n="1">¹School of Biological Sciences, North-West University, Potchefstroom, South Africa</footnote>

<footnote n="2">²School of Geo- & Spatial Sciences / Soil Science, North-West University, Potchefstroom, South Africa</footnote>
# HEAVY METALS ACCUMULATION IN *CHLAMYDOMONAS REINHARDTII* AND *thalassiosira weissflogii* CELLS

Louiza Raisi, Maria Goniotaki, Nikos Lydakis-Simantiris

Laboratory of Environmental Chemistry and of Biochemical Processes, Department of Environmental and Natural Resources Engineering, Technological Education Institution of Crete, PO BOX 89, KDS Chania, 73135 Chania, Crete, Greece

Corresponding author email: lydakis@chania.teicrete.gr

## ABSTRACT

Photosynthetic algae play a crucial role on environmental sustainability, being in the basis of the nutrition chain [1]. Exposure of algae to environments polluted with heavy metals results the accumulation of these toxic elements in them. Accumulation occurs via both adsorption of the metals on the cell walls and insertion into the cells. Examination of the accumulation of the heavy metals in the algae is important both for the information it gives about the condition of the ecosystems, and for the potential of exploitation of these organisms for the remediation of natural waterbodies, or as biofilters with a wide range of applications [2,3,4].

In this study, Cd, Cr, and Pb accumulation in two unicellular algae was investigated. One freshwater microalga (*Chlamydomonas reinhardtii*) and one marine microalga (*Thalassiosira weissflogii*) were cultivated in media polluted with several concentrations of the abovementioned heavy metals, up to lethality levels. Tolerance of these organisms to the heavy metals under examination was examined and the growth curves for the different pollution levels were constructed. The percentage of the adsorbed vs. the total accumulated metal for each experimental condition was estimated by atomic absorption spectrometry, and the biochemical impact of the inserted metals in the cells was examined.

Cells, grown in both unpolluted and polluted media were lysed and differential centrifugation was applied on lysates in order to isolate the different cellular fractions and organelles. Heave metal-induced proteins were searched by SDS PAGE Electrophoresis.

**Keywords:** *Chlamydomonas reinhardtii*, *Thalassiosira weissflogii*, heavy metals, cadmium, chromium, lead, heavy metal-induced proteins, bioindicators

## References:

1. R. Leon, E. Fernandez, (2007) Ch. 1, Transgenic Microalgae as Green Cell Factories. Landes Biosciences and Springer Science & Business Media.

2. Q. Zhou, J. Zhang, J. Fu, J. Shi, G. Jiang, *Analytica Chimica Acta*, (2006) 606, 135-150.

3. R.B. Nessim, A.R. Bassiouny, H.R. Zaki, M.N. Moawad, K.M. Kandeel, *Chemistry and Ecology*, (2014) 27, 579-594.

4. S. Rajamani, S. Siripornadulsil, V. Falcao, M. Torres, P. Colepicolo, R. Sayre, (2007) Ch. 9, Transgenic Microalgae as Green Cell Factories. Landes Biosciences and Springer Science & Business Media.

**Acknowledgements:** This research is implemented through the Operational Program "Education and Lifelong Learning" and is cofinanced by the European Union (European Social Fund) and Greek national funds.

<figure><img src="image_177.png" /></figure>
# STRATEGIES FOR ENHANCING THE PHYTOREMEDIATION OF HEAVY METALS-CONTAMINATED INDUSTRIAL SOILS BY NATIVE SPECIES OF PRINCIPADO DE ASTURIAS (SPAIN)

Ana Bertrand<fnref n="1" />,<fnref n="3" />, Daniel Fernández<fnref n="1" />,<fnref n="3" />, Javier Alfaro<fnref n="1" />, Alejandro Navazas<fnref n="1" />, Victoria Mesa<fnref n="2" />, Anabel Peláez<fnref n="2" />,<fnref n="3" />, Aida González<fnref n="1" />,<fnref n="3" />

Corresponding author email: aidag@uniovi.es

## ABSTRACT

Phytoremediation of heavy-metal polluted soils resulting from industrial and mining activities is a useful technology to solve this important environmental problem. The selection of plants that will be used in phytoremediation is one of the most important factors to consider in order to achieve successful results. The use of species adapted to these soils can improve decontamination programs and in the case of trees increase the effectiveness of metal uptake by plants due to the deeper root system and greater biomass. Furthermore, the use of symbiotic mycorrhizal fungus increases the survival and development rates of trees, although the effect on metal uptake seems to be fungus and plant specific. On the other way, interactions plant-bacteria have strong impact in metal phytoextraction. Bacteria produce substances (siderophores, plant growth hormones, ACC deaminase) that improve the growth and increase the plant biomass. Besides, bacteria capable of tolerating high concentration of metals can affect plant metal uptake by altering the mobility and bioavailability through soil pH, release of chelators and/or oxidation/reduction reactions.

The objective of this study was to investigate the phytoremediation potential of different plant species (Dittrichia viscosa, Melilotus alba, Eupatorium cannabinum, S. atrocinerea, Salix caprea and Betula celtiberica) using different strategies for enhancing phytoremediation and to identify the effectiveness of these strategies for its application in decontamination of heavy metal-polluted soils "in situ" on a field scale. The plants used were clones which showed high metal accumulation capacity, exceeding the hyperaccumulation threshold in a previous *in vitro* screening. Plantlets were micropropagated by *in vitro* culture, acclimated in a greenhouse and transferred to soil. *Salix atrocinerea, S. caprea* and *B. celtiberica* plants were also mycorrhized by an isolated ectomycorrhizal fungus *Paxilus ammoniavirescens* collected in a metal-polluted site. The experimental site was a 600 m2 field located within the industrial zone of Asturias (Spain) which presented high concentration of As, Cd, Cu, Fe, Hg, Pb and Zn. The field soil was ploughed to homogeneity prior to experimentation and was divided in plots, each of 2 m2 size with at least 2 m between plots. Three different strategies were assayed: planting density, application of fertilizer and mycorrhization effect.

Results showed that planting density and fertilization had a significant influence on plant growth and therefore on total biomass obtained. The mycorrhization of *B. celtiberica* increased the length and diameter of shoots and Zn accumulation in leaves reached hyperaccumulation levels in both mycorrhized and non-mycorrhized plants. The highest levels of Pb and As accumulation were obtained in mycorrhized *B. celtiberica*. The extraction efficiency of *B. celtiberica* is even higher than that of other accumulator species as *D. viscosa*. To analyze interactions plant-bacteria, we isolated arsenic and other heavy metals resistant bacteria from inside the roots (endophytic bacteria) and the rhizosphere of *Betula* and *Salix*. The effects of these bacteria in *B. celtiberica* plantlets were assayed "in vitro" individually and in the form of a three-members consortia, in the presence of 300 µM sodium arsenate. The results showed a positive effect on the metal accumulation in the above ground parts of the plants, although the shoot and root length and number of leaves decreased. To check the penetration of added bacteria in the plant, sections of roots (10 µm) were stained with vital dyes (LIVE/DEAD® Viability Assays) and their presence confirmed by "in situ" hybridization (FISH) in a Confocal Laser Scanning Microscope.

In conclusion, the results from this field study show that *D. viscosa* and mycorrhized *B. celtiberica* have an interesting potential application for phytoextraction of heavy metals from contaminated soils.

**Acknowledgements:** The financial support by UE-12-IDARTS-LIFE-11-000547 project is greatly appreciated.

<figure><img src="image_178.png" /></figure>

<footnote n="1">¹ Área de Fisiología Vegetal, Dpto. de Biología de Organismos y Sistemas, University of Oviedo, Oviedo, Spain</footnote>

<footnote n="2">² Área de Microbiología, Dpto. de Biología Funcional, Universidad de Oviedo, Mieres, Spain</footnote>

<footnote n="3">³ Instituto Universitario de Biotecnología de Asturias</footnote>
# PRELIMINARY ANALYSIS FOR A MINING WASTE PHYTOSTABILIZATION PROJECT WITH NATIVE PLANTS IN SW-SARDINIA (ITALY)

Mauro Casti, Mario Cabriolu, Roberto Enne

IGEA S.p.A., Iglesias, Italy

Corresponding author email: maurocasti@hotmail.com

## ABSTRACT

SW-Sardinia (Italy) had been a very important metalliferous mining region through the centuries since the Phoenician-Punic age. The main extracted metals were, among others, zinc, lead, silver and copper. Exploitation closed down at the end of the XX century, leaving hundreds of waste heaps spread across the whole region. The investigated mine area, called San Giorgio, is located in the municipality of Iglesias and it is characterized by several dumps, mainly composed of fine-grained materials derived from metallurgical treatments, leaved at the end of the last century. Because of the high levels of toxic elements, the colonization by plants is slow and the main surface of the waste is still bare. The risk assessment, carried out on the basis of the Italian regulation, indicated that the restoration actions are compulsory.

With the aim to planning the site reclamation using the phytostabilization technology, pioneer plants and soils were analysed in 6 waste deposits in part colonised by various plant communities. The research was focused on 7 garrigue communities, respectively dominated by: *Euphorbia pithyusa* ssp. *cupanii*, *Cistus salviifolius*, *Cistus monspeliensis*, *Limonium merxmuelleri*, *Helichrysum microphyllum* ssp. *tyrrhenicum*, *Dittrichia viscosa* and *Genista corsica*. 15 uniform vegetation patches were examined in the studied dumps, furthermore the soil of the bare areas and the soil of the non-contaminated areas was sampled. In every patch, the soil and the dominant plant species were sampled in three different point. The plant communities have been analysed by the phytosociological method. In order to investigate the soil properties, in every recorded site, the soils taken in correspondence of the plant rhizospheres were analysed to determine: skeleton, texture, pH, conductivity, CEC, exchange complex, C, N, Fe, organic matter (SOM), total content of Cd, Cr, Hg, Pb and Zn and bioavailable fraction of Cd, Pb and Zn.

Analytical results show that most of the bare areas have pH soil of about 7 and SOM <10 g/kg. The rhizosphere soils have, in most of the sampled points, basic pH and SOM from 10 up to 90 g/kg.

Among the investigate species, *E. cupanii* forms the most pioneer garrigues, on very poor soils, comparable to those of the bare areas. On the contrary, the soils with the highest content of SOM are colonized by *G. corsica*, *C. monspeliensis*, *C. salviifolius* or *H. tyrrhenicum* communities. Among these, *Cistus* species seem to prefer clay soils, *G. corsica* grow in soils from clay to loam and *H. tyrrhenicum* can develop also on sandy-loam soils. Between the two *Cistus* species, *C. monspeliensis* is able to grow on soils very skeleton-rich, if compared with *C. salviifolius*. If considered for their tolerance to the heavy metals, the most of the studied species are wide spread in soils with more than 6.000 mg Pb/kg and 1.000 mg Zn/kg as total content. However, *L. merxmuelleri* and *H. tyrrhenicum* show a higher tolerance to Cd (>1000 mg/kg) and Zn (up to 60.000 mg/kg). On the other hand, *L. merxmuelleri* is the only specie not found on soils with more than 40 mg Cr/kg.

The analysis of plant communities allow to observe that the development of the garrigues dominated by deciduous shrubs cause the formation of a leaf litter and promote the growth of annual herbs. However, therophytes are absent on the soils not yet colonised by chamaephytic or nanophanerophytic species. The development of the garrigues or low maquis is not strictly related with the decreasing of toxic metals in soil, however it is possible observe the increasing of the contents of N and SOM at the same time as the plant cover evolves. The pioneer dwarf shrub contribute to create a continuous herbaceous layer that probably help to reduce the transport of the metals in the environment, reducing the risk for the human health. More investigations will be necessary to improve the present state of knowledge and verify how it would be possible carry out a restoration plan based on phytostabilization using native plants. Among the most metal tolerant species, those having low translocation factor could be selected (e.g. *C. salviifolius*).

**Acknowledgements:** This study was supported by Regione Autonoma della Sardegna: PO Sardegna FSE 2007-2013, L.R.7/2007 "Promozione della ricerca scientifica e dell'innovazione tecnologica in Sardegna". The authors are very grateful to all staff of the chemical laboratory of IGEA.

<figure><img src="image_179.png" /></figure>
# PHYTOEXTRACTION POTENTIAL OF FOUR BLACK LOCUST HALF-SIB FAMILIES IN THE PRESENCE OF HEAVY METALS IN NUTRIENT SOLUTION

Milan Župunski<fnref n="1" />, Milan Borišev<fnref n="1" />, Nataša Nikolić<fnref n="1" />, Saša Orlović<fnref n="2" />, Danijela Arsenov<fnref n="1" />, Marko Kebert<fnref n="2" />, Andrej Pilipović<fnref n="2" />, Slobodanka Pajević<fnref n="1" />

Corresponding author email: [milan.zupunski@dbe.uns.ac.rs](mailto:milan.zupunski@dbe.uns.ac.rs)

## ABSTRACT

Phytoremediation of toxic metals has been widely studied during past decades. The possibility for uptaking and accumulating these non-essential elements was examined with different plant species, both perennial and woody tree species. Black locust is characterized as high biomass productive species, adaptive to different types of soils and climates. In relation with that, it represents a good model plant for phytoextraction tests with heavy metals. Present work examines phytoextraction potential of four black locust half-sibs (families 54, 56, 115 and 135), grown hydroponically under semi-controled conditions in greenhouse. Plants were treated separately with 6 ppm of cadmium (Cd), 100 ppm of nickel (Ni) and 40 ppm of lead (Pb) added in Hoagland nutrient solution, accompanying with mixed treatment of all three elements. Presence of Cd, Ni and Pb was observed in all investigated treatments in tissues of the root, stem and leaves of four black locust half-sib families. High accumulation rates of nickel and cadmium were determined in all four half-sib families. Higher amounts of lead were accumulated at individual treatment, compared with mixed treatment, where lead accumulation was potentially inhibited with presence of nickel and cadmium in nutrient solution. A significant decrease in morphological and physiological parameters was present at all treatments in relation to control. Decrease in photosynthetic activity, transpiration rates and water use efficiency at nickel, cadmium and mixed treatments could be the consequence of higher accumulation rates of these two elements in plant tissue. Chlorosis, necrosis and stunted growth of the youngest leaves were visible at both nickel and mixed treatment, accompanying with decrease in chlorophyll pigment contents. Differences in terms of visible symptoms like chlorosis and necrosis were not noticeable among four analysed half-sib families.

**Keywords:** black locust, heavy metals, accumulation, photosynthesis, transpiration

**Acknowledgement:** This research was conducted as a part of the project "Investigating the climate changes and their impact to environment: tracking impact, adaptation and reduction" (43007) financed by the Ministry of Education and Science of the Republic of Serbia within the framework of integrated and interdisciplinary research for the period 2011-2014.

<figure><img src="image_180.png" /></figure>

<footnote n="1">¹University of Novi Sad, Faculty of Sciences, Department for Biology and Ecology, Serbia</footnote>

<footnote n="2">²University of Novi Sad, Institute of Lowland Forestry and Environment, Novi Sad, Serbia</footnote>
# PHYTOTOXICITY OF SODIUM FLUORIDE AND UPTAKE OF FLUORIDE IN WILLOW TREES

Lauge Peter Westergaard Clausen<fnref n="1" />, Ulrich Gosewinkel Karlson<fnref n="2" />, Stefan Trapp<fnref n="1" />

Corresponding author email: lpwc@env.dtu.dk

## ABSTRACT

The willow tree (*Salix viminalis*) toxicity test and a cress seed germination test (*Lepidium sativum*) were used to determine uptake and phytotoxicity of NaF. Concentrations in hydroponic solutions were 0-1000 mg F/L and 0-400 mg F/L in the preliminary and definitive test. A third test was done with soils collected from a fluoride-contaminated site at Fredericia, Denmark. The EC₁₀, EC₂₀ and EC₅₀-values for inhibition of transpiration were determined to 38.0, 59.6 and 128.7 mg F/L, respectively. The toxicity test with soil showed strong inhibition for the sample with the highest fluoride concentration (405 mg free F per kg soil, 75 mg F per L soil solution). The seed germination and root elongation test with cress gave EC₁₀, EC₂₀ and EC₅₀-values of 61.4, 105.0 and 262.8 mg F/L, respectively. At low external concentrations, fluoride was taken up more slowly than water and at high external concentrations at the same velocity. This indicates that an efflux pump becomes overloaded at concentrations above 210 mg F/L. Uptake kinetics were simulated with a non-linear mathematical model, and the *Michaelis-Menten* parameters were determined to half-saturation constant K_M near 2 g F/L and maximum enzymatic removal rate v_max at 9 g/(kg d).

