# HIGH CAPABILITY OF *PTERIS VITTATA* IN ARSENIC REMOVAL FROM CONTAMINATED WATER USING NOVEL HYDROPONICS CULTIVATION SYSTEM

Yi Huang<fnref n="1" />, Keisuke Miyauchi<fnref n="1" />, Chihiro Inoue<fnref n="2" />, Ginro Endo<fnref n="1" />

Corresponding author email: nklocalking@mail.tohoku-gakuin.ac.jp

## ABSTRACT

*Pteris vittata*, the first identified arsenic-hyperaccumulator land plant, can accumulate more than 22,000 mg/kg arsenic in fronds also it can produce 1-2 kg/m<fnref n="2" /> biomass [1]. Utilization of *P. vittata* for the remediation of arsenic contaminated soil is effective and has been widely applied. However, treatment of the arsenic water pollution using this arsenic hyper-accumulator plant has not been fully investigated. Most of research for arsenic phytoextraction by using *P. vittata* still treated it as the land plant. In our research, we tried to cultivate *P. vittata* as a water plant, and develop the novel cultivation system for *P. vittata* hydroponics to get the optimum performance of arsenic phytofiltration.

To confirm the best water level condition for *P. vittata* for hydroponic cultivation, we set up 3 different hydroponic cultivations of *P. vittata*. ⌀: fronds, rhizomes and parts of roots were above the water, ⌀: fronds and rhizomes above the water, ⌀: only fronds above the water. No aeration was applied in all 3 cultivation experiments, and 2ml of 0.2 X HG solution was add directly to rhizomes every 2 days. After one month, ferns grew very well in ⌀ and ⌀ conditions, the fern did not grow and died in ⌀ condition. These results indicate that keeping rhizomes above the water surface level was needed, aeration was not necessary and also low nutritional condition was required for *P. vittata* in hydroponic cultivation.

For arsenic phytofiltration experiment, two types of *P. vittata* were used. After 2 months growth in soil, the ferns were transplanted to the hydroponics (Fern L), and the fern seedlings were directly used for the hydroponics (Fern S). During 4 months hydroponics cultivation of the both type ferns, no aeration and very low nutrition were applied to the hydroponic cultivation. The roots elongated to 40-50 cm and the biomasses of roots were 5-10 times more than the fronds. After the roots elongation, the arsenic phytofiltration experiments were started using tree serial vessels continuous recirculation hydroponics systems. The recirculation flow rate was controlled 48 Lpd.

At first 50 µg/L arsenate (AsV) was added to water, after the AsV concentration reached below measurement limitation, the As concentration conditions were shifted to 500 µg/L AsV and then 1000 µg/L AsV water. The ferns (Fern L & Fern S) were living during this whole experiment. Water samples were analysed by ICP-MS. Arsenic concentration in water reduced to below 10 µg/L (WHO As water standard) within 1 day (50 µg/L AsV), 6 days (500 µg/L AsV), 10 days (1000 µg/L AsV). The arsenic concentrations in water were still kept at under the measurement limitation. These results demonstrated that the ferns have not only high-efficiency of arsenic removal but also hyperaffinity of arsenic uptake from water.

## Reference:

1. Ma LQ, Komar KM, Tu C, Zhang WH, Cai Y, Kennelley ED (2001) A fern that hyperaccumulates arsenic - a hardy, versatile, fast-growing plant helps to remove arsenic from contaminated soils. *Nature* 409, 579-579

**Acknowledgements:** The financial support by Grant-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (no. 24404018), 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), a and the Project from the Mitsui & Co., Ltd. Environment Fund.

<figure><img src="image_81.png" /></figure>

<footnote n="1">¹Faculty of Engineering, Tohoku-Gakuin University, Miyagi, Japan</footnote>

<footnote n="2">²Graduate School of Environmental Studies, Tohoku University, Miyagi, Japan</footnote>
# MICROALGAE BIOTECHNOLOGY FOR WASTEWATER TREATMENT. OPPORTUNITIES AND CHALLENGES

**José A. Perales, M. Carmen Garrido-Pérez**

Department of Environmental Technologies, Centro Andaluz de Ciencia y Tecnología Marinas (CACYTMAR),
Campus de Excelencia Internacional del Mar (CEIMAR), Universidad de Cádiz, Spain

Corresponding author email: joseantonio.perales@uca.es

## ABSTRACT

There are a number of wastewater nutrient removal processes available. Some systems are designed to remove only nitrogen (i.e. Bardenpho) or phosphorous (i.e. AO), while others remove both (i.e. A2O). Although these technologies have well a proven efficiency removing nutrients from wastewater, they consume significant amounts of energy and chemicals and therefore are costly.

More than 50 years ago, several authors [5] suggested the use of microalgae for wastewater treatment; because of they are highly effective in reducing simultaneously nitrogen and phosphorous concentration in wastewater as well as raising the oxygen concentration in wastewater. Nevertheless the use of microalgae biotechnology in wastewater treatment is still not a technological alternative to conventional processes, Why?. In this work, advantages and disadvantages of microalgae biotechnology compared with conventional chemical and biological nutrient removal processes are analysed. Finally, some of the main gaps to be solved for the viability of microalgae biotechnology in wastewater treatment are outlined.

## Results and Conclusions:

The main disadvantages of conventional nutrient removal technologies could be resumed as follows

**The complexity of the processes, particularly biological processes:** Conventional Technology: for nitrogen and phosphorous removal, many key factors influence nutrient removal efficiency; an adequate supply of carbon, sufficient alkalinity, sludge age and maintenance of a deep sludge blanket in the secondary clarifier, an adequate supply of VFAs etc. Microalgae photobioreactors are less complex processes as they can remove simultaneously nitrogen and phosphorous by means of a single type of reactor [1].

**The high consumables costs associated with chemicals,** especially in the case of conventional physicochemical processes. In the case of microalgae the chemicals used include inorganic carbon supply (CO₂ which source could be flue gas), and flocculants-coagulants needed to harvest algal biomass [6].

**Sludge production.** Conventional biological nutrient removal treatments, as well as microalgae biotechnology, produces sludges to be handled. In the case of microalgae the range of possibilities for biomass reuse seems to be much broader [4].

**WWTP retrofitting.** In the case of conventional biological nutrient removal processes, WWTP retrofit implies deep changes in the layout and design of the existing treatment processes. In the case of microalgae photobioreactors as well as some conventional chemical processes, they are end-of pipe technology that could be implemented after secondary treatment with minimal modifications in the original WWTP flowchart [2].

**Greenhouse Gases.** As primary producers, the retrofit of a WWTP with microalgae biotechnology could reduce significantly the ecological footprint of the whole process [3].

Nevertheless, despite these microalgae biotechnology advantages, there are two aspects to be optimized for this technology to be considered competitive: increase the photosynthetic efficiency of the photobioreactors in order to reduce the surface requirements and improve the harvesting technology towards a less energy consuming process.

## References:

1. Arbib Z., Ruiz J., Álvarez-Díaz, P; Garrido-Pérez, C., Perales, J.A. (2013) Int. J. Phytoremediation, 15(8), 774-88.

2. Arbib Z., Ruiz J., Álvarez-Díaz, P; Garrido-Pérez, C., Perales, J.A. (2013) Ecological Engineering, 52, 143-153

3. Arbib Z., Ruiz J., Álvarez-Díaz, P; Garrido-Pérez, C., Perales, J.A. (2014). Water Research, 49(1): 465-474

4. Cabanelas, I.T.D., Arbib, Z., Chinalia, F.A., Souza, C.O., Perales, J.A., Almeida, P.F., Druzian, J. I., Nascimento I. A. (2013) Applied Energy 109, 283-290

5. Oswald, W. J. and Gotaas, H. B. (1957) Transactions. American Society of Civil Engineers, 122, 73-105.

6. Ruiz J., Arbib Z., Alvarez-Díaz, P; Garrido-Pérez, C., Perales, J.A. (2014) Journal of Biotechnology, 178, 32-37

<figure><img src="image_82.png" /></figure>
# SEASONAL DYNAMICS OF Cd AND Cr IN ABOVEGROUND BIOMASS OF PHALARIS ARUNDINACEA IN A CONSTRUCTED WETLAND

Tereza Březinová, Jan Vymazal

Czech University of Life Sciences Prague, Faculty of Environmental Sciences, Prague, Czech Republic

Corresponding author email: brezinova.t@seznam.cz

## ABSTRACT

Constructed wetlands are very complex systems, which are used for wastewater treatment, especially for domestic and municipal wastewater. Currently, constructed wetlands are monitored with focus on the removal of organics, suspended solids, nutrients or microorganisms, whereas sufficient information about the heavy metals removal is quite limited. Only few studies on the accumulation of heavy metals in plants of constructed wetlands have been completed. Information about the seasonal dynamics of heavy metals in the vegetation in constructed wetlands is entirely missing.

The aim of this work was to evaluate the seasonal dynamics of Cd and Cr in the biomass of *Phalaris arundinacea* in a constructed wetland in South Bohemia. The plant biomass from the constructed wetland designed for 400 inhabitants was harvested in one or two-month intervals during the period May 2011 - March 2012. Biomass was separated into steams, leaves and flowers and dried at 60 °C into constant weight. In samples of biomass and also wastewater Cd and Cr were determined using ICP-OES. Total amounts of both metals in biomass per square meter (standing stock) in each month were evaluated (Fig. 1).

<figure><img src="image_83.png" /><figcaption>Fig. 1: Average standing stocks for Cd and Cr in the aboveground biomass of *Phalaris arundinacea* depending on the period of the year.</figcaption></figure>

The results should revealed the maximum accumulation of Cr and Cd in the biomass and consequently the optimal time for vegetation harvesting in order to achieve maximum removal when heavy metals are the treatment target. The obtained results show that elements are probably transported to aboveground biomass of *Phalaris* during different periods of the year. Whereas maximum amount of Cd in the biomass was determined at the top of growing season, the highest amount of Cr was captured at the end of the growing season. In addition concentrations of Cr were highest at the end of growing season in leaves. It seems that *Phalaris* may have special mechanisms for detoxification typical for each metal. This knowledge of the seasonal dynamics of heavy metals in the aboveground biomass may contribute to more effective removal of heavy metals from wastewater.

**Acknowledgements:** The research was supported by the Grant No. 03030400 "Development of technologies for road and other paved areas storm water runoff cleaning" from the Technological Agency of The Czech Republic (TAČR) project.

<figure><img src="image_84.png" /></figure>
# SELECTION OF SUITABLE PLANT SPECIES IN SEMI ARID CLIMATIC CONDITIONS FOR QUALITY IMPROVEMENT OF SECONDARY TREATED EFFLUENT BY USING VERTICAL CONSTRUCTED WETLAND

Gargi Sharma<fnref n="1" />, Urmila Brighu<fnref n="1" />, Pravin K. Mutiyar<fnref n="2" />

Corresponding author email: gargis.mnit@gmail.com

## ABSTRACT

Constructed wetlands are becoming popular as decentralized wastewater treatment for smaller communities, where land is easily available and frequent power tripping prohibits the advocacy of highly mechanized plants. Constructed wetlands have been proved for their treatment performance for physico-chemical and microbiological contaminants removal. The wetland plant species play the most critical role in determining the performances of the wetland systems. The wetland plants govern multiple roles in constructed wetlands during biological treatment of wastewater. They acts as biological pumps controlling the oxygen transfer, provide shelter to micro fauna (microbes) governing the pollutants biodegradation, uptake of recalcitrant contaminates etc. Thus selection of suitable plant species for vegetation in treatment wetland units is of great importance to enhance the efficiency of system. The present research aims to identify the suitable plant species for constructed wetlands in the dry/warm climates of Rajasthan (India). The performance of two widely used Indian wetland plants, *Phragmitis australis* and *Canna indica* were evaluated in vertical up-flow constructed wetland using secondary treated effluents. Performance efficiency of both the plants species was evaluated for physico-chemical and microbial contaminants removal. The study highlighted the comparative as well as significant suitability of *Canna indica* plantation over the *Phragmitis australis* under semi arid climatic conditions.

The unit planted with *Canna indica* showed 37.33% and 54.08% removal for total kjeldhal nitrogen (TKN) and ammonia-nitrogen respectively. Nitrate nitrogen in the treated effluents has a significant increment of 3.8 times higher than influents. Importantly, the indicator organism coliform reduction was observed as 1.87 log (MPN/100ml) in the effluent of *Canna indica* planted unit as against 1.01 log (MPN/100ml) in the effluent of *Phragmitis australis* planted wetland.

<figure><img src="image_85.png" /></figure>

<footnote n="1">¹Department of Civil Engineering, Malaviya National Institute of Technology, Jaipur, 302017 India</footnote>

<footnote n="2">²Department of Civil Engineering, Indian Institute of Technology, Delhi, 110016 India</footnote>
# PERFORMANCE OF YOUNG CONSTRUCTED WETLAND SYSTEM AT DIFFERENT SUBSTRATE CONFIGURATIONS

Rakesh Kadaverugu, Komal Selokar, Azeem Uddin Siddiqui, Karthik Raghunathan, Prashant R. Thawale, Asha A. Juwarkar

CSIR-National Environmental Engineering Research Institute, Nagpur-440020, Maharashtra, India
*Corresponding author email:* Asha A. Juwarkar, Chief Scientist & Head, Ecosystem Division, CSIR-National Environmental Engineering Research Institute, aa_juwarkar@neeri.res.in

## ABSTRACT

Engineered constructed wetlands (ECWs) are designed with greater control to improve the water quality. Numerous research has already been done across the globe to understand the processes occurring in the ECWs. Yet, ECWs pose challenges to the researchers in quantifying the processes.

The degradation of BOD, COD and removal of Nitrate, Phosphate and TSS from the domestic wastewater has been studied with respect to the time at two different substrate configurations. One with a mix of coarse sand (<2 mm) and marble chips (10-15 mm) and another with a mix of marble chips and gravel (50-100 mm). Each treatment is tested with and without the plant *Typha latifolia*. The experiments were carried out in batches, in specially designed columns having filled with the designated substrate media. The results indicate that, the effect of substrate is significant (p<0.01) than the presence of vegetation in the

<figure><img src="image_86.png" /><figcaption>Figure: The columns filled with different substrate media</figcaption></figure>

removal of Phosphate and TSS. Whereas, for the removal of BOD and COD; the contribution of both substrate and vegetation is found to be significant (p<0.05). Further, the treatment having coarse sand, marble chips, and vegetation is found to have higher degradation rate-constants for the pollutants, and the removal percentages of BOD, COD, Phosphate and TSS are measured to be 91%, 98%, 96.7% and 96.2% respectively, after 3 days of retention time.

**Keywords:** engineered constructed wetlands, substrate, *Typha latifolia*, rate kinetics, domestic wastewater

## Abbreviations:

BOD : Biological Oxygen Demand

COD : Chemical Oxygen Demand

ECWs : Engineered Constructed Wetland system

TSS : Total Suspended Solids

<figure><img src="image_87.png" /></figure>
# HYDRAULICS OF HORIZONTAL SUBSURFACE FLOW CONSTRUCTED WETLANDS

Rosa Aiello<fnref n="1" />, Vincenzo Bagarello<fnref n="2" />, Massimo Iovino<fnref n="2" />, Alessia Marzo<fnref n="1" />, Attilio Toscano<fnref n="1" />

Corresponding author email: attilio.toscano@unict.it

## ABSTRACT

The aims of this study was to analyze the hydraulic aspects of horizontal subsurface flow (H-SSF) constructed wetlands (CWs), and, in particular, to investigate the clogging phenomena. Experiments were carried out in three full-scale CW beds, functioning in parallel with different operational life, that receive secondary effluent from a conventional wastewater treatment plant (WWTP) in Eastern Sicily. One CW has been in operation since 2006, while other two have been in operation since summer 2013. Two beds have an almost equal surface area (about 2,000 m<fnref n="2" /> each bed), treat an equal wastewater flow (about 2 L s⁻<fnref n="1" /> per bed) and were planted with *Phragmites australis*. *Typha* was use in the smallest one, that has a surface area of about 1,000 m<fnref n="2" />.