<figure><img src="image_181.png" /></figure>

<footnote n="1">¹Department of Environmental Engineering, Technical University of Denmark, Kgs Lyngby, Denmark</footnote>

<footnote n="2">²Department of Environmental Science, Aarhus University, Roskilde, Denmark</footnote>
# BORON, A HIDDEN TOXIN IN FLUE GAS DESULFURIZATION WATER

Joni Mengarelli<fnref n="1" />, M. B. Gulkaduwa<fnref n="2" />, L. C. Davis<fnref n="1" />, G. Hettiarachchi<fnref n="2" />

Corresponding author email: ldavis@ksu.edu

## ABSTRACT

Coal fired power plants commonly produce a complex mix called flue gas desulfurization (FGD) water containing precipitated sulphur from the flue gases. Usually ground limestone slurry is reacted with the hot gases under oxidizing conditions to precipitate calcium sulphate. The precipitate may have utility in production of gypsum board or for soil amendment, but the remainder is usually treated as wastewater. Such FGD water is poorly defined because it depends on the source of coal, conditions of combustion, and limestone source used for the precipitation. After removal of the gypsum, FGD water is typically high in calcium, magnesium, chloride, sodium, and sulphate. Often it is supersaturated with calcium sulphate. Some elements, including selenium, arsenic, mercury, fluoride and boron are present in much lower concentrations, but at levels exceeding standards for surface water to be used for drinking, or irrigation, or for aquatic life in streams, lakes or rivers. Reported boron levels vary widely (up to >80 mg/L). This exceeds general irrigation limits (~5 mg/L as B) many-fold. FAO data indicate damage occurs at levels of 5 mg/L in soil water for most crops. High B levels are also inhibitory of growth of aquatic plants, including algae, with reported effects at concentrations below 5 mg/L for some species.

Methods for removing mercury to very low levels are in use. Selenium and arsenic can be precipitated under reducing conditions as selenide and arsenide. Simple anions (e.g. F⁻) and cations are removable by ion exchange techniques if necessary. Boron, present as borate, has very low affinity for ion exchangers. It can be sorbed by vicinal hydroxyls of some sugars, but that is a very expensive process.

Tests were conducted with a local source of FGD water to determine its toxicity to hydroponically grown sunflowers, a moderately tolerant crop. Hoagland's solution and simulated FGD water (Hoagland's solution supplemented with sodium chloride and magnesium sulphate) were compared to FGD water. Water collected at the outflow of a constructed wetland used for Se removal, and lake water from the same facility, were also examined. Essential nutrients were added to all solutions to approximate an optimal hydroponic culture solution. Two week old sunflower seedlings of matched weights were used in three replications, with their water use followed for 14 days. While the added salt, approximating that of FGD water, definitely was inhibitory of sunflower water use (>30 %) compared to Hoagland's solution, there was little decrease (~10 %) in net dry matter accumulation. In contrast, inhibition by FGD water was ~40 % for dry matter production, with > 50 % decrease of water uptake. Increasing levels of boron caused further growth inhibition, whether supplied in FGD water, wetland outlet water or lake water. A 20 mg/L level of B gave ~40 % inhibition of growth in the lake and wetland outlet waters. Inhibitory effects of added B were less obvious in the FGD water where growth was ~40 % less than Hoagland's solution, even without added boron.

These results have major implications for disposal strategies used for FGD water. Boron levels in receiving water bodies must be kept below about 5 mg/L of B to avoid significant inhibition of plant growth when that water is used for irrigation, or discharged to zones where aquatic plants grow.

**Acknowledgments:** Burns and McDonnell's Kansas City Office is thanked for discussions and financial support. Supported in part by the Kansas Agricultural Experiment Station

<figure><img src="image_182.png" /></figure>

<footnote n="1">¹Dept. of Biochemistry and Molecular Biology, Kansas State University, Manhattan, KS, USA</footnote>

<footnote n="2">²Dept. of Agronomy, Kansas State University, Manhattan, KS, USA</footnote>
# METAL CONTENT IN SOIL AS A FUNCTION OF DISTANCE FROM SOURCE OF EMISSION, WIND DIRECTION AND METAL SPECIFIC GRAVITY

Mariola Wrochna, Robert Popek, Helena Gawrońska, Stanisław W. Gawroński

Laboratory of Basic Research in Horticulture, Department of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences, Poland

Corresponding author email: stanislaw_gawronski@sggw.pl

## ABSTRACT

Transportation is, apart from industry, one of major sources of environmental pollution. Elevated levels of various contaminants including metals, are recorded in soil in the vicinity of roads with intensive traffic. Metals, which origin mainly from car exhaust, lubricants, tire abrasion and broke pads, accumulate in soils neighboring the roads edge in considerable amounts. Polluted road dust can be easily blown out by wind for further distances causing metal deposition on soils and this would depends on wind direction. Accumulation of heavy metals in urban soils becomes a source of serious hazard to environment and human health. Although literature on the accumulation of metals as a consequence of increasing car traffic is reach data on effect of distance from source of emission is quite limited and often controversial.

The aim of this study was to assess the impact of: (i) the distance from the roadway, a (ii) the most frequent wind direction on the accumulation and (iii) specific gravity of selected metals content in the soil nearby road of intensive traffic.

Study area was the strip along the Przyczółkowa Street (Warsaw, Poland) characterized by intensive car traffic. Samples of soil were collected at the depth of 15 cm from windward and leeward sides of the road from ten distances starting from the road edge (the source of emission) up to 128 m. Collected soil samples were dried, triturated in ceramic mortar and sieved at 0.75 mm sieve. Then sub-samples were placed in certified XRF cups (10 cm³) and were placed in the measuring window of the XRF spectrometer Alpha 4000 for content of 28 selected metals determination. The analysis of each sample was set at measurement lasting 420 second. Active acidity (pH) and electrical conductivity (EC) of every soil samples were also measured. Analysis was performed in three replications for every point of collected samples.

Content of metals in most cases was significantly higher in samples collected from windward than from the leeward sites of the road. At the close distances from road edge out of the 28 measured metals content of four (Pb, Cr, Ba and Ti) were exceeded the background soil values for central Poland. Moreover, content of Pb and Ba exceeded permissible levels for agriculturally used land but were far below the level permitted for industrial use. The distance at which the metals at higher levels were recorded depends on specific gravity being greater for lighter metals i.e. Ti and Ba were recorded in higher amount at further distances (up to 128m) than Pb and Cr (16 m).

**Acknowledgements:** The work was financially supported by Warsaw Plant Health Initiative of REGPOT in the frame of 7FP EU # 286093.

<figure><img src="image_183.png" /></figure>
MATHEMATICAL MODELLING OF RHENIUM PHYTOEXTRACTION WITH INDIAN MUSTARD

Viviani C. Onishi<fnref n="1" />, Luís González<fnref n="2" />, Claudio F. Mahler<fnref n="3" />, Luís A. B. Novo<fnref n="3" />

Corresponding author email: novo@coc.ufrj.br

ABSTRACT

Rhenium (Re) is one of the scarcest elements on Earth's crust (7 x 10⁻⁸%). Due to its rarity, Re is also one of the most expensive metals and an interesting target for phytomining. This work introduces a new mathematical model for Rhenium (Re) phytoextraction with Indian mustard [Brassica juncea (L.) Czern], a species recurrently used in phytomining studies involving other valuable metals such as gold and silver.

Firstly, a theoretical model is proposed to explain the metal accumulation process in the plant. The theoretical model formulation is based on a simplified mathematical model for complex dynamic systems, by assuming several parameters to describe the uptake mechanism of Re and its effect on plant growth. The parameters were set into simpler groups in order to facilitate validation. In addition, the model relates the concentration of Re in the soil and the concentrations of Re in the root and shoot of the plant. The resulting theoretical model is calibrated and tested using two sets of data (two different harvesting periods, 50 and 80 days), considering five concentrations (5, 10, 20, 40 and 80 mg kg⁻<fnref n="1" />) of Re in the substrate. The model accurately describes the experimental results, demonstrating a non-linear relationship between metal concentrations in the soil, root and shoot and the plant biomass yield.

*Acknowledgements:* The authors gratefully acknowledge financial support from the National Council for Scientific and Technological Development of Brazil (CNPq) under processes No. 150084/2014-5 and 151132/2014-3.

<figure><img src="image_184.png" /></figure>

<footnote n="1">¹Department of Chemical Engineering, State University of Maringá, Brazil</footnote>

<footnote n="2">²Department of Plant Biology and Soil Science, University of Vigo, Spain</footnote>

<footnote n="3">³Department of Civil Engineering, COPPE, Federal University of Rio de Janeiro, Brazil</footnote>
# THE EFFECTIVENESS OF USING RADISH (RAPHANUS SATIVUS L.) FOR PHYTOREMEDIATION OF INCREASED LEVELS OF LEAD-CONTAMINATION IN SOIL

Ait Hamadouche N., Aoumeur H., Aoues Aek

University Es-senia, Department of Biology, Laboratory of Experimental Biotoxicology, Biodepollution and Phytoremediation, Oran, Algeria

Corresponding author email: naithamadouche@live.fr

## ABSTRACT

Phytoremediation is an emerging technology that employs the use of higher plants for the clean up contaminated environment. Phytoextraction, the use of plants to extract toxic metals from contaminated soils, has emerged as a cost-effective, environment-friendly clean up alternative. Pot culture experiments using radish (Raphanus sativus L.) was performed to investigate lead (Pb) phytotoxic effects on antioxidant enzymes and other early warning biomarkers of soil Pb exposure. The study included an assessment of heavy metal accumulation in root, shoot and leaf, effect of lead stress on growth parameter (root length, root and shoot dry weight), photosynthetic pigment content, bioaccumulation coefficient (BAC) and the activity of anti-oxidant enzymes. Results demonstrated that efficient Pb uptake was observed by the roots in contaminated plants. Root growth was higher in control plants, as compared to the contaminated. Lead exposure also influenced biochemical and physiological parameters.

Administration of excess of lead was followed by an increase of Pb accumulation in leaves, and associated symptoms of toxicity. Typical symptoms of Pb toxicity developed 30 days after the beginning of treatment. Chlorophyll concentration was decreased in response to heavy metal toxicity. Activity of anti-oxidative enzymes e.g. peroxidase and catalase were increased in response to oxidative stress. Atomic absorption spectrophotometer (AAS) was used for analysis of heavy metal in soil and plant samples. The results of this research showed that radish are hyperaccumulator plants that can concentrate heavy metals in their different parts, thus they can be used for remediation of polluted area. Study also showed that potential of metal accumulator plants for extraction of metal from soil occur up to a certain level of concentration, after that when the concentration of metal increased the phytoextraction rate of metal or bioaccumulation coefficient (BAC) were decreased.

**Keywords:** oxidative stress, peroxidase, phytoremediation, radish

<figure><img src="image_185.png" /></figure>
# ELECTROPHORETIC PROTEIN PROFILING OF MACROPHYTES EXPOSED TO CHROMATE STRESS

Paweł Kaszycki<fnref n="1" />, Aleksandra Dubicka-Lisowska<fnref n="1" />, Barbara Piwowarczyk<fnref n="2" />, Joanna Augustynowicz<fnref n="2" />, Ewa Hanus-Fajerska<fnref n="2" />

Corresponding author email: p.kaszycki@ogr.ur.krakow.pl

## ABSTRACT

Macrophytes can serve as good models for the study of heavy metal interactions with assimilative plant organs. They are also preferable candidates for efficient phytoremediation of heavy metal contamination in aqueous environments. Anthropogenic pollution with chromium has become a worldwide problem due to the wide use of this metal in a number of industrial applications. Cr may appear at various oxidation states forming chemical agents of different toxicity, solubility and stability, and among these the Cr(VI) compounds are the most hazardous to living organisms. Chromate can easily enter cells *via* anion transporters and then it interacts with cell constituents as a strong oxidant. It undergoes reduction to the final Cr(III) form due to both biotic (enzymatic, non-enzymatic) and abiotic reactions. Cr(VI) and many of the intermediates that appear upon chromate metabolism are thus agents causing severe physiological stress.

The aim of the work was to reveal whether the incubation with chromate caused any changes in protein profiles of three common macrophytes which might be used as Cr efficient phytoremediators. Among the studied plants were: water-submersed macrophytes *Callitriche cophocarpa* and *Elodea canadensis* as well as the free-floating *Spirodela polyrhiza*. The source of the Cr(VI) anions was potassium chromate (K₂CrO₄). The plants were incubated for 24 h with chromate levels evaluated experimentally to be sublethal (1, 1, and 0.5 mM, respectively), that is at conditions causing initial symptoms of growth inhibition and chlorosis, and thereafter enabling full recovery within several days after washing out the Cr(VI) solution and further cultivating in an MS medium (i.e. at conditions same as for the untreated control plants). Protein extracts were obtained after mechanically homogenizing the plant material in a mortar with liquid nitrogen. Then, an optimized technique of protein precipitation with acetone and phenol was elaborated to eliminate all the non-protein impurities that severely interfered with the electrophoretic analyses.

The Figure shows exemplary results obtained with the SDS-PAGE electrophoresis. As it can be seen chromate supplementation did not result in changes of proteomic profiles for *C. cophocarpa* (the path marked “CCr”) and *S. polyrhiza* (“SCr”). *E. canadensis* (“ECr”), in turn, revealed only two protein bands induced by Cr(VI) treatment (M<sub>w</sub> of approx. 43 and 48 kDa). Paths “ST” represent molecular weight standards and “C” – electrophoregrams of the respective control experiments.

Abiotic stress has been shown to alter proteomic patterns in many organisms. Our reference studies involving bacterial and yeast proteomes further proved that the Cr-induced stress resulted in protein profile changes where both suppressed and induced proteins as well as down- and up-regulated ones were revealed. So far, little or none has been done with regard to macrophytes with the aim to study their response to chromate at the level of detectable proteomes. Such research is of high interest since these plants can accumulate or interact with chromium compounds directly *via* their photosynthetically active organs.

In this context, the observed lack of changes or only subtle alterations in electrophoretic proteinograms upon Cr(VI) treatment are intriguing phenomena and will launch a detailed study to reveal biochemical and physiological background of the macrophyte adaptation to Cr(VI).

**Acknowledgements:** The financial support by NCN project DEC-2011/03/B/NZ9/00952 is greatly appreciated.

<figure><img src="image_186.png" /></figure>

<figure><img src="image_187.png" /></figure>

<footnote n="1">¹ Department of Biochemistry, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Krakow, Poland</footnote>

<footnote n="2">² Department of Botany and Plant Physiology, Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Krakow, Poland</footnote>
# EFFECTS OF Cd, Pb AND Ni ON THE ROOT HISTOLOGICAL CHARACTERISTICS OF SALIX ALBA L. AND SALIX NIGRA MARSH.

Luković Jadranka<fnref n="1" />, Hrkić Ilić Zorana<fnref n="2" />, Borišev Milan<fnref n="1" />, Zorić Lana<fnref n="1" />

Corresponding author email: jadranka.lukovic@dbe.uns.ac.rs

## ABSTRACT

Species of the genus *Salix* are nowadays increasingly used in phytoremediation projects. Therefore, in this research we focused our attention on the changes of anatomical characteristics of willow root in the presence of elevated concentrations of Cd, Ni and Pb.

Plant material consisted of two willow clones, clone of species *Salix alba* L. and clone of species *Salix nigra* Marsh. Plants were grown in semi-controlled conditions, using a water culture method. After 35 days of the beginning of the experiment, the treatment of plants with two concentrations (10⁻⁴ and 10⁻⁵ M) of all three heavy metals was initiated, for a period of 45 days. Anatomical analysis was performed on cross sections of the roots, that are made in three zones: 1 cm from the apical region, in the middle part and in the base (neck) of root, using the Image Analyzing System. Detailed anatomical analysis included root cortex (number of cell layers, exodermis area and area of parenchimatic cells in the fourth to fifth parenchyma layer) and root central cylinder (percentage of the central cylinder, number and area of the vessels).

Treatments with heavy metals did not cause necrotic changes of the root, regardless of the applied concentration of heavy metals and clone. However, they caused significant changes in the values of analyzed root parameters in comparison with control samples. The largest number of negative effects on the parameters of root anatomical structure exhibited Cd. Significant reduction in the root cross section area of *Salix alba* clone under the influence of Cd is caused by significant reduction of the number of parenchimatic cells layers and area of exodermal cells. The total area of the vessels is significantly higher in the presence of both concentrations of Cd, compared with the control sample. The minimal number of negative effects were caused by Pb. Deposits of heavy metals were observed in the root tissues, mainly in the walls of cortical parenchimatic cells, rarely in the lumen of parenchimatic cells and in the walls of the xylem elements. Nickel and lead caused even positive effects on the *Salix nigra* clone, in terms of increased values of measured parameters, compared with the control, which could mean that applied concentrations had a stimulatory effect on this clone. Generally observing, clone of species *Salix nigra* shows greater tolerance of heavy metals.