Clogging indicators, such as accumulated solids, hydraulic conductivity and drainable porosity were determined and compared among CW systems. Synthetic informations on hydraulic properties of the CWs both in terms of the efficient use of its volume and of mixing patterns were extracted from the wastewater residence time distributions (RTDs). These were obtained by field tracer tests, as a breakthrough curve of a non-reactive tracer (NaCl) instantaneously injected into the entrance of H-SSF CWs and observed in the outflow from the treatment systems (Figure 1). Tracer analyses were also conducted inside the wetlands to determine flow patterns. Preliminary correlations were highlighted among the solids accumulated and both saturated hydraulic conductivity (Ks) and drainable porosity (DP) reduction over time. The saturated hydraulic conductivity of H-SSF CWs was estimated by using three different in situ methods: Darcy's Law, Drainage Equation and Falling Head (FH) method. Darcy's Law and Drainage Equation were applied during the same drainage experiments; the mean hydraulic conductivity value of each bed, obtained by the drainage equation was not significantly different from the value obtained with the Darcy's Law method (4,000-6,500 m day⁻<fnref n="1" /> in H-SSF3 bed). To apply the FH method, in each constructed wetland, hydraulic conductivity was measured in several points along three longitudinal transects of the wetland. During the FH tests negative exponential curves were produced, which represent a decrease in the water height inside sampling points over time; the slope of each curve is linked to hydraulic conductivity (Figure 2). The mean hydraulic conductivity of H-SSF2 gravel is lower because the constructed wetland is in operation by 6 years earlier than the other two beds.

<figure><img src="image_88.png" /><figcaption>Figure 1. Effluent tracer measurements (Electrical Conductivity, EC) over time in tracer test</figcaption></figure>

<figure><img src="image_89.png" /><figcaption>Figure 2. Decrease in water height column over time in three sampling points at H-SSF2 (a), H-SSF3 (b) and H-SSF4 (c).</figcaption></figure>

Evapotranspiration (ET) rates and removal efficiencies of chemical and physical contaminants were also assessed in the H-SSF CWs.

**Acknowledgements:** The financial support by FP-7 project Water4Crops contract No. 311933 is greatly appreciated.

<figure><img src="image_90.png" /></figure>

<footnote n="1">¹Department of Agri-food and Environmental Systems Management, University of Catania, Italy</footnote>

<footnote n="2">²Department of Agri-environmental Systems, University of Palermo, Italy</footnote>
# INNOVATION IN CONSTRUCTED WETLAND DESIGN - NOVEL SYSTEM OF FLOATING PLANT MATS

Kristina Ziegler, Diana Claus, Markus Weiss, André Gerth

Vita 34 AG / Biotech Branch BioPlanta, Leipzig, Germany

Corresponding author email: info@bioplanta-leipzig.de

## ABSTRACT

Artificial floating treatment wetlands are an innovative variant of the more traditional constructed wetland and pond technologies. For intentional designs floating matrices are introduced to enable non-floating plants moving freely at the water surface. In this way, certain characteristics of plants in environmental services could be combined for waste water quality improvement [1].

The task embedded in the project "Water4Crops" is designated for developing innovative biotechnologies for the treatment of municipal waste water to be reused in agriculture, e.g. for irrigation. The solutions will be particularly suited for decentralized application at small communities. In context to plant-based systems one of the project objectives is aimed at testing the efficiency in pathogen reduction of novel floating plant mats.

In preliminary tests different plant species will be investigated for their potential to reduce coliform bacteria, e.g. acute sedge (Carex acuta), hare's-tail cottongrass (Eriophorum vaginatum), yellow flag (Iris pseudacorus), soft rush (Juncus effusus), common reed (Phragmites australis). These plants are promising to have an effect in reduction of coliforms based on root exudates, oxygen enrichment and formation of root associated biofilms [2,3,4]. In greenhouse experiments slightly moving water

bodies based on hydroponic systems will be simulated to show an effect according to the planting density (Figure 1). The contact time of the plants with pre-treated municipal waste water will be varied according to the plants behaviour as some of them are known to have longer adjustment period to have an antibacterial effect. Mixture of best performing plants will be tested in the same way.

<figure><img src="image_91.png" /><figcaption>Figure 1: Scheme of experimental setup</figcaption></figure>

An important fact while developing an intentional floating plant mat is the application of suitable floating matrices. There are several variants present on the market all with more or less advantages and disadvantages. A novel system might be established.

During the 11th International Conference in Phytotechnologies we will present the results and conclusion of the experiments done during summer 2014. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2012-2016) under the grant agreement number 311933.

## References:

1. Kadlec, R. H. & Wallace, S. (2008) Treatment Wetlands, 2nd Edition

2. Seidel, K. (1964) Abbau von Bacterium coli durch höhere Wasserpflanzen. Naturwissenschaften 51, pp 395

3. Seidel, K. (1971) Wirkung höherer Pflanzen auf pathogene Keime in Gewässern. Naturwissenschaften 58, pp 150

4. Ferrando, L. D. (2010) Evaluation of different methods to reduce microbial risk from treated wastewater, Master-Thesis

<figure><img src="image_92.png" /></figure>
# PHYTO PROCESS PREDICTABILITY TREATS FOOD PROCESSING WASTEWATER YEAR-ROUND

Louis A. Licht<fnref n="1" />, Marshall J. English<fnref n="2" />

## ABSTRACT

A phyto waste water reactor is designed to be a rhizosphere with a predictable volume, pollutant dose, reaction rate and dwell time. Regulatory agencies understand this approach when approving conventional lagoons or in-tank treatment systems for industry and towns.

Port of Morrow (POM) at Boardman OR, USA is in the fourth year of a waste water phyto treatment research program. Pom discharges 21 million liters (5.8 million gallons) waste water per day from food processors averaging 114 mg nitrogen per liter. Current effluent treatment during the growing season requires 5,000 acres of irrigated food & feed crop. Controllable variables include waste water dose, pretreatment, crop variety, irrigation equipment, and cropping techniques. Uncontrollable variables include native soils, past farming practices and weather.

Effluent irrigation water chemistry is analyzed and tabulated. Ground water recharge is estimated using a water balance analysis, with inputs from both soil moisture sampling and lysimeters. Soil moisture content is monitored by neutron probe and real-time sensors

Winter waste water potentially can be irrigated onto deep-rooted poplar. Demonstration has started using 23 hectares (60 acres) of poplar trees with an alfalfa understory irrigated with effluent. Phyto nitrogen treatment will be managed using Irrigation Management ~ Online (IMO), an irrigation management program that explicitly models the spatial distribution of soil water movement and percolation in irrigated fields. IMO has potential for operating daily effluent irrigation at full-scale. At full-scale, 500 acres of deep-rooted poplar will be irrigated during the dormant season. The University of Iowa is determining nitrogen removal mechanisms from a poplar rhizosphere during tree dormancy. The field and laboratory data combine to better quantify nitrogen transport and cycling dynamics as POM irrigates waste water.