## References:

1. Borišev, M., Pajević, S., Nikolić, N., Pilipović, A., Krstić, B., Orlović, S. (2009) Phytoextraction of Cd, Ni, and Pb using four willow clones (Salix spp.). *Polish Journal of Environmental Studies*, 18(4), 553–561.

2. Luković, J., Merkulov, Lj., Pajević S., Zorić, L., Nikolić N., Borišev, M. Karanović D. (2012) Quantitative Assessment of Effects of Cadmium on the Histological Structure of Poplar and Willow Leaves. *Water Air Soil Pollut*, 223, 2979–2993.

**Acknowledgements:** A research presented in this paper was supported by the Ministry of Education and Science, Republic of Serbia, grant no. OI-173002 and III-043007.

<figure><img src="image_188.png" /></figure>

<footnote n="1">¹ University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg D. Obradovića 2, Novi Sad, Serbia</footnote>

<footnote n="2">² University of Banja Luka, Faculty of Forestry, Vojvode Stepe Stepanovića 75 a, Banja Luka, Bosnia and Herzegovina</footnote>
# INTERANNUAL AND INTRA-ANNUAL MERCURY VARIATION IN A TREE SPECIES
## (QUERCUS ILEX L.)

R. Millán, J. Rodriguez-Alonso, M.J. Sierra

CIEMAT, Departamento de Medio Ambiente, Unidad de Conservación y recuperación de suelos, Avenida Complutense, 40. E-28040 Madrid, Spain

Corresponding author email: rocio.millan@ciemat.es

## ABSTRACT

Plants which are exposed to environmental pollutants are able to accumulate them in their organs depending on specie of plant, type of organ or its age among other factors. The evergreen plants can hold in the same branch stems and leaves which are one, two or more years old. Thus, the amount of contaminants of these former organs could vary with the exposure time to contaminating source.

Dehesa de Castilseras is a forestry area located in Almadén mining district (Ciudad Real, Spain). In this district mercury mining and metallurgical activities have been carried out for centuries. Within Dehesa de Castilseras, close to Almadenejos village, the leaves and the stems of the main evergreen plant species (Quercus ilex L.) have been analyzed with the aim of studying how the mercury concentration varies between consecutive years and during the same year. For that reason, three different trees were selected and two branches were taken from each of them, one facing north, and the other facing south.

Considering each branch and each year, the results show that leaves have higher mercury concentration than stems on average. Furthermore, the lowest mercury concentration was measured in fruit (acorn). The obtained values are similar to mercury concentrations measured in acorns from oak crops that live under free mercury conditions.

With regard to ageing effect on mercury concentration and taking into account each branch separately, the results show the older leaves have higher mercury concentration than younger ones. Nevertheless the older stems not always have higher mercury concentration than younger ones. Moreover, the mercury concentration of the leaves and stems which have recently sprouted is very low and very similar while if these fractions are analyzed six months after its sprout, mercury concentration values are much higher and heterogeneous. This fact leads us to suggest that these kind of young organs are sampled between 6 to 9 months after their sprouting.

<figure><img src="image_189.png" /></figure>
# RESISTANCE OF PLANTS TO SOIL CONTAMINATION BY CHROMIUM (VI)

Agata Borowik<fnref n="1" />, Jadwiga Wyszkowska<fnref n="1" />, Mirosław Wyszkowski<fnref n="2" />

Corresponding author email: miroslaw.wyszkowski@uwm.edu.pl

## ABSTRACT

Soil contamination by heavy metals, including chromium, leads to the gradual degradation of soils in many parts of the world; in some cases the damage can be permanent, so that the affected soil becomes infertile and its plant cover disappears. Heavy metals may also have a negative effect on the growth and development of plants, their chemical composition and the biological properties of soil, such as the enzymatic activity and counts of microorganisms. These considerations have encouraged us to undertake the current study, in which our aim was to determine the resistance of plants, including their chemical composition, to soil contamination by hexavalent chromium. In addition to this, activity of some enzymes engaged in the transformations of carbon, nitrogen and phosphorus has been studied as well as the physicochemical properties of soil.

An experiment, conducted in an unheated greenhouse at the University of Warmia and Mazury in Olsztyn (northeastern Poland), was set up on soil material collected from the arable humus horizon of proper brown soils (Eutric Cambisol). According to the soil classification based on grain-size distribution, proposed by the United States Department of Agriculture, this soil represented loamy sand. It had the following properties: pH in 1 mol KCl dm³ - 6.2; $C_{organic}$ - 6.50 g kg⁻<fnref n="1" />; Cr - 11.35 mg kg⁻<fnref n="1" />; $N_{total}$ - 0.76 g kg⁻<fnref n="1" />; hydrolytic acidity - 14.10 mmol(+) kg⁻<fnref n="1" />; sum of exchangeable bases $Ca^{++}$, $Mg^{++}$, $K^+$, and $Na^+$ - 80.00 mmol(+) kg⁻<fnref n="1" />; cation exchange capacity - 94.00 mmol(+) kg⁻<fnref n="1" />; base saturation - 85.11%. The experiment included 5 replicates. Two variables were examined: the degree of contamination by chromium (VI) in mg $Cr^{6+}$ kg⁻<fnref n="1" /> of soil (0, 15, 30, 45 and 60 and several species of crops (oat, spring barley, spring oilseed rape, white mustard, yellow lupine and horse bean). All the treatments received the same fertilization composed of macro- and micronutrients. During the whole period of growing the plants, the soil moisture was maintained on the level of 60% water capillary capacity. After harvest (on day 50), the volume of yields and their chemical composition were determined (content of nitrogen, phosphorus, potassium, calcium, magnesium, sodium and chromium). Also, the activity of the following soil enzymes was determined: dehydrogenases, urease, acid phosphatase and alkaline phosphatase. Based on the analytical results, values of the resistance index for all the plants to chromium (VI) in soil as well as the activity of individual enzymes in response to this soil contaminant and the biochemical index of soil potential fertility were calculated. The results were submitted to the Tukey's test at the level of significance p=0.05. The principal component analysis (PCA) method was applied to assess the effect of chromium on plant yields and activity of soil enzymes.

Hexavalent chromium had a very toxic effect on the plants, which was confirmed by a highly significant negative correlation between the dose of Cr(VI) and yield of aerial parts of horse bean ($r$ = -0.993), spring barley ($r$ = -0.981), spring oilseed rape ($r$ = -0.968), yellow lupine ($r$ = -0.961), while mustard ($r$ = -0.960) and oat ($r$ = -0.929). The values of the correlation coefficients themselves suggest that the tested crop species were variously tolerant to the pollution. Toxic symptoms, such as water balance disorders (wilting), chlorosis of new leaves or damage to the growth apex and roots, appeared in yellow lupine in all treatments, including the ones with the smallest chromium dose (15 mg · kg⁻<fnref n="1" /> of soil) and becoming more intensive under higher doses. The smallest dose of $Cr^{6+}$ also caused a significant decrease in the yields of spring oilseed rape and white mustard. Such a change in yields was manifested by the resistance indices (RS) of individual crops in response to the heavy metals. For all the crops, the RS was the lowest in soil polluted by 60 mg $Cr^{6+}$ kg⁻<fnref n="1" /> of soil. The most chromium-resistant plant proved to be horse bean (RS = 0.500). The resistance of the other crops was much lower. The RS of white mustard was 0.337, oat - 0.247, spring barley - 0.168, yellow lupine - 0.097 and spring oilseed rape - 0.062. Assessment of the effect of chromium cannot be limited to plant yields but should also consider the chemical composition of plants. The results of our analyses demonstrated that the soil contamination by chromium induced larger changes in the concentrations of nitrogen, phosphorus or potassium than magnesium, calcium and sodium in aerial organs of the plants. Another aspect that must not be neglected is the accumulation of chromium by plants. Its elevated content in a plant is one of the consequence of the presence of chromium in the environment due to anthropogenic activity. Chromium was also found to have inhibited the activity of dehydrogenases, urease, acid phosphatase and alkaline phosphatase. Moreover, it reduced the value of the potential biochemical index of soil fertility. Dehydrogenases proved to be the most sensitive to the excess of chromium in soil, in contrast to alkaline phosphatase, which was the most tolerant of all the analyzed enzymes.

<figure><img src="image_190.png" /></figure>

<footnote n="1">¹Department of Microbiology, University of Warmia and Mazury in Olsztyn, Poland</footnote>

<footnote n="2">²Department of Environmental Chemistry, University of Warmia and Mazury in Olsztyn, Poland</footnote>
# LABORATORY AND PILOT-SCALE PHYTOFILTRATION OF URANIUM-CONTAMINATED WATER

João Pratas<fnref n="1" />,<fnref n="3" />, Paulo J.C. Favas<fnref n="2" />,<fnref n="3" />

Corresponding author email: pjcf@utad.pt

## ABSTRACT

The objective of the present study is: 1) developing a phytofiltration prototype system; 2) to test the potential of the species *Callitriche stagnalis*, *Potamogeton natans* and *Potamogeton pectinatus* for the decontamination of U-contaminated waters.

The installed prototype consists of a closed circuit of channels. The system was initially contaminated with 500 µg/L of U as uranyl.

The aquatic plants tested in the U phytofiltration experiments showed a high ability for U accumulation and, therefore, they can be useful choices for future *in situ* applications. The results described for the phytofiltration prototype show a reduction of U concentration, in the water, from 500 to 72.3 µg/L of U, thus representing an efficiency of 85.5%. At the end of 15 days of the test the U concentration in *C. stagnalis* increased from 0.98 to 1567 mg/kg, in *P. natans* increased from 3.46 to 271 mg/kg and in *P. pectinatus* increased from 2.63 to 1588 mg/kg. All these species showed a rapid U uptake and the results highlight the effectiveness of the selected plants to remove uranium from the water.

The advantage of the phyto-system proposed in this study is related to the high tolerance of the species tested to U contamination and with their abundance and natural occurrence in the Portuguese rivers which prevent the invader factor for an *in situ* application. In fact, the native character of these species represents a very important factor for the establishment of this technology taking into account the sociability relationships with other aquatic species and their ecosystems adaptation. The combination of several species, resembling the natural environment, can prove more effective than the usual mono-specific culture option.

The selected plant species are a promising choice for the remediation of low to medium U contaminated waters. However, to assess the real phytotechnological potential of the proposed system, and before an *in situ* application, laboratory tests are needed to determine the toxicity level, the biomass productivity and what enhancement strategies of U uptake can be used. The issue of the final disposal of the generated residues also has to be addressed.

*Acknowledgements:* This study was partially supported by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE) and National Funds through the Portuguese Foundation for Science and Technology (PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).

<figure><img src="image_191.png" /></figure>

<footnote n="1">¹ University of Coimbra, Department of Earth Sciences, 3001-401 Coimbra, Portugal</footnote>

<footnote n="2">² University of Trás-os-Montes e Alto Douro, UTAD, School of Life Sciences and the Environment, Quinta de Prados, 5000-801 Vila Real, Portugal, www.utad.pt</footnote>

<footnote n="3">³ IMAR-CMA Marine and Environmental Research Centre, University of Coimbra, Portugal</footnote>
# OPTIMIZATION OF HARVEST TERM OF PHYTOEXTRACTION SHORT ROTATION COPPICE

Pavla Zárubová, Pavel Tlustoš, Jiřina Száková

Dept of Agro-environmental Chemistry and Plant Nutrition; Czech University of Life Sciences, Czech Republic

Corresponding author email: tlustos@af.czu.cz

## ABSTRACT

Soil contamination by risk elements belongs to significant contemporary ecological problems. Following this reason, the interest for economically reasonable methods of soil decontamination, as phytoextraction is increasing. Fast growing trees from *Salix* spp. and *Populus* spp. are suitable plants for phytoextraction of risk elements, especially on medium and weakly contaminated soils [1]. The majority of risk elements is usually accumulated in leaves than in twigs [2] therefore a biomass harvest can be preferable realise within vegetation period.

The aim of our study was to optimize rotation period and term of harvest of fast growing trees in field conditions and to improve the phytoextraction potential. The experimental short rotation coppice plantation near Příbram town (49°42'24"N, 13°58'32"E), was planted in April 2008 and 2009 with clones of *Salix* and *Populus* species: (*Salix schwerinii* x *S. viminalis*) x *S. viminalis* (S1), *S. smithiana* (S2), *Populus nigra* x *P. maximowiczii* (P1) and *P. nigra* (P2). Both plantings were completely harvested in February 2012. Other samples were taken after next 8 months of growth in September 2012. The yield parameters and the content of risk elements were determined at all samples. We investigated the differences between three years (planting in 2008) and four years harvest rotation (planting in 2009) as well as differences between winter (February) and summer (September) harvest of trees.

The biomass yield at three and four years rotation was similar. The differences between biomass yield from winter and summer harvest weren't significant, clones reached the same yields 8 months after winter harvest as after 3 and 4 years of growth. In September, the trees were harvested with leaves. The ratio of leaves to twigs weight was different at individual clones: S1=0.28, S2=0.36, P1=0.61, P2=0.50. The dry biomass yield decreased in sequence: P1>>S2>P2≥S1 (433-97g plant⁻¹).

The contents of observed risk elements weren't different in trees harvested after 3 or 4 years. The significant differences were found between winter and summer harvest. The highest concentration of nutrients K, Ca and Mg were determined at all clones in leaves (K 10.09-13.85 g kg⁻¹, Ca 13.76-22.43 g kg⁻¹, Mg 3.49-5.91 g kg⁻¹) and consequently in twigs in September, while the lowest concentration of these elements were detected in twigs harvested in February (K 0.63-2.29 g kg⁻¹, Ca 1.29-2.79 g kg⁻¹, Mg 0.10-0.32 g kg⁻¹). Also the other elements (P, Cd, Fe, Mn, Pb and Zn) showed the highest concentrations in leaves (e.i.: Cd 12.67-51.43 mg kg⁻¹), and very low concentrations in twigs harvested in September (e.i.: Cd 4.04-9.78 mg kg⁻¹) and high concentrations in twigs harvested in February. Copper content in twigs harvested in February was higher than content in leaves harvested in September. This is related with changes of element concentration in xylem sap [3]. The element uptake was significantly influenced by biomass production. The highest uptake was determined at the most productive clones P1 and S2. The concentration of elements was also highly affected their uptake, as Fig. 1 and 2 show. Higher amount of Ca, K and Mg was removed at summer harvest, their high concentrations were determined in leaves and twigs as well; conversely Cd, Cu, Fe, and Pb were predominantly removed by winter harvest.

Our study showed the shortening of rotation period by one year does not influence both the biomass yield and element uptake of older trees. Summer harvest is less suitable for phytoextraction of contaminants: the risk elements removal was low and nutrient removal high at September compare to February harvests.

## References:

1. J. K. Jensen, et al. (2009) Environ Pollut 157, 931 - 937

2. A. P. Maxted, et al. (2007) Plant Soil 290, 157 - 172

3. I. Laureysens, et al. (2005) Environ Pollut 133, 541 - 551

Acknowledgements: The financial support by project CIGA contract No. 20142005 is greatly appreciated.

<figure><img src="image_192.png" /><figcaption>Figure 1 and 2: Mean (± SE) removal amount of Cd and K</figcaption></figure>

<figure><img src="image_193.png" /></figure>
# ACCUMULATION OF RISKY METALS IN SEEDS AND ABOVEGROUND BIOMASS OF AMARANTH

**Alena Vollmannova<fnref n="1" />, Eva Margitanova<fnref n="1" />, Tomas Toth<fnref n="1" />, Radovan Stanovic<fnref n="1" />, Tatiana Bojnanska<fnref n="1" />, Iveta Cicova<fnref n="2" />, Michaela Benkova<fnref n="2" />**

Corresponding author email: alena.vollmannova@uniag.sk

## ABSTRACT

Amaranth is an 8000 year old cultivated crop originating on American continent. It is not demanding on soil and climatic conditions, is resistant to stress and high temperatures and produces a lot of biomass. A few years ago amaranth was rediscovered as a multipurpose crop applying high nutritional quality seeds and leafy vegetables for food and animal feed. In some parts of the world amaranth seed is considered as a staple food rich in lipids, proteins, carbohydrates, dietary fiber and also another components such as squalene, tocopherols, polyphenols, phytates and vitamins. In West Africa amaranth is cultivated also for its edible leaves rich in vitamins and dietary minerals. The objective of the study is to compare the degree of accumulation of risk metals by amaranth seeds and aboveground biomass and to evaluate the safety of consumption parts of five different amaranth cultivars from the aspect of heavy metal content. Soil as a starting place for input of risk substances into a human food chain was analysed, too.

In soil the agrochemical characteristics (pH/KCl, content of K, Mg, P, Ca and humus) were determined. Pseudotal content of risk metals (Zn, Cu, Ni, Cr, Pb and Cd) including all the form besides residual metal fraction was assessed in soil extract by aqua regia and content of mobile forms of selected heavy metals in soil extract by NH₄NO₃ (c=1mol/dm³). Gained results were evaluated according Law No. 220/2004 (valid in the Slovak Republic) as well as threshold values proposed JRC EC (2006). Used analytical method was flame AAS (AAs Varian AA Spectr DUO 240 FS/240Z/UltrAA). Seeds and aboveground biomass of five amaranth cultivars (Golden Giant, Rawa, Annapurna, Oscar Blanco, Koniz) were manually separated, dried at 105°C to constant weight and powdered. After previous microwave decomposition of plant samples the contents of risky metals were determined by AAS method (AAS Varian AA Spectr DUO 240FS/240Z/UltrAA) and expressed as mg/100 DM (dry matter). Gained results were evaluated according to FC SR (Food Codex of the Slovak Republic valid in the Slovakia) as well as according Commission Regulation (EC) 1881/2006.

The soil of amaranth cultivation was uncontaminated. Only determined Cd content was by 40% and Pb content by 10% higher than limit values given by Law No. 220/2004 for the soil extract by aqua regia and for the soil extract by NH₄NO₃, respectively. But on the other hand the determined Cd content did not exceed the threshold value given by EC (2006). In aboveground amaranth biomass the followed values of heavy metal content were determined (in mg/kg DM): Zn: 26.20-30.38; Cu: 6.20-16.84; Ni: 0.63-1.75; Cr: 3.90-8.00; Pb: 0.70-2.00; Cd: 0.69-1.87. In amaranth seeds these values were measured (in mg/kg DM): Zn: 10.33-20.20; Cu: 2.37-4.43; Ni: 0.45-0.68; Cr: 0.40-1.70; Pb: 0.25-0.60; Cd: 0.15-0.20. In seeds of all investigated amaranth cultivars the maximal allowed amounts (given by FC SR as well as EC 1881/2006) for Cd and Pb were by 60-100% and 25-200% (respectively) exceeded. In aboveground amaranth biomass 0.17 - 12.25 fold higher amounts of heavy metals were determined in comparison to amaranth seeds. The statistically significant differences in values of heavy metal content measured in seeds as well as in aboveground biomass between investigated amaranth cultivars were confirmed. Our results confirm, that amaranth seeds and leaves as food raw materials could represent a risk to the health of the consumer from the aspect of high Cd and Pb amounts.

Because of ability to produce abundance of biomass and at the same time to accumulate high amounts of risk heavy metals amaranth could be used as a potential plant for a soil phytoremediation. The results confirmed the ability of amaranth to accumulate risk metals even from relatively „clean“ soil.

**Acknowledgements:** This work was co-funded by European Community under project No. 26220220180: Building Research Centre "AgroBioTech" and also supported by project: VEGA 1/0308/14.

<figure><img src="image_194.png" /></figure>

<footnote n="1">¹ Dept of Chemistry, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic</footnote>

<footnote n="2">² National Agriculture and Food Centre, Plant Production Research Centre in Piestany, Slovak Republic</footnote>
# EFFECT OF URANIUM ON PHOTOSYNTHETIC PARAMETERS IN *PISUM SETIVAM* IN CONJUGATION WITH ANTIOXIDANT DEFENSE

D. K. Gupta, F. Tawussi, L. Lütke, C. Walther

Institut für Radioökologie und Strahlenschutz (IRS), Leibniz University of Hannover, 30419 Hannover, Germany
*Corresponding author email: gupta@irs.uni-hannover.de*

## ABSTRACT

Large uranium accumulations in vegetable foodstuffs may present risks of human health if they are consumed. The toxic effects of U in living organisms depend on uranyl ion speciation and their solubility. In our present study we studied the impact of U on photosynthetic pigments (chlorophyll/carotenoid contents) and some antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and the cysteine and non-protein thiol (NP-SH), as well as lipid peroxidation as oxidative-damage markers. Disturbances in nutrient status induced by U are another feature previously noted in other plants. It has been also observed that ion transporters could be the target of ROS hence, U and macro/micronutrients uptake was also studied by analyzing the content of those elements in roots and leaves of *Pisum setivam* plants.

In tested plants photosynthetic pigments significantly altered depending on the concentration of uranium uptake in comparison to control, the decrease in net photosynthesis as a consequence of reduced absorption of essential mineral nutrients is an indirect reason for plant chlorosis. CAT and GR activity was lower in roots and shoots in compare to control at higher concentration but it was interesting to note that both cysteine and NP-SH was higher in root as well as in shoots in higher concentration U treated plants after 5 days. The decline in CAT activity might be due to the inhibition of the enzyme by post-transcriptional alterations or down-regulation of gene expression. The increase in the level of thiols and cysteine may be due to stimulation of enzymes of sulfate reduction pathway such as APS reductase and Serine acetyltransferase. The enhancement in NP-SH concentration possibly also reflects a defense reaction to enhanced production of ROS. Increment in lipid peroxidation indicates the prevalence of oxidative stress and perhaps this may be one of the possible mechanisms by which toxicity due to U could be manifested in the plant tissues. However, the level of lipid peroxidation was lower in roots as compared to that of shoots. This was attributed to low U load in shoots that probably resulted in low oxidative stress and membrane damage. Among all tested macro and micro elements (Ca, Fe, K, P, S, Mn, Zn and Cu) only Ca uptake is higher in shoots rather than roots. U accumulation is also higher in roots rather in shoots after 5 days of treatment. Our observation suggests that U severely disturbed the nutritional status of the experimental plant, a situation that could induce alterations in cell metabolism and thereby stunt growth. It has been hypothesized that the metal detoxification mechanism was more pronounced at the site of its uptake, which is restricting the mobility of metals to shoot from root. The detail of this work is going to be discussed in the meeting.

<figure><img src="image_195.png" /></figure>
RELATIONSHIPS OF FREE AMINO ACIDS AND FATTY ACIDS WITH CADMIUM IN
NOCCAEA CAERULESCENS ECOTYPES

Veronika Zemanová<fnref n="1" />, Daniela Pavlíková<fnref n="1" />, Milan Pavlík<fnref n="2" />, Pavlína Kyjáková<fnref n="3" />, Jana Najmanová<fnref n="1" />

ABSTRACT

Metal hyperaccumulation is a physiological process that enables the plant to store large quantities of metal
ions in above-ground tissues without suffering toxic effects. Cadmium (Cd) is one of the most toxic metals for
living organisms and Noccaea (previously named Thlaspi caerulescens, J. & C. Presl) is one of the four identified
Cd hyperaccumulators identified up to now. The uptake of Cd from contaminated soil affects the plant
metabolism of amino acids and lipids. This effect is indicated by change in the content of free amino acids and
fatty acids in tissues of hyperaccumulators. The aim of the study was to determine the changes in free amino
acids and fatty acids content in selected ecotypes of N. caerulescens.

A pot experiment was conducted in a vegetation hall under controlled conditions (temperature day/night 24 °C/18 °C, relative humidity 60 %, light intensity: day/night 16 h/8 h) using three ecotypes of Noccaea caerulescens (F.K. Mey): "Ganges" (France), "Mežica" (Slovenia) and "Redlschlag" (Austria) and four Cd level 0, 30, 60 and 90 mg.kg⁻<fnref n="1" /> applied as Cd(NO₃)₂·4H₂O. Plants in growing period of four leaves were placed in 3 L plastic pots filled soil (chernozem modal: habitat Prague-Suchdol; pHₖ₄ = 7.1 ± 0.3; Cₓₓ = 3.1 ± 0.7 %; KVK = 225 ± 19.5 mmol(l₄)·kg⁻<fnref n="1" />; contents of Cd = 0.42 ± 0.05 mg.kg⁻<fnref n="1" /> ) with nutrients and contaminations. The dose of nutrients was: 0.3 g N, 0.10 g P, and 0.24 g K applied in the form of NH₄NO₃ and KH₂PO₄. The first harvest was performed 90 days and the second harvest 120 days after planting of N. caerulescens into soil. The Cd concentrations in plant biomass were determined by ICP-OES with axial plasma configuration (VarianVistaPro, Varian, Mulgrave, Australia). Total free amino acids (AA) compounds were determined using an EZ-faast amino acid analysis procedure (Phenomenex, Santa Clara, USA). Samples were analyzed for AA contents by the GC-MS (Agilent Technologies, Torrance, USA). The contents of methyl esters of fatty acids after transesterification were measured by GC-MS (Thermo Fisher Scientific, USA). The statistical analyses were performed using hierarchic analyses of variance (ANOVA) with inteactions at 95% (P < 0.05) significance level with subsequent Tukey's HSD test and linear correlation (R<fnref n="2" />) by using Statistica 12.0 software (StatSoft, Tulsa, USA).

Uptake of Cd was the highest in the Cd-accumulating ecotype, Mežica, than in the low Cd-accumulating ecotypes, Ganges and Redlschlag. The free AA content in the biomass of ecotype Mežica declined progressively with increasing Cd concentrations during both harvests (R<fnref n="2" />=0.99-1 and R<fnref n="2" />=0.90-1). The same trend, decrease of free AA content was observed in biomass of ecotype Redlschlag. Results confirmed the significant relationship between contents of free AA and Cd content in biomass (R<fnref n="2" />=0.93-1 for both harvests). Opposite trend, increase of free AA content was observed in biomass of ecotype Ganges. The significant relationships between free AA content and Cd content in biomass was confirmed using linear regression (R<fnref n="2" />=0.84 and R<fnref n="2" />=0.62). The major free AA determined in all ecotypes of N. caerulescens were glutamic and aspartic acid, glutamine, asparagine and proline. The Cd soil contamination did not significantly modify the total contents of saturated and unsaturated fatty acids in biomass of ecotypes Ganges and Redlschlag. Different results were showed in biomass of ecotype Mežica. Linear correlations confirmed closed relation of saturated and unsaturated fatty acid contents and Cd content in biomass. Content of saturated fatty acids decreased with concentration of Cd (R<fnref n="2" />=0.92-1 and R<fnref n="2" />=0.99-1). On the other hand content of unsaturated fatty acids increased with concentration of Cd (R<fnref n="2" />=0.92 and R<fnref n="2" />=0.99).

Results of the pot experiment showed Cd effect on total content free AA and fatty acids playing an important role in plant adaptation to stress. Analyses of free AA and fatty acids confirmed the higher cadmium tolerance of ecotype Mežica in contrast to the ecotypes Ganges and Redlschlag. These results indicate that the mechanisms of Cd hyperaccumulation and metabolism of free AA and lipids are not identical in this species.

Acknowledgements: The financial support by CULS project CIGA contract No. 20142004 is greatly appreciated.

<figure><img src="image_196.png" /></figure>

<footnote n="1">¹ Department of Agro-Environmental Chemistry and Plant Nutrition, Czech University of Life Sciences, Czech Republic</footnote>

<footnote n="2">²Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Czech Republic</footnote>

<footnote n="3">³Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Czech Republic
*Corresponding author email: zemanovav@af.czu.cz*</footnote>
# INFLUENCE OF ESSENTIAL METALS ON CADMIUM UPTAKE BY SORGHUM PLANTS

Petrová Šárka, Soudek Petr, Vaněk Tomáš

Laboratory of Plant Biotechnologies, Joint Laboratory of Institute of Experimental Botany AS CR and Crop Research Institute, Rozvojová 263, 165 02 Prague 6, Czech Republic

Corresponding author email: petrova@ueb.cas.cz

## ABSTRACT

Increased concentrations of toxic metals in agricultural soils can cause death of plants or reduce their production. Contaminants accumulated in food crop may also cause danger to human health. On the other hand, plants grown on contaminated soil can reduce the pollution through a transfer of the contaminant from soil to harvestable plant parts. Phytoextraction can be a solution how to prevent the spread of pollution while ecologically enhanced the site with economical aspect. The method is suitable for low contaminated sites and it is used for its low cost and wide public acceptance. Appropriate plant species should be tolerant to pollutants toxicity and it should have also high biomass production. This condition meets energy plants such as woody plants, and grasses.

Sorghum bicolor is an important crop due to its wide use. Due to a high biomass production and known agronomic techniques, sorghum can be used as an energy crop and at the same time it can clean up the environment. The aim of this study was to expand knowledge about protection mechanisms of different species of sorghum (S. bicolor var. eusorghum, S. bicolor x S. sudanese) under cadmium stress. The effects of combination of cadmium with essential metals such as copper, iron, and zinc were studied.

<table><thead><tr><th>Cd (mM)</th><th>Cu, Fe, or Zn (mM)</th><th>Cd (mM)</th><th>Cu, Fe, or Zn (mM)</th><th>Cd (mM)</th><th>Cu, Fe, or Zn (mM)</th><th>Cd (mM)</th><th>Cu, Fe, or Zn (mM)</th></tr></thead><tbody><tr><td>0.01</td><td>0</td><td>0</td><td>0.01</td><td>0.01</td><td>0.2</td><td>0.2</td><td>0.01</td></tr><tr><td>0.05</td><td>0</td><td>0</td><td>0.05</td><td>0.05</td><td>0.2</td><td>0.2</td><td>0.05</td></tr><tr><td>0.1</td><td>0</td><td>0</td><td>0.1</td><td>0.1</td><td>0.2</td><td>0.2</td><td>0.1</td></tr><tr><td>0.5</td><td>0</td><td>0</td><td>0.5</td><td>0.5</td><td>0.2</td><td>0.2</td><td>0.5</td></tr><tr><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0.2</td><td>0.2</td><td>1</td></tr><tr><td>5</td><td>0</td><td>0</td><td>5</td><td>5</td><td>0.2</td><td>0.2</td><td>5</td></tr></tbody></table>

Results showed that cadmium toxicity depended on chosen sorghum variety. EC₅₀ value range was from 1.2 to 2.0 mM for cadmium, 1.2 - 2.2 mM for copper, 3.5 - 8.2 mM for iron, and 5.4 - 12.6 mM for zinc. Essential elements had an influence on toxicity of cadmium to sorghum plants and also an effect of cadmium on copper, iron, and zinc toxicities was evident.

Acknowledgement: This work was supported by projects LD14106 and LD14107.

<figure><img src="image_197.png" /></figure>
HYPERACCUMULATOR *THLASPI CAERULESCENS* (GANGES ECOTYPE) RESPONSE
TO INCREASING LEVELS OF DISSOLVED CADMIUM AND ZINC

Saoussen Benzarti<fnref n="1" />, Helmi Hamdi<fnref n="2" />, Shino Mohri<fnref n="1" />, Yoshiro Ono<fnref n="3" />

ABSTRACT

Phytoextraction or phytoremediation of heavy metals has been thought to be an environmentally
friendly, cost-effective strategy to clean-up soils or waters contaminated with these toxic pollutants.
Efficient metal extraction relies basically on the use of hyperaccumulator plants with exceptional
metal-accumulating capacity, known as natural phytoextraction. Hyperaccumulator plants possess
several exceptional characteristics, such as the ability to take up and translocate exceedingly large
amounts of metals and hypertolerate their phytotoxic effects. In this regard, *Thlaspi caerulescens*
(Alpine pennycress) is considered as one of the best known examples of hyperaccumulator plants,
being able to primarily hyperaccumulate Zn and Cd as well as Ni in some accessions.

In the current study, *Thlaspi caerulescens* (Ganges ecotype) was separately exposed to Cd and Zn at 0, 50, 100 and 200 µM for 7 d to monitor plant physiological response in hydroponics. Significant dose dependent accumulation was observed for both metals, mainly in roots (up to 3.2 and 9.2 mg g⁻<fnref n="1" /> for Cd and Zn, respectively). However, Cd was more phytotoxic in terms of plant growth and photosynthesis. This higher toxicity was also evidenced by the MetPLATE bioassay applied to exposure solutions. Root exudation was significantly correlated to Cd and Zn translocation (*r* > 0.85) proving its involvement in facilitating metal uptake.