Keywords: food processing, waste water, irrigation, rhizosphere, poplar

<figure><img src="image_93.png" /></figure>

<footnote n="1">¹Ecolotree Inc., North Liberty IA. lou-licht@ecolotree.com</footnote>

<footnote n="2">²Oregon State University, Corvallis OR. marshall.english@oregonstate.edu</footnote>
# SCREENING OF SIX SALIX SPP. (WILLOW) GENOTYPES FOR THE BIOREMEDIATION OF MUNICIPAL EFFLUENT

M. Garofolo<fnref n="1" />,<fnref n="2" />, A. McCracken<fnref n="1" />, J. McGrath<fnref n="2" />

Corresponding author email: Michele.Garofolo@afbini.gov.uk

## ABSTRACT

There is a growing interest in using Short Rotation Coppice (SRC) willows as a means of treating municipal wastewater. Willows have many advantages over other plants included: longer growing season; reducing soil losses; large root system; wide genetic diversity. SRC willow may represent an ideal solution to clean-up polluted water and reduce emission of greenhouse gases. Current studies suggest that the recommended rate of nitrogen (N) application to willow should not exceed 150 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />. This is to minimize potential leaching of N to groundwater.

The aim of this study was to evaluate the response of six *Salix* spp. genotypes when irrigated with high levels of wastewater. Plants were treated using four concentrations (0, 300, 450, 600 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />). Plant height, above ground biomass and root weight were used to assess the effect of prolonged use of wastewater on growth, and chlorophyll content was used as an indication of stress levels in plants. Wastewater irrigation at all levels resulted in increased height. All the genotypes showed the maximum height when irrigated with the highest dosage, with the exception of "Anki 832502" which was tallest when treated with 300 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />. Plants used as control (0 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />) were significantly (p<0.01) smaller than those treated. Above ground weight was significantly (p<0.01) greater in plants receiving wastewater (300, 450, 600 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />) than control, although there was no significant difference between treatments. Root weight showed a different trend. The highest biomass was obtained when genotypes were treated with 300 and 450 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" /> but produced significantly (p<0.01) less biomass at the highest dosage. Chlorophyll content increased significantly in plants both used as control and treated with 300 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" /> while showed a diminution in chlorophyll content at the highest wastewater applications (p<0.01).

Wastewater irrigation resulted in increased height, and biomass production (above and below ground) suggesting that these genotypes studied had a beneficial effect from it. However, most of genotypes treated with the highest wastewater dosages (450, 600 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" />) showed severe toxic effects by the end of the treatment period, which is consistent with data obtained from root weight and chlorophyll content. At the highest dosages (450-600 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" /> for chlorophyll content and 600 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" /> for root weight), willows showed a significant reduction of these two values. Wastewater application estimated at 300 kg N ha⁻<fnref n="1" /> yr⁻<fnref n="1" /> did not have negative effect on plants health and consequently may not pose a risk for the environment, although further studies are required to confirm this study.

**Funding support:** This ANSWER (Agricultural Need for Sustainable Willow Effluent Recycling) project is part-financed by the European Union's European Regional Development Fund (ERDF) through the INTERREG IVA Cross-border Programme, managed by the Special EU Programmes Body (SEUPB).

<figure><img src="image_94.png" /></figure>

<footnote n="1">¹Sustainable Agri-Food Science Division, Agri-Food & Biosciences Institute, Belfast, N. Ireland UK</footnote>

<footnote n="2">²Queen's University Belfast, School of Biological Sciences, Medical Biology Centre</footnote>
# ECO-ENGINEERING PROCESS FOR THE TREATMENT OF CONTAMINANTS FROM SALTED HIGHWAY RUNOFF: ADAPTED WETLAND AND ACTIVE FILTER

Rosa Galvez-Cloutier<fnref n="1" />, Gaëlle Triffault-Bouchet<fnref n="2" />, Samuel Roy<fnref n="1" />

Corresponding author email: rosa.galvez@gci.ulaval.ca

## ABSTRACT

Highway and road de-icing operations provide safe driving conditions during winter in Canada, north of US and Europe. However, the runoff from salted roads adversely affects aquatic and terrestrial ecosystems by increasing salts and other contaminants (heavy metals and hydrocarbons) concentrations. Salt may accelerate eutrophication in water bodies and may reduce biodiversity of flora and fauna or encourage dominance of salt-tolerant species. Therefore, salted road runoff should be treated before discharge into water bodies in order to protect natural resources and to benefit from ecosystem services. A series of laboratory and greenhouse assays were conducted as means to develop two treatment methods. Based on these results a pilot treatment station was constructed near Quebec City on a heavily urbanized watershed. This compact, innovative, economically viable and ecologically sustainable treatment system includes 2 unit eco-processes: an adapted constructed wetland (ACW) that uses salt-tolerant and halophytes plants, and an active filter bed (AFB) that uses calcite as reactive filter media. This paper presents the research findings that resulted in the design and construction of this innovative system.

<table><caption>Table 1: Contaminants found in used snow and highway trenches - 1: Acceptable quality criteria for fresh water</caption><thead><tr><td>Contaminants</td><td>Concentrations (mg/l)</td><td>Thresholds for surface water (mg/l) <sup>(1)</sup></td></tr></thead><tbody><tr><td>Suspended matter</td><td>1209</td><td>&lt; 30</td></tr><tr><td>Oil and Grease</td><td>105</td><td>5</td></tr><tr><td>Sodium (Na<sup>+</sup>)</td><td>350</td><td>200</td></tr><tr><td>Calcium (Ca<sup>2+</sup>)</td><td>-</td><td>-</td></tr><tr><td>Chloride (Cl<sup>-</sup>)</td><td>3851</td><td>250</td></tr><tr><td>Lead (Pb<sup>2+</sup>)</td><td>85</td><td>0,01</td></tr><tr><td>Cadmium (Cd<sup>2+</sup>)</td><td>-</td><td>0,005</td></tr><tr><td>Zinc (Zn<sup>2+</sup>)</td><td>-</td><td>7,4</td></tr><tr><td>Iron (Fe<sup>3+</sup>)</td><td>913</td><td>0,3</td></tr><tr><td>Phosphorous (PO<sub>4</sub><sup>2-</sup>)</td><td>-</td><td>0,02-0,03</td></tr></tbody></table>

This study used one salt-tolerant and three halophytic plants natives of the south shore of the St. Lawrence River estuary (natural saline wetlands of Kamouraska, Quebec): *Atriplex patula*, *Salicornia europea* and *Spergularia Canadensis*. Some results are presented in figure 1 below.

<figure><img src="image_95.png" /><figcaption>Figure 1. Bioaccumulation of Na and Cl by halophyte and salt resistant plants</figcaption></figure>

<figure><img src="image_96.png" /></figure>

<footnote n="1">¹Civil and water engineering department, Laval University, Quebec, Canada</footnote>

<footnote n="2">²Centre d'expertise en analyse environnementale, MDDEP, Quebec, Canada</footnote>
# COMPARISON OF ENERGY AND WATER BALANCE METHODS TO ESTIMATE EVAPOTRANSPIRATION IN A CONSTRUCTED WETLAND

Salvatore Barbagallo, Giuseppe L. Cirelli, Simona Consoli, Mirco Milani, Attilio Toscano

Department of Agri-food and Environmental Systems Management, University of Catania, Via Santa Sofia 100, Catania 95123, Italy

Corresponding author email: attilio.toscano@unict.it

## ABSTRACT

**Introduction:** The analysis of soil-plant-atmosphere (SPA) interactions within a constructed wetland may allow the comprehension of complex mechanisms acting within the system and indispensable for reliable design procedures and hydraulic modelling of pollutant removal. In this study, the fluxes of energy (solar radiation) exchanged within the complex SPA environment of a full-scale Horizontal Subsurface Flow Constructed Wetland (H-SSF CW) were monitored through the energy balance approach; the results of the method in terms of evapotranspiration (ET) fluxes were compared with the ET estimated by the water balance of the CW working as a lysimeter.