As for antioxidative responses, plants reacted to Cd and Zn by broadly exhibiting an elevation of
glutathione reductase (GR) activity before declining at 200 µM due to higher phytotoxicity. In
contrast, superoxide dismutase (SOD) activity was unlikely to be affected by both metals. Root-to-
shoot apoplastic flow was traced using a fluorescent dye (PTS), whose concentration in leaves
increased to a certain extent with Cd and Zn accumulation indicating that heavy metals have a
comparable effect to drought or salinity in promoting the passive diffusion of water and solutes.
Nevertheless, Cd at 200 µM hindered the diffusion of PTS and consequently affected the apoplastic
transport in plants by damaging root cells.

Keywords: *Thlaspi caerulescens*, cadmium, zinc, exudation, antioxidation, apoplastic flow

<figure><img src="image_198.png" /></figure>

<footnote n="1">¹ Graduate School of Environmental Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan</footnote>

<footnote n="2">² Water Research and Technology Center, University of Carthage P.O. Box 273, Soliman 8020, Tunisia</footnote>

<footnote n="3">³ Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan</footnote>
# ASSESSMENT OF EFFICACY OF EDTA AND CITRIC ACID IN RHIZOFILTRATION ENHANCEMENT OF CADMIUM USING LEMNA MINOR

R. Aravind, V. S. Bharti, M. Rajkumar, P. K. Pandey, C. S. Purushothaman, A. Vennila, S. P. Shukla

Aquatic Environment and Health Management Division (AEHMD), Central Institute of Fisheries Education (CIFE), Mumbai, Maharashtra, India

Corresponding author email: aravindbfsc@gmail.com

## ABSTRACT

Free floating aquatic macrophyte namely *Lemna minor* (Linnaeus, 1753) was exposed to different concentration of cadmium (1, 5 and 10 mg L⁻¹) for a period of 30 days to evaluate its cadmium (Cd) accumulation capability in the presence of chelating agents such as EDTA and citric acid. The chelating agents were added at the rate of 1, 2 and 3 mg L⁻¹ separately and the experiment was conducted in triplicate. The water and plant samples were collected at 15 days interval for the analysis of cadmium. There was a significant difference in the Cd uptake (P<0.05) by the plant in the presence of chelating agents when compared to the control. Bioconcentration factor (BCF) of cadmium by the plants showed an increasing trend in the presence of chelating agents. The percentage uptake of cadmium by *L. minor* in the presence of EDTA was significantly higher than that of citric acid (P<0.05). The overall results suggest that EDTA can be effectively used to enhance phytoremediation efficiency of cadmium by *L. minor* in the contaminated water.

<figure><img src="image_199.png" /><figcaption>Fig. 1, 2 - Cadmium accumulation (µg g⁻¹) by L. minor on 15 and 30th day from 1 mg Cd L⁻¹ respectively</figcaption></figure>

*Acknowledgements:* The financial support by Central Institute of Fisheries Education, Mumbai, India is greatly appreciated.

<figure><img src="image_200.png" /></figure>
# THE CURRENT STATUS OF BISCUTELLA LAEVIGATA L. (BRASSICACEAE) SPECIMENS TESTED FOR THE SUITABILITY TO PHYTOREMEDIATION TECHNOLOGY

Ewa Muszyńska<fnref n="1" />, Ewa Hanus-Fajerska<fnref n="1" />, Krystyna Ciarkowska<fnref n="2" />, Tomasz Czech<fnref n="3" />

Corresponding author email: emuszynska@ogr.ur.krakow.pl

## ABSTRACT

The well known facts are that phytoremediation has become an advantageous way to recover degraded lands, and that numerous *Brassicace* members are easy accommodating into almost every kind of habitats. Many species are even known to perform well on waste areas as they are well adapted to recently formed, non-structural substrates. *Biscutella laevigata* L. is perennial herbaceous species, which has developed different ecotypes, and in Europe is recorded on rocky, often alpine stands, metal enriched soils, and anthropogenic substrates like waste heaps polluted with heavy metals. Simple leaves are gathered in a basal rosette with a woody root-stock. The leaved inflorescence has branched stalk with numerous bright yellow flowers. The fruit dehiscent is short-ovate with false-septum. In Poland *B. laevigata* has been described from two different stands: first is located in the Tatry Mountains, and second in the Zn-Pb ore-mining region in district of Olkusz located in southern part of the country, where it occur as an endemic metallophyte typical of calamine flora. There is a strong possibility that this calamine ecotype will be useful to reclamation of waste material polluted with Zn, Pb and Cd. Therefore, experiments were undertaken to investigate the fitness of *B. laevigata* specimens representing the calamine ecotype from the Olkusz ore-bearing district, during culture under vegetation hall conditions on wastes after zinc and lead ores flotation.

Pot experiment was set up in April 2011, so until now it was conducted for 3 years. Pots with a diameter of 22 cm were filled with substrate in an amount of 5.5 kg per pot. The substrate was wastes after flotation of zinc and lead ores collected from the settling of tailings materials, belonging to metallurgical and mining plant situated in Bukowno near Olkusz (the southern part of Poland). The experiment was conducted in six replications, and included three treatments: control, without fertilization; mineral fertilization at a dose of 1.2 g N; 0.4 g P₂O₅, 1g K₂O per pot, using a mixture of aqueous solutions: NH₄NO₃, KH₂PO₄ and KCl; fertilization with municipal sewage sludge of 135g per pot, and enriched with potassium dose 0.8 g K₂O/pot. The dose used was the maximum permissible dose of sewage sludge, which can be used for reclamation purposes, in accordance with applicable Polish Regulation. Substrate moisture was maintained at 35% of capillary water capacity. Observations of *B. laevigata* growth and development started a month after planting, and detailed biometrical measurements were conducted every four weeks. After each growing season one replication of each fertilizing objects was taken for analyses of the plant material and the substrate.

Flotation material used as a substrate for growing *Biscutella* was characterized by alkaline reaction, trace contents of organic carbon and total nitrogen, and very low contents of available phosphorus and potassium. The content of total forms of zinc, lead and cadmium (respectively: 10690 mg/kg, 8058 mg/kg and 78,8 mg/kg) several times exceeded the permissible contents of these metals as defined in the Regulation of the Minister of Environment for industrial and post-mining lands. Cultivation of plants with fertilization contributed to the slight increase in the content of organic carbon and total nitrogen in each object with respect to the contents of these elements in the initial flotation material. The increase of the carbon and nitrogen contents was set in the following range: control object < object fertilized with mineral fertilizers < object fertilized with sewage sludge. This

<figure><img src="image_201.png" /></figure>

<footnote n="1">¹ Unit of Botany & Plant Physiology, Institute of Plant Biology & Biotechnology, Faculty of Horticulture, University of Agriculture, Krakow, Poland</footnote>

<footnote n="2">² Department of Soil Science & Soil Protection, Faculty of Agriculture & Economics, University of Agriculture, Krakow, Poland</footnote>

<footnote n="3">³ Department of Agricultural & Environmental Chemistry, Faculty of Agriculture & Economics, University of Agriculture, Krakow, Poland</footnote>
arrangement was maintained throughout the whole period of the experiment, and the observed
changes intensified with time. During the experiment changes in zinc lead and cadmium contents in
the substrate were also observed. After each growing period contents of Zn, Pb and Cd in the
substrate gradually lowered. The decreases in contents for Pb and Zn were similar in all experimental
treatments, and amounted to 35,9-40,4% and 35,9-36,5% respectively. However in the case of Cd
the phytoremediation activity of *Biscutella* specimens, it was strongly related to particular treatment,
and it was the lowest in the object fertilized with sewage sludge in spite of the fact that plants
developed the biggest biomass in this substrate. The contents of Zn and Pb were the lowest in the
substrate of the object fertilized with sewage sludge, and the largest in the substrate of the object
without fertilization.

Thus the investigated ecotype was proved to accumulate relatively high doses of Zn, Pb and Cd
without any visible symptoms of phytotoxicity. Detailed morphological features will be presented
during poster session. The next planed step is the investigatation of the mechanisms responsible for
accumulation of metallic elements, using *Biscutella laevigata* as model plant species.

<figure><img src="image_202.png" /></figure>
# POTATOES – A CROP RESISTANT AGAINST INPUT OF HEAVY METALS FROM THE METALLICALY CONTAMINATED SOIL

Janette Musilova<fnref n="1" />, Judita Bystricka<fnref n="1" />, Jaromir Lachman<fnref n="2" />, Lubos Harangozo<fnref n="1" />, Pavol Trebichalsky<fnref n="1" />, Beata Volnova<fnref n="1" />

Corresponding author email: janette.musilova@uniag.sk

## ABSTRACT

Cultivation of potatoes in Slovakia has a long tradition, determined by suitable climatic conditions and the development of agriculture. On the other hand, its development together with the development of industrial activity is related to the contamination of the environment. Enhanced levels of heavy metals in soil present a risk not only for cultivated crops, but especially for consumers. The ability of plants to accumulate heavy metals varies depending on the species: pea < cereals < potato < sugarbeet < corn < clover and their penetration influences soil-ecological conditions such as soil types, soil pH, concentrations and bonds of heavy metals, humus content in soil, oxidative-reductive conditions around root system connecting with microbial decomposition of inorganic and organic substances, soil moisture, temperature, utilized fertilizers and preparations for the plant protection.

The objective of our study was to assess the extent of accumulation of cadmium, lead and zinc in potato tubers depending on the concentration of these heavy metals in soil and to compare the resistance of 12 cultivars of potato (Laura, Red Anna, Marabel, Manitou, Cicero, Silvana, Rosara, Mozart, Cicero, Desiree, Volumisa, Adora), cultivated in 5 localities of the Slovakia (Odorin, Ivanka pri Nitre, Spissky Stvrtok, Belusa, Imel) against input of these heavy metals into the consumption part of potato.

Soils on which potatoes were grown, can be characterized as acidic to strongly alkaline (pH / KCl = 5.15 - 7.86), with low to moderate supply of humus (% hum. = 1.62 - 2.85), poor to good phosphorus content (P = 35.85 - 82.76 mg/kg) and moderate to very high potassium and magnesium contents (K = 189 to 456 mg/kg, Mg = 135-368 mg/kg). In soil extracts by ammonium nitrate (NH₄NO₃ c = 1 mol/dm³) and *aqua regia* contents of Pb, Cd and Zn using method of flame atomic absorption spectrometry (AAS Varian Spectr AA DUO 240FS/240Z/UltrAA) were determined (mean values of determined Pb, Cd and Zn content in all sampling sites were as follows: 0.146 - 0.640; 0.030 to 0.188 and 0.035 to 0.238 mg/kg in soil extract by NH₄NO₃, and 14.0 - 24.90; 0.92 - 2.72 and 35.0 - 72.60 mg/kg in soil extract by *aqua regia*).

The contents of Cd, Pb and Zn were determined in potatoes in extracts of freeze-dried samples. Mineralization of the samples was performed by microwave digestion (MARS X-press, CEM USA). Contents of Pb, Cd and Zn were determined at a wavelength of 217.0, 228.8 and 213.9 nm (respectively) using AAS method and expressed in mg/kg of fresh matter (FM). Determined contents of heavy metals were in the range of 0.020 – 0.630 mg Pb/kg FM, 0 – 0.058 mg Cd/kg FM, resp. 2.265 - 3.457 mg Zn/kg FM. Enhanced levels of heavy metals in soil partially corresponded to their accumulation in potatoes. Moderate statistically significant correlations between the content of heavy metal in soil and that in potato tubers were confirmed as follows: Zn: soil – potato tubers (cv. Marbel – loc. Odorin, Belusa, cv. Red Anna – loc. Odorin, cv. Volumia – loc. Imel), Pb: soil – potato tubers (cv. Red Anna – loc. Odorin, cv. Cicero and cv. Volumia – loc. Imel), resp. Cd: soil – potato tubers (cv. Laura – loc. Spissky Stvrtok). According to EC No 1881/2006 the maximal level 0.01 mg/kg FM for Cd as well as for Pb (for potatoes the maximum level applies to peeled potatoes) are given. The maximum limit for Zn is not given by this regulation. According Food Codex valid in the Slovak Republic (in which the maximum level for Zn in potatoes 10.0 mg Zn/kg FM is given) as well as according EC No 1881/2006 the allowed maximum values for Zn and Cd in potatoes were not in any cultivar exceeded. The maximum allowed amount of Pb was exceeded in cultivars Marabel (loc. Odorin, Belusa), Cicero (loc. Ivanka pri Nitre), Laura (loc. Spissky Stvrtok) and in all potato cultivars from locality Imel.

Varietal dependence as the most significant factor participating on the production and quality of potatoes (content of starch and other chemical compounds such as carbohydrates, organic acids, minerals, vitamins, amino acids, glycoalkaloids), was also reflected in a different extent of accumulation of heavy metals in the tubers, when in 80% of investigated cultivars statistically significant differences were confirmed (Multiple Range Tests for heavy metal by a variety Method: 95.0 percent LSD).

Acknowledgements: The financial support by project VEGA1/0456/12 is greatly appreciated.

<figure><img src="image_203.png" /></figure>

<footnote n="1">¹ Dept. of Chemistry, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic</footnote>

<footnote n="2">² Dept. of Chemistry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic</footnote>
# EVALUATION ABILITY TO ACCUMULATE Cd AND Zn OF ARABIDOPSIS HALLERI SSP. GEMMIFERA IN FIELD AND HYDROPONIC STUDY

Kazuki Sugawara<fnref n="1" />, Xia Wen<fnref n="1" />, Yi Huang<fnref n="2" />, Keisuke Miyauchi<fnref n="2" />, Ginro Endo<fnref n="2" />, Nobuyuki Kitajima<fnref n="3" />, Chihiro Inoue<fnref n="1" />

Corresponding author email: sugawara@er.kankyo.tohoku.ac.jp

## ABSTRACT

Cadmium (Cd) is one of the most toxic heavy metal and concerned for human health in the world. Cd contamination of agricultural field and water causes serious problem in human health (ex. Itaiitai disease) through the food chain. Therefore, it is required that to clean them up from an environment at minimal cost and environmental load. *Arabidopsis halleri* ssp. *gemmifera*, known as Cd and Zinc (Zn) hyperaccumulator, has been researched for using phytoextraction in recent years, however detail of Cd and Zn accumulation pathway and characteristics remains obscure. In the present study, we conducted that field experiment in Cd and Zn contaminated site using *A. halleri* to evaluate Cd and Zn accumulation from the field in Miyagi Prefecture, Japan. In addition, characteristics of Cd and Zn accumulation were investigated under hydroponic condition.

Field trial of Cd and Zn phytoextraction was conducted from September, 2012 to May, 2013. *A. halleri* ssp. *gemmifera* was planted in the field by seed sowing and seedling transplantation, and then harvested after 9 months. During the experiment, plant samples (n=5) and soil core samples (n=4) were collected from each plot in December 2012, March, April and May, 2013. Harvested plant was washed with tap and milli Q water, then dried for 2 days in 60°C. Dried sample was digested with HNO₃ using wet ashing method. Soil sample was screened 2 mm sieves then digested using inverse aqua regina. Cd and Zn concentration of each sample was determined by ICP-MS. In the hydroponic study, *Arabidopsis halleri* ssp. *gemmifera* was conducted pre-cultivation using 1/5 Hoagland solution for 2 weeks. The plant was transplanted to 5 mM MES buffer which contained 5 µM of Cd and 5, 50, 500 µM of Zn and cultivated for 24 hours then harvested. During cultivation, part of a solution was collected in 1, 2, 4, 6, 8, 12, 24 hours from the start. Cd and Zn concentration of plant and solution was determined same way of the field trial.

In the field study, the biomass of seedling transplantation was 2.1 t/ha and it was three times higher than seed sowing. Cd and Zn concentration of the plant was maintained constant during the experiment. Final Cd concentration of seedling and sowing was about 1,400 mg/kg DW and 850 mg/kg DW (Fig.1) and Zn concentration was both of 12,500 mg/kg DW (Fig.2). Cd and Zn removal by plant was 1.8 kg/ha (23% of soil content) and 27.4 kg/ha (3% of soil content). Bioaccumulation factor (BF) of Cd showed almost 200, it was 10 times higher than that of Zn. These results suggested that the plant has independent pathway of Cd and Zn, additionally plant more accumulates Cd actively than Zn. In the 24 hours hydroponic experiment, Cd concentration of the solution was declined until 12 hours. However, decline ration was depended on Zn concentration (Fig.3). It was suggested that Cd and Zn adsorption to surface of the root was competitive. Interestingly, Cd concentration of shoot was raised with Zn concentration of solution. This result represented *A. halleri* ssp. *gemmifera* accumulated much Cd even Zn existed 100 fold higher in solution.

In conclusion, in the field experiment, *A. halleri* ssp. *gemmifera* showed 10 times higher BF values of Cd than its Zn. In hydroponics, it was suggested Cd and Zn accumulation pathway was independent.

**Acknowledgments:** The financial support by Mitsui Co. Ltd. Environment fund, Japan (Grant No. R11-F1-011).

<figure><img src="image_204.png" /><figcaption>Fig.1 Changes of Cd concentration in shoots of *Arabidopsis halleri* ssp. *gemmifera*</figcaption></figure>

<figure><img src="image_205.png" /><figcaption>Fig.2 Changes of Zn concentration in shoots of *Arabidopsis halleri* ssp. *gemmifera*</figcaption></figure>

<figure><img src="image_206.png" /><figcaption>Fig.3 Changes of Cd concentration in solution in 24h.</figcaption></figure>

<figure><img src="image_207.png" /></figure>

<footnote n="1">¹Graduate School of Environmental Studies, Tohoku University, Japan</footnote>

<footnote n="2">²Faculty of engineering, Tohoku Gakuin University, Japan</footnote>

<footnote n="3">³Fujita Co., Japan</footnote>
# LOCALIZATION OF SOYBEAN HMA8 TRANSPORTER AND ITS IMPLICATION IN COPPER HOMEOSTASIS

**Diana Sancho**<fnref n="1" />, **Marinus Pilon**², **Rafael Picorel**<fnref n="1" />, **María Bernal**<fnref n="1" />,#

Corresponding author email: diana.sancho@eead.csic.es

## ABSTRACT

Copper (Cu) is an essential micronutrient in living organisms. In plants, the Cu physiological content is generally in the range of 5–20 µg g⁻<fnref n="1" /> in the vegetative organs [1]. However, the Cu concentration in soil has a wide variation. Concentrations less than 5 µg g⁻<fnref n="1" /> were observed in different countries, such as Belgium, Croatia or Greece, whereas values higher than 100 µg g⁻<fnref n="1" /> were also found regularly in several countries as Italy, Norway or Spain [2]. An appropriate Cu concentration in the plant is essential for its correct development, therefore, the study of Cu status in crops has an important agronomical interest. Furthermore, we have seen in previous works that Cu limiting conditions also induced a decrease in iron (Fe), another essential element [3]. To overcome this disadvantage, plants have developed a complex homeostatic network to maintain the Cu concentration in the tissues under physiological values. In that network, there are several proteins involved in the uptake and distribution of Cu, such as the heavy metal ATPase 8 (HMA8), responsible for Cu (I) transport across the thylakoid membranes [4].

In the model plant *Arabidopsis* (A.) *thaliana*, there is only one gene encoding for HMA8, whereas in soybean (Glycine max (L.) Merr), there are two different genes subjected to alternative splicing, *HMA8-1* and *HMA8-2*. *HMA8-1* encodes a complete membrane protein and *HMA8-2* encodes a putative small truncated protein. The expression of both genes has been studied by quantitative real time PCR in soybean plants grown hydroponically under different Cu regimes. In mature leaves, the *HMA8-1* expression is downregulated by Cu, whereas the *HMA8-2* expression is upregulated. These results suggest that *HMA8-1* and *HMA8-2* could perform complementary functions related to Cu regulation. To further investigate the function of the HMA8-2 protein, its cellular localization was analysed using a C-terminus YFP tagged construct in combination with transient expression experiments in A. *thaliana* protoplasts. Preliminary results indicated that the HMA8-2 protein was localized in the chloroplast. It is thought that the chloroplast is the organelle that contains most of the cellular Cu pool in plants [5], suggesting that the localization of HMA8-2 may be in agreement with a role of this protein in Cu homeostasis. The study of the Cu homeostasis in soybean, an important crop plant, may be of agronomical importance. Therefore, the study of the regulation and localization of the Cu-related genes and proteins HMA8-1 and HMA8-2 will improve our understanding of plant Cu homeostasis, facilitating the selection of plants that may be able to compete in limiting nutrient soils as well as heavy metal tolerant plants used in phytoremediation.

## References:

1. Marschner, H. (1995) Ion Uptake Mechanisms of Individual Cells and Roots: Short-distance Transport. In Mineral nutrition of higher plants, Elsevier Press, pp. 344-346.

2. Heijerick, D. G., Van Sprang, P. A., Van Hyfte, A. D. (2006) Ambient Copper Concentration in Agricultural and Natural European Soils: an Overview. Environmental Toxicology and Chemistry, 25, 858-864.

3. Bernal, M., Casero, D., Singh, V., Wilson, G. T., Grande, A., Yang, H., Dodani, S. C., Pellegrini, M., Huijser, P., Connolly, E. L., Merchant, S. S., Krämer, U. (2012) Transcriptome Sequencing Identifies SPL7-Regulated Copper Acquisition Genes FRO4/FRO5 and the Copper Dependence of Iron Homeostasis in Arabidopsis. The Plant Cell, 24, 738-761.

4. Abdel-Ghany, S. E., Müller-Moulé, P., Niyogi, K. K., Pilon, M., Shikanai, T. (2005) Two P-type ATPases are required for copper delivery in *Arabidopsis thaliana* chloroplasts. The Plant Cell, 17, 1233-1251.

5. Pilon, M., Ravet, K. (2013) Copper and Iron Homeostasis in Plants: the Challenges of Oxidative Stress. Antioxidants & Redox Signalling, 19, 919-932.

<figure><img src="image_208.png" /></figure>

<footnote n="1">¹Estación Experimental de Aula Dei-CSIC, Zaragoza, Spain</footnote>

<footnote n="3">³Department of Biology, Colorado State University, Fort Collins CO, USA</footnote>

<footnote>#Present address: Department of Plant Physiology, RUHR, Bochum, Germany</footnote>
# HEAVY METAL EFFECTS ON SUNFLOWERS: BIOMASS PRODUCTION AND ACCUMULATION CAPACITY

Xin Zhao<fnref n="1" />, Dongwook Kim<fnref n="2" />, Jae Young Kim<fnref n="1" />

Corresponding author email: jaeykim@snu.ac.kr

## ABSTRACT

Phytoremediation is an emerging technology using selected and engineered metal-accumulating plants for environmental clean-up. Sunflower (*Helianthus annuus* L.) has been known as one of the metal-accumulating plants. In this study, biomass productions and heavy metal accumulation capacities of sunflowers were monitored in the heavy metal contaminated soils and the correlation was analyzed.

Sunflower seeds were obtained from a commercial market, and the specie and cultivar is frequently and widely cultivated in the Republic of Korea. Soil specimen was collected from a farmland in the Gyeongsangnam-do, the Republic of Korea was used after taking a series of treatments. It was air-dried, mixed thoroughly and then screened through a 2 mm (10 mesh) sieve to remove large particles. The soil was then amended with the heavy metals: Pb as PbCl₂; Cd as CdCl₂·2.5H₂O and Cu as CuCl₂·2H₂O. Each heavy metal concentrations of soil used in this study were 250, 1,000, and 2,000 mg-Pb/kg; 50, 100, and 600 mg-Cd/kg and 100, 300, and 450 mg-Cu/kg. The sunflowers were grown for 98 days in a greenhouse. After the 30 days, 65 days and 98 days, the sunflowers were harvested. The biomasses of sunflowers were measured and Pb, Cd and Cu in sunflower was analyzed by an inductively coupled plasma optical emission spectrometer (ICP-OES).

All values are presented as average values ± standard deviation (SD) obtained with at least three replicates. Student t-tests were used to assess differences between the sunflower biomass of 65 days and that of 98 days. Differences of heavy metal mass in sunflower among heavy metal treatments were determined using ANOVA, according to Duncan's multiple range test. A significance level of P < 0.05 was used throughout the study. The IBM SPSS statistics (Version 21.0) analysis program was used for the statistical analyses.

<table><caption>Table 1. Heavy metal accumulation by sunflower after 98 days growth</caption><thead><tr><th>Heavy metal</th><th>Conc. (mg/kg)</th><th>Mass (mg)</th><th>Heavy metal</th><th>Conc. (mg/kg)</th><th>Mass (mg)</th><th>Heavy metal</th><th>Conc. (mg/kg)</th><th>Mass (mg)</th></tr></thead><tbody><tr><td rowspan="3">Pb</td><td>250</td><td>90.40 ± 10.16a</td><td rowspan="3">Cd</td><td>50</td><td>225.29 ±<br/>112.69b</td><td rowspan="3">Cu</td><td>100</td><td>91.28 ±<br/>31.49a</td></tr><tr><td>1,000</td><td>362.06 ± 23.69a</td><td>100</td><td>305.81 ±<br/>40.65b</td><td>300</td><td>192.06 ±<br/>41.46b</td></tr><tr><td>2,000</td><td>719.71 ± 264.72b</td><td>600</td><td>0.00 ± 0.00a</td><td>450</td><td>40.69 ±<br/>15.09a</td></tr></tbody></table>

Values followed by the same letters are not significantly different at (P > 0.05), Means ± SD, n=3

The biomass productions of sunflower in 65 days and 98 days were not significantly changed. The accumulated Pb in sunflower was increased as Pb concentrations increased in soil. There was no harvest in 600 mg-Cd/kg contaminated soil because of high toxicity. Accumulated Cd in 50 and 100 mg-Cd/kg were not significantly different from each other. Sunflower has the larger accumulation capacity in 300 mg-Cu/kg than those in 100 and 450 mg-Cu/kg.

According to the results, the sunflower has a relatively stronger tolerance to Pb contaminated soil than that to Cd and Cu contaminated soil. That means sunflower is a suitable remediation tool in high Pb contaminated soil.

## References:

1. Kumar, P.B.A.N., Dushenkov, V., Motto, H., Raskin, I., 1995. Phytoextraction-the use of plants to remove heavy metals from soils. Environ. Sci. Technol. 29, 1232-1238.

2. IBM SPSS statistics, 2013. Statistical Software ☐ Datasolution Korea Inc.

Acknowledgements: This work is supported by the Korea Ministry of the Environment as "The GAIA (Geo-Advanced Innovative Action) Project".

<figure><img src="image_209.png" /></figure>

<footnote n="1">¹ Department of Civil and Environmental Engineering, College of Engineering, Seoul National University, Seoul, Republic of Korea</footnote>

<footnote n="2">² Phygen Inc., Daejeon, Republic of Korea</footnote>
# ELEUSINE INDICA - A NEWLY DISCOVERED ANTIMONIC HYPERACCUMULATOR PLANT

Yuan YiNing

School of aeronautical mechanical manufacturing engineering of Changsha Aeronautical Vocational and
Technological College, Changsha 410014, China

Corresponding author email: 271312819@qq.com

## ABSTRACT

China is the main reserves area of antimony. According to the review about the world's mineral resources issued by U.S. Geological Survey in 2011, there are 52.8 percent of the world's proven reserves of antimony in China; it ranks first in the world. The antimony reserves area of china is mainly distributed in Xi Kuang Shan in Leng Shui Jiang city of Hu Nan province. Xi Kuang Shan is called as the stibium capital of the world. To know the antimonic pollution status in the mine of Xi Kuang Shan, the antimonic concentration in the plants and the soils in the mine of Xi Kuang Shan were investigated. Different varieties of plants in the antimony mine area of Xi Kuang Shan including the factory area and the residential area are collected random. The topsoils of the plants grown are also collected. The plant samples were washed and divided into overground parts and underground parts and then cutted into pieces (<1 cm²) then air dried. The soil samples were air dryed and grinded and sieved with 100 mesh. The air dryed plant and soil samples were digested with HNO₃ and HClO₄, antimonic concentration in the digestive solution was measured with inductively coupled plasma atomic emission spectrometry (ICP-AES). Unexpected, it was found that *Eleusine indica* is an antimonic hyperaccumulator plant! The investigation results show that the average antimonic mass fraction in the overground parts of *Eleusine indica* reaches 1864.7 kg mg⁻¹, the range is about 933.8~2638.9 kg mg⁻¹; the average antimonic mass fraction in the underground parts of *Eleusine indica* is only 321.7 kg mg⁻¹, the range is about 163.8-502.6mg mg kg⁻¹; the average antimonic concentration in the soil is 102.3 mg kg⁻¹, the range is about 81.2-110.0 mg kg⁻¹. It is the first time that *Eleusine indica* is discovered as an antimonic Hyperaccumulator plant and it is the first time that the antimonic hyperaccumulator plant is discovered in China, this discovery fills in gaps on antimonic hyperaccumulator plant in china, it supply a new plant resource for phytoremediation of antimonic contaminated soil. Howerever *Eleusine indica* is only proved as an antimonic hyperaccumulator plant by field investigation. Artificial cultivation experiments have not been executed yet. Antimonic hyperaccumulation mechanism and characteristics of *Eleusine indica* have not been researched yet, especially that whether antimony is hyper-accumulated through underground parts of *Eleusine indica* from the soil or it is hyper-accumulated by overground parts of *Eleusine indica* from the air. These are the further research contents.

<figure><img src="image_210.png" /><figcaption>Fig.1 The collected place of Eleusine Indica in Xi Kuang Shan in Leng Shui Jiang city of Hu Nan province in China</figcaption></figure>

<figure><img src="image_211.png" /></figure>
# EFFECTS OF CADMIUM STRESS IN STRAWBERRY PLANTS

Hatice Gulen<fnref n="1" />, Zeynep Zaimoglu<fnref n="2" />, Fuat Budak<fnref n="2" />, Muge Kesici Zengin<fnref n="1" />, Sergul Ergin<fnref n="3" />, Sevgi Demir<fnref n="1" />, Atilla Eris<fnref n="4" />

## ABSTRACT

The effects of water-applied cadmium (Cd) treatments on antioxidative enzyme activities were investigated in strawberry (Fragaria x ananassa cv. Camarosa) plants. Cold stored seedlings were grown in perlite:torf:soil (1:1:1) mixture in a greenhouse. Four weeks after planting, plants were treated by Actagro (7-7-7) nutrient solution containing 0, 500 and 1000 mg/l Cd (CdSO₄) during 8 weeks. Fully expanded leaf and roots were collected from the plants at the end of the treatments for the analyses of cell membrane injury, lipid peroxidation (Malondialdehyde, MDA content) and chlorophyll content. In general, Cd treatments caused a linear increase in cell membrane injury and MDA content in both leaf and root issues with increasing Cd concentrations. Root tissues were more injured than leaf tissues by Cd treatments. The accumulation of Cd in root of the plants was determined higher than leaf, which were parallel to cell membrane injury and MDA content. In addition Cd treatments significantly increased the chlorophyll contents of the leaf tissues in comparison to control plants.

<figure><img src="image_212.png" /></figure>

<footnote n="1">¹Horticulture Department, Faculty of Agricultural Engineering, Uludag University, Bursa, Turkey</footnote>

<footnote n="2">²Environmental Engineering Department, Faculty of Engineering and Architecture, Cukurova University, Adana, Turkey</footnote>

<footnote n="3">³Department of Agricultural Biotechnology, Faculty of Agriculture, Eskisehir Osmangazi University, Eskisehir, Turkey</footnote>

<footnote n="4">⁴Department of Genetic and Bioengineering, Faculty of Engineering, Istanbul Bilgi Universiy, Istanbul, Turkey
*Corresponding author email: hsgulen@uludag.edu.tr*</footnote>
# IMPORTANT ROLE OF ENHANCED LEVEL OF GLUTATHIONE IN CRAMBE ABYSSINICA TO DETOXIFY Ag NPs AND Ag⁺ IONS

Chuanxin Ma<fnref n="1" />, Craig Musante<fnref n="2" />, Stephanie Long<fnref n="3" />, Rakesh Minocha<fnref n="3" />, Jason C. White<fnref n="2" />, Baoshan Xing<fnref n="1" />,
Om Parkash Dhankher<fnref n="1" />

Corresponding author email: parkash@umass.edu

## ABSTRACT

Widespread use of silver nanoparticles (AgNPs) in the environment has raised some serious concerns about their adverse effects on the environmental and human health. However, the biochemical mechanisms that plants uses to detoxify the nanotoxicity are not understood well. *Crambe abyssinica* (a member of Brassicaseae), which is capable of tolerating abiotic stresses, such as cold, salt, and heavy metal, is an ideal industrial oil seed crop for phytoremediation of toxic metals, biofuels, and bioproducts. The importance of glutathione (GSH) in heavy metals and xenobiotic detoxification is well established. In this study, we engineered crambe plants overexpressing *E. coli* γ-glutamylecysteine synthase (γ-ECS), which is critical in GSH biosynthesis pathway. Several-folds higher levels of GSH were shown in all transgenic plant tissues compared to wild type control plants. In order to demonstrate that enhanced levels of GSH can protect plants from Ag nanotoxicity, two independent transgenic lines of crambe were exposed to various concentrations of Ag NPs and AgNO₃ (Ag⁺ ions). Compared to WT plants, γ-ECS crambe showed higher tolerance and attain significantly higher fresh biomass along with the increase in total chlorophyll contents and transpiration rate. Moreover, transgenic lines can take up more Ag and translocate in shoots. Analysis of the levels of cysteine, γ-EC, GSH and its derivatives phytochelatins (PCs), showed significantly higher levels of these peptides. Additionally, exposure of transgenic lines to Ag NPs and Ag⁺ ions further increased the levels of γ-EC, GSH, and PCs in transgenic lines compared to WT plants. These results indicate that metabolic pathway for GSH biosynthesis was strongly induced to detoxify Ag nanotoxicity. On the other hand, analysis of the important nutrient elements (such as Ca, K, Fe, Zn, Mg, P, and Mn) in plants showed that the levels of both exchangeable P and Zn in transgenic lines treated with Ag NPs and AgNO₃ were significantly higher than control groups. All other elements were not affected. These results clearly show that GSH is involved in the detoxification of Ag NPs and AgNO₃ (Ag⁺ ions). Last, but not the least, polyamines (PAs) biosynthetic pathway can protect plants from abiotic stresses. Analysis of the regulations of putrescine (Put), spermidine (Spd) and spermine (Spm), showed that Put level was strongly induced in transgenic lines treated with Ag NPs and Ag⁺ ions compared to transgenic control. There were no significant changes of the level of Put in WT plants. Also, Spd/Put ratio in transgenic lines treated with Ag NPs and Ag⁺ ions was specifically higher than WT plants. However, no significant changes of the levels of Spm were found in both WT and transgenic lines. These results suggest that PAs biosynthetic pathway may also respond to detoxification of Ag NPs. This study is highly helpful to understand the fate, transport and toxicity of NPs in plants and could provide a useful way for remediation NPs in the environment.