**Methods:** The CW, located in San Michele di Ganzaria (Eastern Sicily), has been operating since December 2012 and receives part of secondary effluent (about 2 L/s) of the conventional wastewater treatment plant (Imhoff tank, trickling filter and a secondary sedimentation tank) of the village. The wetland, with a surface of about 2,000 m², was planted in autumn 2012 with *Phragmites australis* (Cav.) Trin. at density of four rhizomes m⁻² and it was completely covered in summer 2013. Electronic flow meters were installed at the CW inlet and outlet and connected to a control panel for recording the measured volumes. Rainfall data were measured by a weather station located at the experimental site. Data monitoring allowed to directly determining the amount of water loss by ET from the constructed wetland through a simple water balance method. Infiltration losses were neglected since the wetland is lined with an impermeable membrane and the CW water storage capacity was considered, when relevant, by measuring the water level inside the bed. The outputs of the water balance method were compared with the ET fluxes estimated as residual term of the energy balance through the SPA system. To solve the energy balance, the CW was equipped by a micrometeorological station, located inside the CW, allowing adequate fetch conditions. The station was provided by one net radiometer for directly measuring the net radiation (Rn), three heat flux plates to measure the heat flux (G) coming from the substrate of the CW, and two fine-wire thermocouples for estimating the sensible heat flux (H) via the Surface Renewal technique [2]. Data on H from Surface Renewal were calibrated by H data directly measured by one Eddy Covariance sonic anemometer. ET rates (or latent heat fluxes, LE) were finally obtained as residual term of the energy balance equation as: ET=Rn-G-H.

**First Results:** The preliminary assessment of the research is referred to the first months of 2014, when both the methods (water balance and energy balance) worked simultaneously. ET rates obtained by the energy balance were in the order of 3.0 mm d⁻¹, around 27 mm on ten-day periods (i.e. time step more reliable for the water balance application). The comparison between the two methods in terms of ET losses was reasonably good, with differences lower than 10% during the whole monitoring. The crop coefficient of the *Phragmites australis* (Cav.) Trin., calculated by the mean Kc approach as suggested by the FAO-56 paper, had a mean of 1.4, with peaks of 4.5, as confirmed by similar studies [1]. Plants inside the CW were able to use more than 80% of the available energy (Rn-G) for evapotranspiring; this rate is quite higher that that of evergreen crops.

**Conclusions:** First results of the study have proved the reliability of micrometeorological methods based on energy balance to estimate ET fluxes from CW systems. However, effective conclusions regarding the performance of the techniques will require a wider data set.

## References:

1. Milani M., Toscano A. (2013). Evapotranspiration from pilot constructed wetlands planted with *Phragmites australis* in a Mediterranean environment. J Environ Sci Heal A, 48(5), 568-580.

2. Paw U. K. T., Qiu J., Su H.B., Watanabe T., Brunet Y. (1995). Surface renewal analysis: a new method to obtain scalar fluxes without velocity data. Agric. For. Meteorol., 74, 119-137.

**Acknowledgements:** The financial support by FP-7 project Water4Crops contract No. 311933 is greatly appreciated.

<figure><img src="image_97.png" /></figure>
# CAPABILITY OF HIGH RATE ALGAL PONDS FOR REMOVING EMERGING CONTAMINANTS FROM DOMESTIC WASTEWATER

**Víctor Matamoros**<fnref n="1" />, **Yolanda Rodríguez**<fnref n="1" />, **Raquel Gutiérrez**<fnref n="2" />, **Ivet Ferrer**<fnref n="2" />, **Joan García**<fnref n="2" />, **Josep M Bayona**<fnref n="1" />

Corresponding author email: victor.matamoros@cid.csic.es

## ABSTRACT

High rate algal ponds (HRAPs) are a promising wastewater technology that have receiving a lot of interest during the last years due to the resource recovery of algal biomass, for use as fertilizer, protein-rich feed or biofuel, and an effluent treated water of high quality [1]. HRAPs are shallow raceway reactors, where microalgae and bacteria grow in symbiosis. In these systems, organic matter is degraded by heterotrophic bacteria, which consume oxygen provided by microalgal photosynthesis and, therefore, no aeration is needed [3]. Although the capability of microalgal systems for wastewater treatment has already been studied for removing nutrients, heavy metals, bacteria, helminth eggs, phenolic compounds, surfactants, biocides and polycyclic aromatic hydrocarbons, little attention has been paid to its effectiveness for removing emerging contaminants (ECs). ECs are comprised of a broad spectrum of compounds belonging to different types of chemical products such as pharmaceuticals, personal care products, plasticizers, flame retardants, surfactants, and certain pesticides among others, and whose ecotoxicological effects are relatively unknown. Therefore, the occurrence of ECs in the aquatic environment is nowadays a well established issue and has become a matter of both scientific and public concern.

Our aim in this study was to evaluate for the first time the effect of hydraulic residence time (HRT) and temperature on the removal efficiency of 25 ECs in two HRAP pilot plants feed with real domestic wastewater. The HRAP pilot plants were located at the NE of Spain (see picture). Samples from the influent and effluent of both systems were collected daily over a week in summer (July 2013) and winter (December 2013). Samples were analyzed for ECs as described previously [2]. Finally, hazard quotients (HQs) were calculated for ecotoxicological risk assessment.

<figure><img src="image_98.png" /></figure>

The removal efficiency of ECs ranged from no removal to up to 90%.

HRAPs performed slightly better removal efficiencies of ECs than conventional activated sludge wastewater treatment plants. ECs were classified in three groups in accordance with their overall average removal efficiency in HRAPs: high removal (>90%, caffeine, acetominophen, ibuprofen, methyl dihydrojasmonate and hydrocinnamic acid), moderate-high removal (from 60% to 90%, oxybenzone, ketoprofen, 5-methyl benzotriazole, naproxen, galaxolide, tonalide, tributylphosphate, triclosan, bisphenol A and octylphenol), moderate-low removal (from 40 to 60%, diclofenac, benzotriazole, triphenyl phosphate, cashmeran, diazinone, celestolide and atrazine) and poor or no removal (<30%, carbamazepine, benzothiazole, methyl paraben, OH-benzotriazole, tris(2-chloroethyl) phosphate, and 2,4-D). The elimination of ECs in HRAPs was only affected by the HRT during cold season, whereas no removal differences where observed in warm season. Cold season decreased the removal efficiency of most of the compounds due to the lower temperature and sun-light irradiation. The compounds with the highest occurrence (caffeine, acetominophen and ibuprofen) presented removal efficiencies up to 90% with low seasonally and HRT effect. The ecotoxicological risk assessment study revealed that the HQ at the affluent wastewater was removed by up to 90%, which indicates no environmental risk of water effluents.

## References:

1. Craggs, R., Sutherland, D., Campbell, H., (2012) Journal of Applied Phycology, 24, 329-337

2. Matamoros, V., Jover, E. and Bayona, J.M., (2010) Analytical Chemistry 82(2), 699-706

3. Passos, F., Mariona, H.-M., García, J., Ferrer, I., (2014) Water Research, 49, 351-359

**Acknowledgements:** The financial support by the Spanish Ministry of Economy and Innovation through project CTM2012-33547 is greatly appreciated.

<figure><img src="image_99.png" /></figure>

<footnote n="1">¹Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain</footnote>

<footnote n="2">²Department of Hydraulic, Maritime and Environmental Engineering, Universitat Politècnica de Catalunya-BarcelonaTech, Barcelona, Spain</footnote>
# TRANSFORMATION OF CHLOROFORM IN SUBSURFACE-FLOW CONSTRUCTED WETLANDS: A MASS BALANCE STUDY

**Yi Chen, Jan Vymazal**

Dept of Landscape Ecology, Czech University of Life Sciences in Prague, Prague, Czech Republic

Corresponding author email: chenyi8574@gmail.com

## ABSTRACT

Chlorination of water has been reported to produce disinfection byproducts (DBPs), some of which are carcinogenic and are consequently of health and regulatory concern. Chloroform was the most frequently detected DBPs in drinking water treatment plants, and it is urgent priority of regulation by the government of many countries due to its potential human-health concerns. Thus, how to efficiently remove chloroform in water is potentially of much practical value. Our previous studies showed that the use of planted or litter added CWs as tertiary treatment systems resulted in a good removal efficiency for chloroform, and its half-lives in CWs are much shorter than those in control CWs and other systems. However, the main mechanism for chloroform removal in CWs is still not clear, because the contributions of biodegradation, sorption and other elimination processes are scarce to date. The objective of this study was to evaluate the fate of chloroform in SSF CWs, including planted and unplanted CWs with or without litters.

In this study, Six SSF CW microcosms (0.3 m×0.3 m×0.5 m) were located in a controlled greenhouse, and the wetlands were W1 (control), W2 (100 g cattail litter), W3 (200 g cattail litter), W4 (22 plants m⁻²), W5 (40 plants m⁻²) and W6 (22 plants m⁻², 100 g cattail litter). All the microcosms were filled with gravel (8–13 mm, porosity = 0.4), and W4, W5 and W6 were planted with cattail (*Typha latifolia*). The feed water of the CWs was the secondary effluent from a wastewater treatment plant with chlorine disinfection, and the concentration of the chloroform was 60 ± 18 µg L⁻¹. The wetlands operated as a batch system with pulse loading, which were filled with wastewater at the start of each batch and were gravity drained within 1 h prior to the next batch. All the treatments were triplicated, and there were 18 batches (each batch lasted for 5d).

As shown in Fig 1, sorption is the dominant pathway for chloroform removal in litter added wetlands, accounting for 73.5-81.2% of the total removal. Sorption to litters and biofilms might be an important mechanism that contributes to the removal of chloroform. Furthermore, biodegradation was another important removal pathway in litter added CWs, which accounted for 17.6%-26.2% of the chloroform removal. In the planted CWs, sorption on gravel was the primary route for chloroform removal (53.6%-66.1%), and plant uptake was the second route for chloroform removal (25.3%-36.2%), and densely planted unit more efficient than the sparsely planted one through plant uptake. As shown in Fig 1, biodegradation and volatilization only accounted for 6.5%-7.6% and 2.0%-2.5% of the chloroform removal in planted CWs. The large contribution of plant uptake (25.3-36.2%) but the marginal volatilization (2.0%-2.5%) for chloroform removal suggested that most of the chloroform have accumulated in the plant tissues after the uptake.

<figure><img src="image_100.png" /><figcaption>Fig 1 The fate of chloroform in W1-W6 wetlands</figcaption></figure>

Further study showed that the removed chloroform was stored in both belowground and aboveground of plant, and chloroform content in roots (1079-1750 µg) was significantly higher than that in shoots (292-530 µg) and leaves (284-486 µg). This suggested that about 40% of the chloroform transported from the roots to shoots and leaves, higher than the ratio of other organic pollutants reported in previous study. Considering the low hydrophobicity of chloroform (log Kow = 1.97), it might be bound loosely to the roots surface that it can be easily translocated within the plant via the transpiration stream. The high plant transpiration (31-35% of inflow) positively correlated to the efficient uptake (25-36% of chloroform removal) in planted CWs, suggesting that transpiration could be the main driver for plant uptake of chloroform in wetlands.

In conclusion, our mass balance study of chloroform phytoremediation suggests that both the active and senescent cattail (*Typha latifolia*) litter can effectively remove the chloroform. In litter added CWs, sorption was the primary route for chloroform removal, and biodegradation was another important removal pathway; in the planted CWs, sorption and plant uptake was the main route for chloroform removal, and the volatilization was negligible.

**Acknowledgements:** The financial support by ESF & MEYS (CZ.1.07/2.3.00/30.0040) is greatly appreciated.

<figure><img src="image_101.png" /></figure>
FACTORS INFLUENCING BACTERIA REMOVAL IN CONSTRUCTED WETLANDS

Jochen A. Müller<fnref n="1" />, Matthias Kästner<fnref n="1" />, Peter Kuschk<fnref n="1" />, Mareike Braeckevelt<fnref n="1" />, Helmut Wand<fnref n="1" />,
Gabriela Vacca<fnref n="1" />, Jaime Nivalla<fnref n="2" />, Roland A. Müller<fnref n="2" />

Corresponding author email: matthias.kaestner@ufz.de

ABSTRACT

Treated domestic wastewater is an important water resource for irrigation purposes. A major
challenge for its reuse is the reduction of pathogen counts to safe levels. Constructed wetlands (CWs)
are able to lower the concentration of pathogens in domestic wastewater by several orders of
magnitudes but perform best in two-stage plants. However, despite already extensive research
activities the CW's inherent pathogen removal processes are still not sufficiently understood.

Here we report on several laboratory-scale and pilot-scale experiments targeting factors that may influence bacteria removal from model- and domestic wastewater. In laboratory-scale simulated vertical down-flow CWs loaded with *Escherichia coli* cell suspensions or domestic sewage, the presence of particular macrophytes (*Juncus effusus* or *Phragmites australis*) had no significant influence of on bacteria cell removal. Likewise, bacterial cell adsorption to the sand filter material was of low significance. In contrast, disinfection of plant roots with sodium hypochlorite hampered *E. coli* removal, and a negative correlation of protozoa and *E. coli* cell numbers was observed. Beside protozoa also predatory bacteria of the group *Bdellovibrio* showed an increased number while bacteria cell number of *E. coli* suspension or domestic sewage decreased. The relationship between bacterial pathogen removal and community structures of microbial predators was further investigated in two types of horizontal flow CWs. The research results strongly support the assumption that microbial predatory activities play an important role in wastewater hygienization in CWs. The role of bacteriophages should be characterized in future investigations.

In general, a better understanding of the microbial predatory activities in CWs could be helpful for a
more directed optimization of their running conditions and design for wastewater hygienization.

<figure><img src="image_102.png" /></figure>

<footnote n="1">¹ Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany</footnote>

<footnote n="2">² Center for Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Permoserstr. 15, 04318 Leipzig, Germany</footnote>
# UNEXPECTED NET EXPORT OF FAECAL INDICATOR BACTERIA FROM A TYPHA WETLAND INTERCEPTING AGRICULTURAL DRAINAGE

**Rebecca Stott, James Sukias, Lucy A. McKergow, Rob Davies-Colley, Chris Tanner**

National Institute of Water and Atmospheric Research (NIWA), Hamilton

Corresponding author email: [rebecca.stott@niwa.co.nz](mailto:rebecca.stott@niwa.co.nz)

## ABSTRACT

This paper reports on studies showing that a constructed wetland treating episodic (rain event driven) dairy pasture subsurface drainage can be a *net exporter* of the faecal indicator bacterium, *Escherichia coli*. Monthly monitoring of farm drainage to the wetland during the wetter months of the year indicated a relatively low median inflow concentration of 23 *E. coli*/100mL increasing to 98 *E. coli* per 100mL in the outflow. This is a surprising finding given that constructed wetlands treating steady-flow wastewaters usually achieve net *removal* of *E. coli*. A cautious approach to this unexpected finding was undertaken by investigating if the increase in *E. coli* might be an artefact of a sampling regime biased towards flow recession after rain events. Testing this “hypothesis” entailed considerable flood-chasing work using time-based and flow-proportional sampling with calculation of inflow and outflow fluxes (cfu/s) and loads (cfu/event and cfu/yr) – culminating in the eventual conclusion that the wetland was indeed a net exporter of *E. coli* with increases in *E. coli* export ranging from 2 to 34 fold. The increase in *E. coli* is probably due partly to wildlife deposition, but genetic and other evidence suggests that the main source is growth of this bacterium as environmental ‘naturalised’ populations within the wetland. This raises interesting questions regarding the microbial ecology of *E. coli* and interpretation of *E. coli*-based water quality in relation to waterborne disease and health risk downstream from wetland environments.