**Keywords:** *Crambe abyssinica*, γ-glutamylecysteine synthase, silver nanoparticles, glutathione, putrescine

<figure><img src="image_213.png" /></figure>

<footnote n="1">¹Stockbridge School of Agriculture, Universtiy of Massachusetts Amherst, MA 01003</footnote>

<footnote n="2">²Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA</footnote>

<footnote n="3">³USDA Forest Service, NRS, 271 Mast Road, Durham, NH 03824, USA</footnote>
# THE RELATIONSHIPS BETWEEN CONTENT OF HEAVY METALS IN SOILS AND IN STRAWBERRIES

**Judita Bystricka, Janette Musilova, Pavol Trebichalsky, Jan Tomas, Radovan Stanovic, Daniel
Bajcan, Petra Kavalcova**

Dept. of Chemistry, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Slovak Republic

Corresponding author email: Judita.Bystricka@centrum.sk

## ABSTRACT

Strawberry can grow in a wide range of climatic conditions - from the subtropical regions of the Northern hemisphere to the tropics of the Southern hemisphere. Strawberries are one of the most popular edible fruits in Slovak Republic and their consumption has increased with the development of new varieties available at almost all seasons. Strawberry is a good source of bioactive compounds due to its high level of vitamin C and phenolics. These compounds present antioxidant effects, and therefore a consequent beneficial effect on the maintenance of consumer health [Da Silva Pinto et al., 2008; Giampieri et al., 2012].

The environmental study, carried out in Slovak Republic, was aimed at assessment of quality of strawberry based on the contents of heavy metals (Zn, Cu, Ni, Pb, Cd) as well as the possible correlations between selected heavy metals in soil and strawberries (*Fragaria x ananassa* Duch.). Seven different varieties were analysed (Clery, Sonata, Alba, Korona, Azia, Antea, Joly). Seven sampling sites were selected and the analyses of pH/KCl, P, K, Mg and chosen heavy metals in 2 different extracts were carried out (*aqua regia*, 1 M NH₄NO₃). The soils on which the strawberries were grown, can be characterized as strongly to weakly acidic (pH/KCl = 4.73 - 6.56), with low to moderate supply of humus (% Hum. = 1.27 to 2.24), with low to high phosphorus content (P = 41.89 - 120.41 mg/kg), with good to very high content of potassium (K = 295.7 - 431.70 mg/kg) and good to high content of magnesium (Mg = 192.6 - 300 mg/kg).

The results of our study revealed that from all observed metals in soil determined in *aqua regia* only in the case of cadmium the maximum permissible limit in comparison with the limit resulting from the law no. 220/2004 (valid in the Slovak Republic) as well as threshold values proposed by European Commission (EC) (2006) has been exceeded. In our paper the values of cadmium in the soil ranged in the interval 1.30 - 1.690 mg/kg representing 1.86 to 2.41 times higher values than limit valid in the Slovak Republic (0.7 mg/kg) and 2.6 to 3.38 times higher in comparison to European Commission (EC) (0.5 mg/kg). In our study in 1 M NH₄NO₃ the values of lead ranged from 0.125 to 0.205 mg/kg representing values exceeded the limit valid in Slovak Republic (0.1 mg/kg) about 0.037-0.105 mg/kg. Despite exceeded values of heavy metals in soil, no values above the limit directly in strawberries when compared to Food Codex of Slovak Republic (FC SR) as well as to Commission Regulation 1881/2006 (CR) were recorded. Among the varieties statistically significant differences (P<0.05) in intake of heavy metals were found (Multiple Range Tests for heavy metal by cultivar, Method: 95,0 percent LSD).

**Acknowledgements:** The financial support by project VEGA1/0456/12 and by project VEGA 1/0724/12 is greatly appreciated.

<figure><img src="image_214.png" /></figure>
# STUDY ON As UPTAKE AND REMOVAL BY As HYPER-ACCUMULATOR PTERIS VITTATA AND IT'S RHIZOSPHERE BACTERIA

Mei-Fang Chien<fnref n="1" />,<fnref n="2" />, Kazuki Obata<fnref n="2" />, Yi Huang<fnref n="2" />, Keisuke Miyauchi<fnref n="2" />, Ginro Endo<fnref n="2" />, Chihiro Inoue<fnref n="1" />

Corresponding author email: meifang@tjcc.tohoku-gakuin.ac.jp

## ABSTRACT

Arsenic hyper-accumulators such as *Pteris vittata* have been known applicable for phytoremediation of arsenic-contaminated soil. Previous studies show that *P. vittata* mainly absorbs arsenic as arsenate. Therefore, arsenic uptake by *P. vittata* is largely determined by arsenic speciation, which is strongly influenced by microbial activities. However, little is known about interactions between *P. vittata* and its rhizosphere bacteria. The objective of present study was to characterize the details of arsenic transformation prior to arsenic uptake by *P. vittata* and the participation of the rhizosphere bacteria on arsenic uptake.

Pot experiments of *P. vittata* cultivation and arsenic oxidation in the soil were applied to investigate the transformation and transition of arsenic from the soil to *P. vittata*. The concentration of total arsenic and arsenite in the soil and *P. vittata* was monitored by atomic absorption spectrophotometry. The terminal restriction fragment length polymorphism (TRFLP) targeted to microbial arsenite oxidase gene, *aroA*, was performed to analyze the microbial community in the rhizosphere of *P. vittata*. The results of arsenic analysis showed that the arsenite in the soil was promptly oxidized to arsenate and subsequently absorbed by *P. vittata*. The arsenite oxidation was inhibited by antibiotics, which indicated that bacteria played an important role. The results of TRFLP suggested that four *aroA* containing bacterial species were increased during early phase of cultivation when arsenic oxidation was observed and were subsequently decreased during late phase. The rhizosphere samples of *P. vittata* in the pot experiments were applied to isolate culturable bacteria. Thirty-seven bacterial strains with different morphologies and arsenic tolerance were isolated, and *aroA* gene was detected from four of these isolates. The selected isolate was further investigated about the arsenite oxidative activity, and the results showed that 89.2% of arsenite in the medium was oxidized to arsenate by the isolate after 48 hours incubation. Besides, the rhizosphere re-colonization of this isolate was also confirmed two days after inoculating to *P. vittata*.

According to these results, we concluded that the rhizosphere bacteria play a role in arsenite oxidation prior to arsenic uptake by *P. vittata* and thus may have potential to improve phytoremediation of arsenic-contaminated soil by *P. vittata*.

**Keywords:** arsenic, *Pteris vittata*, phytoremediation, rhizosphere bacteria, arsenite oxidation, *aroA*

**Acknowledgements:** The financial support by Grant-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (no. 25870658), the Project for Promotion of Strategic Establishment of Research Foundation in Private Universities from the Ministry of Education, Culture, Sports, Science and Technology-Japan (MEXT), and the Project from the Mitsui & Co., Ltd. Environment Fund is greatly appreciated.

<figure><img src="image_215.png" /></figure>

<footnote n="1">¹Graduate School of Environmental Studies, Tohoku University, Miyagi, Japan</footnote>

<footnote n="2">²Faculty of Engineering, Tohoku-Gakuin University, Miyagi, Japan</footnote>
# ROOT UPTAKE OF Cs 134 EARLY AFTER RADIOACTIVE FALLOUT

Jan Mihalik

Monitoring, National Radiation Protection Institute, Czech Republic

Corresponding author email: mihalik.radeco@gmail.com

## ABSTRACT

Our investigation is focused on the dynamism of Cs 134 increase in the plant tissues after soil contamination by radioactive fallout. We assume that the harvesting early after fallout could reduce the quantity of the contaminated biomass. Our experiments were carried out with 3 weeks old sunflowers as a model plants in a phytotrone. The 200 ml of the Cs 134 solution with the activity 3 kBq/L was spilt on the top-soil. The first group was harvested after 1 day. The plants were washed. Roots, stems and leaves were separated, dried and finally, their activity was determined by gamma spectrometry.

The main finding is that the harvesting in the first two days could be effective in reduction of the quantity of contaminated biomass. However, the Cs 134 activity in the green biomass was low even at the end of experiment. The leaves of the plants from the first group contained the 5 Bq/kg (dw) or less. From fifth to tenth day the Cs 134 activity concentration in plant tissues still slightly increases but after ten days it remains almost constant. Twenty days after contamination the Cs 134 activity concentration in leaves was 26 Bq/kg (dw). Contrary to leaves, the activity in the roots permanently increased untill the end of the experiment when it reached 430 Bq/kg (dw). It corresponds well with the ability of sunflower roots to adsorb a high quantity of cesium from contaminated water. The lowest activity was catched in the stems.

This approach is applicable in the case when the negligible quantity of radionuclides is retained on plants during rain.

<figure><img src="image_216.png" /></figure>
# VARIOUS SPECIES FOR PHYTOREMEDIATION OF Ag, Cu AND Zn IN DIFFERENT CONCENTRATIONS FROM A SOIL MATRIX

Dominic Desjardins, Frédéric E. Pitre, Werther Guidi Nissim, Michel Labrecque

Institut de recherche en biologie végétale, Montréal, Canada

Corresponding author email: dominic.desjardins@hotmail.com

## ABSTRACT

Transition metals are often present in post-industrial soils in concentrations that require specialized attention because of their potential health risk for nearby biota. Two different soils (S1 and S2) contaminated with high level of Ag (34.6 and 113.6 mg kg⁻¹ respectively), as well as with Cu (47.5 and 20.0 mg kg⁻¹ respectively) and Zn (162.2 and 177 mg kg⁻¹ respectively), were collected from the same brownfield in eastern Montréal (Canada). In a pilot experiment, four plant species (*Brassica juncea*, *F. arundinacea*, *Medicago sativa* and *Salix miyabeana*) were grown in different pots containing the soils to investigate their potential use in phytoremediation of the contaminated site. Analysis showed that the only influence of soil type on plant growth was to reduce aerial biomass production of *F. arundinacea* grown in S2 soil compared to S1. Higher Ag concentrations were found in underground parts of all species grown in S2 substrate compared to S1. The three herbaceous species performed equally well, and all were more effective than *S. miyabeana* in uptake of Ag in both substrates. For Cu and Zn, no difference in root concentrations was recorded between substrates. *F. arundinacea* showed the highest root concentration of Cu, while all species showed similar results for Zn, regardless of substrate. The highest aboveground concentration of Ag was observed in *B. juncea* grown in S2 soil (12.2 mg kg⁻¹); *S. miyabeana* was the only specie translocating Zn aboveground, performing equally in both substrates (mean=118.25 mg kg⁻¹). To our knowledge, this is the first study to demonstrate bioaccumulation and translocation of Ag by higher plants from a soil matrix. These findings suggest that *B. juncea* could be used as a phytoextractor of Ag from a soil matrix, and that the four species have potential for use in complementarity to remediate an Ag/Cu/Zn contaminated brownfields.

<figure><img src="image_217.png" /></figure>
# THE EFFECT OF SUPER ABSORBENT POLYMER ON PHYTOEXTRACTION OF TRACE ELEMENTS

Dongwook Kim<fnref n="1" />, Seong Min Woo<fnref n="1" />, Jaehong Yim<fnref n="1" />, Jongchan Park<fnref n="2" />, Eunjin Lee<fnref n="2" />, Bomin Kang<fnref n="2" />, Gwang Hyun Han<fnref n="2" />

Corresponding author email: dwsoil88@daum.net

## ABSTRACT

In general the mechanism of plant root uptake is classified to root interception, mass flow and diffusion. Plant root distribution is an essential factor to determine the capacity of phytoextraction. However the direct uptake by root interception is not sufficient because most of ions in soils are taken up through mass flow or diffusion. Therefore soil water movement forward to rhizosphere can be more critical. In this study, super-absorbent polymer (SAP) was used to make a matric potential gradient of soil water between rhizosphere and its ambient soil. Sunflower was planted with SAP and without SAP. The concentration of SAP treatment was 0.1% in the soil volume basis. During test period water content was monitored. Sunflower biomass yield and the TE concentration in plants were also analyzed. Water content of soil with SAP remained 2~5% higher than one without SAP. Biomass yield on soil with SAP was estimated higher: in the SAP treated case, the yield of root, stalk, leaf, flower, and seed was 2,125, 6,909, 2,710, 2,435, 2,756 kg ha⁻<fnref n="1" /> respectively, in the SAP non treated case, 1,338, 4,449, 1,906, 1,892, 2,004 kg ha⁻<fnref n="1" />. The result of the analysis of Cd, Cu, Zn, and Pb in each plant part showed some tendency that these ions were accumulated more in the harvestable parts, especially stalks, when SAP was treated. From this test it is expected that SAP treatment can be used as an alternative soil conditioner to enhance the phytoextraction of TE.

**Acknowledgements:** The financial support by Korea Ministry of Environment as "the GAIA Project (2012000550023) is greatly appreciated.

<figure><img src="image_218.png" /></figure>

<footnote n="1">¹Phygen Inc., Daejeon, South Korea</footnote>

<footnote n="2">²Department of Environmental and Biological Chemistry, Chungbuk National University, Cheongju, South Korea</footnote>
# SACCHARIFICATION OF SUNFLOWER STALKS WITH HIGH METAL CONTENT USING LIGNOCELLULASES FROM A FUNGAL CONSORTIUM COMPRISING *PHOLIOTA ADIPOSA* AND *ARMILLARIA GEMINA*

Priyadharshini Ramachandran<fnref n="1" />, Tae-Su Kim<fnref n="1" />, Yun Chan Kang<fnref n="1" />, Dongwook Kim<fnref n="2" />, Jung-Kul Lee<fnref n="1" />

Corresponding author email: jkrhee@konkuk.ac.kr

## ABSTRACT

Lignocellulases from *Armillaria gemina* and *Pholiota adiposa* are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising *P. adiposa* and *A. gemina* were used for the saccharification of sunflower stalks. Sunflower stalks were thermo-chemically pretreated using 2% NaOH at 50°C for 24 h. The saccharification process parameters including enzyme loading, substrate concentration, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3%) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5%, temperature 50°C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme β-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars.

**Acknowledgements:** The financial support by Korea Ministry of Environment as "the GAIA Project (2012000550023) is greatly appreciated.

<figure><img src="image_219.png" /></figure>

<footnote n="1">¹Department of Chemical Engineering, Konkuk University, Seoul, South Korea</footnote>

<footnote n="2">²Phygen Inc., Daejeon, South Korea</footnote>
# PHRAGMITES AUSTRALIS' DEFENSIVE RESPONSE TO METAL CONTAMINATION

A. Cristina S. Rocha<fnref n="1" />, C. Marisa R. Almeida<fnref n="2" />, M. Clara P. Basto<fnref n="1" />, M. Teresa S. D. Vasconcelos<fnref n="2" />

*Corresponding author email: cristinasrocha@gmail.com*

## ABSTRACT

Saltmarshes are complex coastal ecosystems of great productivity and environmental relevance. Nevertheless, these ecosystems are constantly subject to anthropogenic pollutants. Saltmarsh plants have been proven to influence the distribution and bioavailability of metals in rhizosediment being also able to uptake and bioaccumulate considerable amounts of this kind of pollutants [1,2]. For these reasons, saltmarsh plants have been recognised as potential phytoremediators/phytostabilisers. Halophytes seem to present advantages over non-halophytic plants since they are able to survive and reproduce in salt-rich environments [3], feature that may ensure their tolerance to other environmental stresses as metal contamination. This ease to cope with harsh environmental conditions is thought to be associated to efficient internal biophysicochemical mechanisms.