<figure><img src="image_103.png" /></figure>
# THE EFFECT OF SWINE WASTEWATER ON THE ECOPHYSIOLOGY AND STOICHIOMETRY OF TWO OPPORTUNISTIC MACROALGAE

**Olympia Nisiforou**<fnref n="1" />,<fnref n="2" />, **Sotiris Orfanidis**<fnref n="2" />, **Alexandros G.**, **Charalambides**<fnref n="1" />, **Nicolas Kalogerakis**<fnref n="3" />

Corresponding author email: a.charalambides@cut.ac.cy

## ABSTRACT

The effects of swine wastewater stream of high salinity, ammonia, and organic nutrients collected from a pig farm located in Cyprus on the growth, photosynthetic performance and tissue content (C, N, and P) on two macroalgae (*Cladophora* sp. and *Gracilariopsis longissima*) were studied. Two functional indices, the effective quantum yield of photosystem II (Δ*F*/F~m~), and the relative growth rate (RGR, day⁻<fnref n="1" />), under constant laboratory conditions (21°C ±1°C, 33.2-34.5 PSU, 70 µmol photons m⁻²s⁻<fnref n="1" />, 14h light per day), after 9 days cultivation were examined. Various experiments with media renewal and measurements on a daily basis were performed using dilutions from 1/20 to 1/500 (sewage:seawater).

The results were analyzed performing factorial or repeated two-way ANOVA. The two species responded significantly different to the effluents with *Cladophora* sp. showing a better performance than *Gracilariopsis longissima* in higher effluent concentrations. The limit to perform was on dilution 1/40 for *Cladophora* sp. and 1/100 for *G. longissima*. Mean tissue nitrogen content ranged from 7 to 17% (dry weight) and from 7 to 10 % N for *Cladophora* sp. and *G. longissima*, respectively. Maximum N values estimated for *Cladophora* sp. (17%) at dilution 1:80 and for *G. longissima* (10%) at dilution 1:400. The N:P ratio between the species' tissue had no statistical differences, whereas, it was significantly different based on their interaction with the different dilutions. Tissue C content showed no statistical difference amongst the two species and the different dilutions.

**Keywords:** seaweeds, effective quantum yield, relative growth rate, tissue content, N:P ratio

**Acknowledgements:** The financial support by the Cyprus Research Promotion foundation (ΠΕΝΕΚ/0311/21) is greatly appreciated.

<figure><img src="image_104.png" /></figure>

<footnote n="1">¹Department of Environmental Science and Technology, Cyprus University of Technology, Lemesos, Cyprus</footnote>

<footnote n="2">²National Agricultural Research Foundation, Fisheries Research Institute, 640 07 Nea Peramos, Kavala, Greece</footnote>

<footnote n="3">³Department of Environmental Engineering, Technical University of Crete, Chania, Greece</footnote>
# REMOVAL OF Cr(III) AND Cr(VI) IONS FROM AQUEOUS SOLUTION BY CALLITRICHE COPHOCARPA

Joanna Kyzioł-Komosińska<fnref n="1" />, Joanna Augustynowicz<fnref n="2" />, Wojciech Lasek<fnref n="3" />

## ABSTRACT

The aim of this study was to determine the adsorption capacity of the dry mass of *Callitriche cophocarpa* for Cr(III) and Cr(VI) ions. Additionally, the influence of pH in initial solution (3.0 and 5.0) and the kind of anion (chloride and sulfate) on the amount of bonded ions were investigated. The Cr was applied as Cr(III) in the form of $CrCl_3 \cdot 6H_2O$ or $KCr(SO_4)_2$, whereas Cr(VI) was used in the form of $K_2CrO_4$. This particular plant species was chosen due to its excellent Cr(VI)/Cr(III) accumulation capacity in an aquatic environment, that was proved in our earlier works. The sorption process was performed under static conditions contacting the solid phase:solution (batch method), at initial Cr concentration in solution from 0.1 mg/L to 700 mg/L. Solid phase and the solution ratio was 1:100 (i.e. 0.5 g of plant sample + 50 ml of Cr solution at the adequate concentration), and the shaking time was 24 hours.

The adsorption capacity of the *Callitriche* biomass was different for studied Cr ions. It depended on the initial concentration of Cr, kind of an ion and the value of pH in solution. The amount of Cr(III) adsorbed increased in order: Cr(III) – Cl pH 3.0 (22.0 mg/g) < Cr(III) – $SO_4$ pH 3.0 (27.1 mg/g) < Cr(III) – $SO_4$ pH 5.0 (35.8 mg/g) < Cr(III) – Cl pH 5.0 (51.2 mg/g). The amount of Cr(VI) increased in order: Cr(VI) pH 3.0 (6.58 mg/g) < Cr(VI) pH 5.0 (17.61 mg/g). The coefficients in the adsorption isotherms were estimated with the linear and non-linear regression methods. The linear regression method was found that the Freundlich and Dubinin-Radushkevich isotherms described the element sorption much better than the Langmuir model. On the other hand, all three models described well the experimental data in the non-linear regression method. Furthermore, the 1/n value (<1) obtained from the Freundlich equation for all the Cr-sorbent systems indicated the favorable sorption.

Interpretation of experimental results should take into consideration i.a. acid-base equilibrium for Cr(VI) and Cr(III), excellent complexation ability for Cr(III) and redox properties for Cr(VI). Formation of sulphato-complexes with Cr(III) may explain better sorption potential at lower pH (preferable adsorption of greater molecule for complex) and worse sorption potential at higher pH (delayed formation of polymeric hydroxide from hydrolysis of sulphato-complexes). On the other hand, for Cr(VI) at lower pH it is not possible to neglect the equilibrium between chromate $CrO_4^{2-}$ and dichromate $Cr_2O_7^{2-}$. Dichromate which is present at greater quantities at lower pH and due to its larger size than chromate, may exhibit limited diffusion to the deeper parts of the plant tissue. Another yet possibility is related to increasing oxidative power of Cr(VI) in acidic solution, sufficient enough to oxidize primary alkoxy- or phenoxy or aldehyde functional groups, resulting with formation of stable, soluble Cr(III) complexes, staying in solution rather than in an adsorbed state onto biomass.

Experimental constants determined for Freundlich and Dubinin-Radushkevich isotherms indicate, that biomass of *Callitriche cophocarpa* reveals remarkable heterogeneity and that adsorption of chromium ions can proceed as a multilayer process, governed rather by the chemisorption mechanism than the physisorption one.

Sorption capacity found for dry biomass of *Callitriche cophocarpa* is in case of Cr(III) better than other adsorbing agents – including activated carbon, examined earlier by the others and comparable to majority of chromate natural adsorbents in case of Cr(VI).

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

<figure><img src="image_105.png" /></figure>

<footnote n="1">¹ Institute of Environmental Engineering, Polish Academy of Sciences, ul. M. Skłodowskiej-Curie 34, 41-819 Zabrze, Poland</footnote>

<footnote n="2">²Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Kraków, al. 29 Listopada 54, 31-425 Kraków, Poland</footnote>

<footnote n="3">³ChemTech-ProSynTech Chemical Engineering&Technology, Starowiejska 19, 34-340 Jeleśnia, Poland
*Corresponding author email: augustyn@ogr.ur.krakow.pl*</footnote>
# HARNESSING THE INTERACTION OF PLANTS AND BACTERIA IN WETLAND SYSTEMS TO REMEDIATE TRICHLOROETHYLENE CONTAMINATED GROUNDWATER

Camille Warner<fnref n="1" />, Sean Hohm<fnref n="1" />, Amanda Ludlow<fnref n="2" />, David Tsao<fnref n="3" />, Lee A. Newman<fnref n="1" />

Corresponding author email: ciwarner@syr.edu

## ABSTRACT

The Environmental Protection Agency (EPA) has classified trichloroethylene (TCE), a common groundwater contaminant, as a carcinogen. Exposure has detrimental effects on mammalian liver, kidneys, central nervous system and reproductive system. Phytoremediation is the preferred method for remediation in lieu of traditional removal methods that are invasive and expensive. The utility of wetland plants for phytoremediation of TCE has not been extensively researched as is the case for poplar trees and larger plants. To fill that void, this research focuses on wetland plants such as *Carex crinita*, *Iris versicolor*, and *Scripus atrovirens*. Post exposure, the plant tissue will be analyzed by gas chromatography for TCE and its metabolites. Serial dilutions and Denaturing Gradient Gel Electrophoresis (DGGE) will be employed to detect shifts in the soil microbial community population. This research will shed light on the mechanisms underlying the degradation of TCE in wetland systems.