As knowledge on this matter is still limited, a battery of studies was performed with *Phragmites australis*, a perennial macrophyte, commonly found in European temperate saltmarshes. The purpose of this work was to attain a better understanding how metal contamination could condition the liberation of aliphatic low molecular weight organic acids (ALMWOAs) and the production of antioxidant thiolic compounds (as cysteine (Cys), oxidised glutathione (GSSG) and reduced glutathione (GSH)) [4]. Experiments were performed in three complementary media: (I) freshwater from the river (a natural medium simpler than estuarine water); (II) rhizosediments soaked in the respective elutriates; and (III) rhizosediment cores containing *P. australis* stands (a microcosm scale up study).

Results demonstrated that contamination by metals, particularly Cd and Cu, could trigger several specific responses in the marsh plant. For instance, experiment I showed that the liberation of oxalic and citric acids by *P. australis* roots was influenced by the exposure to Cd or Cu contaminated freshwater, being the plant response dependent upon the metal. Likewise, in experiment II, both metals could induce a specific influence on the production of thiolic compounds in roots and leaves cells. A short-term exposure of *P. australis* to metal (Cd or Cu) contaminated rhizosediments soaked in the respective elutriates caused a disequilibrium in cell's homeostasis, which was confirmed in experiment III. In fact, in experiment III, there was a significant decrease of GSSG levels in roots cells and of Cys, GSH and GSSG levels in leaves cells of *P. australis* after short-term exposure to Cd-contaminated sediment. However, after long-term exposure (two months) to this Cd-contaminated sediment, *P. australis* re-established the basal levels (levels in plants not exposed to contaminated sediment) indicating that defensive mechanisms involving thiolic compounds were activated in the organs of the plant. Furthermore, no external toxicity symptoms were observed in the specimens in any of the experiments. Considering the remarkable ability to cope with contaminated environments and harsh conditions allied to its ability to bioaccumulate metals, *P. australis* is therefore a suitable choice for phytoremediation procedures.

Estuarine ecosystems are very sensitive areas, their management and phytoremediation strategies facing severe challenges. For this reason, pursuing research on the dynamics behind salt marsh plants' success to cope with the harsh conditions of marsh areas and to remove pollutants from sediments is utterly important and will contribute for the planning of more efficient remediation projects of impacted marsh areas.

*Acknowledgements:* This work was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT (Portugal) for PTDC/MAR/099140/2008, PesT-C/MAR/LA0015/2011, REEQ/304/QUI/2005 and PhD fellowship (SFRH/BD/38780/2007) co-financed by POPH/FSE.

## References:

1. C. M. R. Almeida, A. P. Mucha and M. T. S. D. Vasconcelos, (2004) *Environmental Science & Technology* 38 (11), 3112-3118

2. C. Padinha, R. Santos and M. T. Brown, (2000) *Marine Environmental Research* 49 (1), 67-78

3. E. Manousaki and N. Kalogerakis, (2011) *Industrial & Engineering Chemistry Research* 50, 656-660

4. A. C. Rocha, PhD Thesis, (2013) Faculty of Sciences of Oporto University, Portugal

<figure><img src="image_220.png" /></figure>

<footnote n="1">¹ CIIMAR / CIMAR and Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007, Porto, Portugal</footnote>

<footnote n="2">² Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR / CIMAR), Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal</footnote>
# INPUT OF RISK METALS INTO FABA BEAN CULTIVATED IN TARGETED CONTAMINATED SOIL IN MODEL CONDITIONS

Mária Timoracká, Alena Vollmannová, Dalaram S. Ismael, Janette Musilová

Dept. of Chemistry, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic

Corresponding author email: maria.timoracka@uniag.sk

## ABSTRACT

Agricultural production is the main source of foodstuffs, it is important to evaluate negative effects of risky elements on quality of agricultural products. Metals, such as iron, zinc and manganese are essential metals, since they play an important role in biological systems. Cu and Zn are essential micronutrients, they can be toxic when taken in excess. Lead and cadmium are nonessential metals as they are toxic, even in trace - these elements are present in the soil above the hygienic limit on most territory of the Slovak Republic. We investigated the effect of accumulation of heavy metals in faba bean grown in model conditions in the targeted contaminated soil with increasing rates of the selected heavy metals (cadmium, lead, zinc and copper, separately).

For cvs. Zobor and Saturn, four variants in each of the experiments were realised: A: control (without Zn or Cu addition), B: 40 mg Zn.kg⁻¹ or 20 mg Cu.kg⁻¹ of soil (hygienic limit), C: 250 mg Zn.kg⁻¹ or 50 mg Cu.kg⁻¹ of soil (half dose of analytically significant contamination, D: 500 mg Zn.kg⁻¹ of soil or 100 mg Cu.kg⁻¹ (analytically significant contamination). Faba beans were harvested in full ripeness. For cv. Merlin were made 4 variants: A-control (with basic ferilization) and cadmium and lead was applied in gradual specific doses with 5 (variant B), 10 (variant C), 15 (variant D) multiple as the limit value by the Law no. 220/2004 Z.z. to assess the state of soil contamination.

Trace elements (Zn, Cu, Co, Ni, Cr, Pb, Cd) - in soil and matter - were determined using a Varian AA240FS atomic absorption spectrometer equipped with a D2 lamp background correction system, using an air-acetylene flame. Pseudototal content of risk metals was assessed in soil extract by *aqua regia* and content of mobile forms of selected heavy metals in soil extract by NH₄NO₃ (c =1 mol.dm⁻³) and in the solution of 2 M HNO₃ was determined. The results were evaluated according to the legislative valid in the Slovak Republic. The soil from two localities Cakajovce (cvs Saturn and Zobor) and Vycapy-Opatovce (cv. Merlin) used in the pot trial was uncontaminated.

Faba beans matter was harvested in full ripeness. The samples of faba bean were analysed after the decomposition by HNO₃ on the microwave digestion. Gained results was evaluated by the content of risky elements in seeds were evaluated according to the Food Codex of the Slovak Republic.

With increased Zn (Cu) doses applied into the soil the strong statistical relationship between soil Zn (Cu) content and Zn (Cu) amount in seeds of both of investigated faba bean cultivars was confirmed. Despite of high Zn doses applied into the soil in model conditions, in all variants the determined Zn amount in seeds of faba bean cv. Saturn was just below the maximal allowed content in foodstuffs given by the legislative. In cv. Zobor the determined Zn content was in C as well as in D variant higher than maximal allowed amount (by 2% and 12%, respectively). In variants with Cu application the determined Cu contents were far below the limit value. Faba bean seeds cvs. Saturn and Zobor in full ripeness accumulated (in all variants higher than hygienic limits) high amounts of Pb and Cd (Zn application into soil) and amounts of Pb and Ni (Cu application). The contents of all other heavy metals were lower than hygienic limits. In variants with Zn application the determined Zn and Cu contents in seeds were increased, Cd content was decreased and contents of Ni, Cr and Pb were changed only slightly. In variants with Cu application the determined elements were changed only slightly.

With increased Cd doses applied into the soil the relationship between soil Cd content and Cd amount (R=0,92) as well as Zn and Cd values in seeds of investigated faba bean cultivar Merlin was confirmed. In all variants the determined Cd and Ni amount in seeds was exceeded the maximal allowed content in foodstuffs given by the legislative.

With increased Pb doses applied into the soil the relationship between soil Pb content and Pb amount as well as Zn, Pb and Cd values in seeds of investigated faba bean cultivar Merlin was not confirmed. The accumulation of lead into seed was lower in comparison to cadmium. The determined Pb content was in D variant higher than maximal allowed amount (by 45%). In all variants the determined Cd amount in seeds was exceeded the maximal allowed content in foodstuffs given by the legislative.

Acknowledgements: This work was co-funded by European Community under project No. 26220220180: Building Research Centre "AgroBioTech" and also supported by project: VEGA 1/0308/14.

<figure><img src="image_221.png" /></figure>
# OPTIMIZING VALORIZATION OF METAL-CONTAMINATED SOILS USING SRC OF WILLOW, PLANT-ASSOCIATED BACTERIA AND FERTILIZERS

Jolien Janssen, Sarah Croes, Nele Weyens, Robert Carleer, Jaco Vangronsveld

Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium

Corresponding author email: jolien.janssen@uhasselt.be

## ABSTRACT

The production of non-food crops on metal-contaminated soils is an economically beneficial and environmentally sustainable land use. In case metal accumulating crops, like short rotation coppice of willow are used, produced biomass does not only serves bioenergy purposes, but also gradually remediates metal contamination by the extraction of metals out of the soil. To improve biomass production (farmer's income) and metal extraction (shortening clean up time) of short rotation willow, we investigated the use of plant-associated bacteria which can have a beneficial effect on plant growth, metal uptake and translocation of metals to harvestable plant parts. Besides, also fertilization of willow was tested as a metal-phytoextraction improving strategy.

Bacteria isolated from a selected *Salix alba x alba* clone were screened for their plant growth-promoting characteristics (e.g. production of indole-3-acetetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase), for characteristics enhancing the uptake of toxic metals (e.g. production of siderophores and organic acids) and for Cd and Zn tolerance. Six strains were selected for a pot inoculation experiment with cuttings of *S. alba x alba* planted in a Cd-Zn-Pb contaminated soil. In a parallel experiment, two fertilization strategies were tested: (i) a commercial slow release fertilizer mixed with the soil and (ii) an adapted solution of N, K and Mg weekly provided to the plants. This work summarizes biomass production of the inoculated or fertilized cuttings and metal concentrations in the produced biomass after 2 months of growth in pots.

Compared to non-inoculated and non-fertilized control cuttings, the production of biomass (roots, shoots and leaves) increased after an inoculation with the bacteria *Bacillus sp.* Both fertilizers did not increase dry weight biomass production. The concentrations of Cd and Zn inside the plant were, compared to control plants, higher for cuttings inoculated with *Sphingobacterium sp.* and after treatment with the slow release fertilizer. Calculating the extracted amount of Cd and Zn after only 2 months of growth in metal-contaminated soil revealed already a slightly increased uptake of both metals after inoculation with *Bacillus sp.* and 2 different *Sphingobacterium sp.* as well as the slow release fertilizer treatment. These results indicate that the valorization of metal-contaminated soil can be optimized by the bioaugmentation of willow with selected plant-associated bacteria as well as the application of appropriate fertilizers.

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# TRANSGENIC LINSEED (LINUM USITATISSIMUM L.) WITH alphaHMT1a: THE EFFECT ON THE ACCUMULATION OF SEVERAL METAL ELEMENTS

**Magdalena Cvečková, Miroslava Vrbová, Michaela Pavelková, Martina Větrovcová, Iva Smýkalová, Miroslav Griga**

Agritec Plant Research Ltd., Plant Biotechnology Department, CZ-787 01 Šumperk, Czech Republic
*Corresponding author email: cveckova@agritec.cz*

## ABSTRACT

The results presented here reassume our long-term research on transgenic linseed (Linum usitatissimum L.) that we generated by transformation with αHMT1 transgene for overproduction of heavy metal-binding recombinant peptide (mammalian metallothionein) and cadmium (Cd)-binding CP peptide. The evidence of improved Cd accumulation was recorded as related to parental, non-GM line [1]. Because this material showed high genetic stability in subsequent generations, based on official EU field trials we focused on testing its tolerance and accumulation potential in relation to further metallic compounds. Transgenic lines of linseed were grown, selected and investigated in controlled field conditions and compared with conventional linseed varieties. Further *in vitro* screening of several GM-lines (homogenous T5 generation) for accumulation potential in relation to selected metallic micro-and macroelements (Se, Mg, Fe) was carried out.

The hypocotyl segments of linseed line AGT917 with partially removed epidermis were infected with *A.tumefaciens* strain EHA105 harboring pBI121-derivatives in the presence of acetosyringone and cellulase. After regenerating transformants *in vitro*, further generations were grown in greenhouse and later in field conditions, respectively. The compositional analysis of transgenic plants from natural field background was conducted. In T5 plants germinated *in vitro*, the presence of αHMT1a::gus and CP::gus fusions and their expression driven by 35S CaMV promoter were confirmed by β-glucuronidase histochemical assay and PCR. In T5 plants, the influence of metallic compounds on growth parameters was evaluated using three *in vitro* systems on MS-based media: a) callus cultures in liquid media, b) cultures of multiple shoots and c) germinating seedlings. The determination of metallic compounds content in the biomass by atomic absorption spectroscopy (AAS) was done.

First results of compositional analysis in GM plants (both αHMT and CP) from field (T3 generation) showed evident increase of Cd and Pb content in above-ground biomass in comparison with parental, non-GM cultivar. The contrasting results were obtained for Se content and no difference was detected in case of Mg content.

*In vitro* experiments with GM-lines (T5 generation) exposed to increasing concentrations of Mg showed differences in growth parameters between GM- and non-GM lines and positive effect of transformation on accumulation of Fe and Cu within the seedlings.

In conclusion: This type of transformation in linseed may be considered as stable. The transgenes used show the metabolic activity in transgenic plants even after the several years of selection in field conditions. The preliminary results can lead us to the idea, that this transgenic linseed can have high phytoremediation potential. Nevertheless, many questions concerning this interesting and unique plant material still remain unanswered and have to be an area for further basic and applied research.

## Reference:

1. Vrbová, M., Kotrba, P., Horáček, J., Smýkal, P., Švábová, L., Větrovcová, M., Smýkalová, I., Griga, M. (2013): Enhanced accumulation of cadmium in *Linum usitatissimum* L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein. Plant Cell, Tissue and Organ Culture, 112: 321-330.

**Acknowledgement:** The financial support by "Institutional support for long-term conceptual development of research organization" - Czech Ministry of Agriculture - ref.No. 21639/2012-MZE-17011, nr. RO0113 is greatly appreciated.

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# THE EFFECT OF HUMIC ACIDS ON CADMIUM ACCUMULATION BY AGROPYRON REPENS L. AND BIOCHEMICAL PARAMETERS

Saule Atabayeva, Ayzhan Beisenova, Batyrbek Sarsenbayev, Saule Kenzhebayeva, Ravilya Alybayeva, Saltanat Asrandina

Al-Farabi Kazakh National University, Almaty, Kazakhstan

Corresponding author email: sauleat@yandex.ru (050040 Al-Farabi str, 71. Almaty, Kazakhstan)

## ABSTRACT

The effect of humic acids (HA) on the uptake of lead by quack grass plants was studied. Cadmium levels in the variant (Cd (250 mg/kg + 2,5 g/m² HA) increased in the roots in 1,6 times and in the shoots – decreased by 12% compared to control with cadmium without HA. With increasing content HA (5 g/m²) in the soil Cd content in the shoots and in the roots increased increased by 2.1 times compared to variant with cadmium without HA. Biochemical parameters were studied with plants grown hydroponically in 5 variants 1-0 (control); 2 – 0.4 mM Cd (Cd1); 3 - 0.8 mM Cd (Cd2);4 - Cd1 + HA (0,5 mM); 5 - Cd2 + HA (0, 5 mM). Chlorophyll (Chl) a content decreased in all treatments, except [Cd1 + HA (0,5 mM)]-variant. Content of carotenoids remained unchanged or increased in variant with HA. Proline content increased significantly in the shoots in all variants, especially in [Cd2 + HA (0, 5 mM)]-variant in 13 times, and in the roots - about in 5-9 times. Proline content in the shoots in variants without HA was lower than in the presence of HA, and in the roots, vice versa. In variant [Cd2 + HA (0, 5 mM)] was observed the most increasing of ABA content: in the shoots in 3 times and in the roots - in 2 times. Thus, HA stimulated Cd uptake by roots and shoots. Cadmium caused decrease of Chl a content, but increased Chl b and carotenides, it might be adaptive reaction. Cadmium significantly increased proline content, this increasing roles as osmotic compatible and osmoprotector under heavy metals stress. ABA content increased in all variants, but the most increasing was observed in variant with high concentration of humic acids.

**Keywords:** cadmium, humic acids, chlorophyll, carotenoids, proline.

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