Acknowledgements: The financial support by Roux Associates is greatly appreciated.

<figure><img src="image_106.png" /></figure>

<footnote n="1">¹SUNY College of Environmental Sciences and Forestry, Syracuse, USA</footnote>

<footnote n="2">²Roux Associates, Islandia, NY, USA</footnote>

<footnote n="3">³BP Corporation North America, Inc. Naperville, IL, USA</footnote>
# AQUATIC MACROPHYTES AZOLLA AND SALVINIA AS BIOFILTERS FOR SURFACTANT DECONTAMINATION

Cinzia Forni<fnref n="1" />, Patrizia Di Cori<fnref n="1" />, Manuela Pintore<fnref n="2" />, Elisha Tel-Or<fnref n="3" />

Corresponding author email: forni@uniroma2.it

## ABSTRACT

The wide range of domestic and industrial applications of surfactants can arise ecological problems when these molecules are present in the aquatic environment in relatively high concentration, exhibiting toxic effects and affecting at different levels the ecosystems. Conventional wastewater treatment plants are not always perfectly designed to remove these contaminants, thus many of them can occur at different concentrations in surface waters and soils. In previous works two species of floating macrophytes, *Azolla filiculoides* Lam. and *Lemna minor* L., have been exploited for phytoremediation of sodium dodecyl sulphate (SDS), a commercially important anionic surfactant, that is the core component of several detergent and cosmetic product formulations, contributing significantly to the pollution profile of sewage and wastewater of all kinds. The uptake rates of SDS by duckweed and water velvet showed a potential and remarkably efficient phytoremediation system [1]. Besides the use of living plants, dried macrophyte biomasses can be used to biosorption of pollutants like a wide range of heavy metals, as demonstrated by Tel-Or and coworkers [2, 3 and references therein]; this biosorption procedure offers a different approach for reclame of metals from mining waste and industrial discharges.

The aim of this work was to verify the applicability of this technology for SDS removal from polluted water in order to offer a possible alternative solution to the use of wetlands and to the burning problem of our industrial society, i.e. to reduce water consumption trying to recycle waste water.

For this purpose, biomasses of *Azolla filiculoides* Lam. and *Salvinia natans* (L.) All. were dried in oven (70-80° C for 48 hrs) or in the sun and stored at room temperatures until use. 100 grams of mildly crushed dried biomass were rewetted in 500 ml distilled water for 1 hr, and packed in the column (Biorad), according to the procedure of Cohen- Shoel et al. [2]. Solutions, containing 40 ppm SDS were passed through the dried biomass by idrostatic pressure at a flow rate of 5 to 10 mL min⁻<fnref n="1" />. The effluent was collected in 250 mL fractions. pH effect on SDS biosorption was tested at pH 2, 7 and 10. The pH of the solution was adjusted by addition of HCl or NaOH. Before starting the filtration, each biofilter was rinsed with double distilled water (DDW) at the same pH of the experiments. The amount of SDS in the eluates was determined according to Forni et al. [1]

The different pH of SDS solution influenced the binding ability of the biofilters, which biosorption rates enhanced along with the increase in pH, being higher at pH 7 than at pH 2. The binding of SDS to insoluble constituents in the fern matrix most probably involves cell wall charged groups, therefore low pH influenced the binding of the anionic surfactant probably because of the negative charged groups of the cell walls. In fact, the dry biomass of both ferns contains very little cellulose and higher amount of pectins, a major component in *Azolla* (5-7%), and lignin [2]. These molecules together with polyphenols and phytates may play a role in biosorption.

Good binding performances were obtained at pH 7 and 10. *Azolla* biofilter was better than *Salvinia*, in some cases 90-100% of SDS removal could be obtained. A good rate of the binding already occurred in the first fractions, suggesting that the initial rinsing with DDW at different pH was necessary to adjust the charges of binding groups and a progressive saturation of the binding sites.

## References

1. Forni C., Giordani F., Pintore M. and Campanella L. (2008). Effects of Sodium Dodecyl Sulphate on the aquatic macrophytes *Azolla* and *Lemna*. *Plant Biosystems* 142/3, 665-668.

2. Cohen-Shoel N., Ilzycer D., Gilath I. and Tel-Or E. (2002). The involvement of pectin in $Sr^{2+}$ biosorption by *Azolla*. *Water, Air, and Soil Pollution* 135, 195-205

3. Tel-Or E. and Forni C. (2011). Phytoremediation of hazardous toxic metals and organics by photosynthetic aquatic systems. *Plant Biosystems* 145 (1), 224-235.

<figure><img src="image_107.png" /></figure>

<footnote n="1">¹ Dept. of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133 Rome, Italy</footnote>

<footnote n="2">² Pa.L.Mer, Via Carrara 12/A loc. Tor Tre Ponti, 04013 Latina Scalo (LT)</footnote>

<footnote n="3">³ R.H. Smith Institute of Plant Science, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel</footnote>
# Cr(VI)-CONTAMINATED GROUNDWATER REMEDIATION BY CONSTRUCTED WETLANDS PLANTED WITH FOUR HALOPHYTIC PLANTS

Helen Dimitroula, Eleni Manousaki, Nicolas Kalogerakis

School of Environmental Engineering, Technical University of Crete, Greece

Corresponding author email: nicolas.kalogerakis@enveng.tuc.gr

## ABSTRACT

Chromium being widely used in the metal and chemical industries is considered one of the world's most useful materials; however, hexavalent chromium is a heavy metal that causes serious environmental pollution to soils, sediments, groundwater and surface waters. In central Greece, groundwater contamination with Cr(VI) due to former industrial activity in the area has been identified and requires immediate attention. The use of constructed wetlands (CWs) as an alternative wastewater or contaminated groundwater remediation method has increased over the past years and in this study, Cr(VI)-contaminated groundwater remediation by constructed wetlands is investigated with the use of halophytes. Halophytic plants thrive in excessive toxic ion environments and are specially considered for this ability in the scientific community while research efforts have revealed that some halophytes through common physiological mechanisms may also tolerate more stresses than salty environments such as heavy metals.

The specific aims of this study are to investigate the potential of halophyte-based CWs for the removal of Cr(VI) from contaminated groundwater and evaluate the Cr(VI) rhizofiltration ability and tolerance of the halophytes *Juncus acutus*, *Tamarix gallica*, *Halimione portulacoides* and *Sarcocornia perennis*. For that purpose, four horizontal, continuous flow and with complete water recirculation wetlands were constructed and planted with each plant species. All experiments were conducted with initial Cr(VI) concentration in the range of 60–160 µg/L and inlet-outlet samples were collected daily and analyzed for total and hexavalent Cr. Furthermore, the plants were examined for their tolerance towards Cr(VI) and finally plant tissues were analysed for Cr accumulation.

The data of the above experiments reviled that all four plants were able to rhizofiltrate Cr(VI) with the highest and fastest efficiency performed by *J. acutus* and the lowest by *T. gallica*. Especially for *J. acutus* it was found that after five days maximum of re-circulating flow, the concentration of hexavalent chromium in the water medium was below the detection limit (Figure). Moreover, Cr removed from the system was found to be accumulated in the plants tissues.

<figure><img src="image_108.png" /><figcaption>Figure. Concentration of Cr(VI) in the influent of the CW in which *J. acutus* plants are grown.</figcaption></figure>

**Acknowledgements:** This work was co-funded by the European Union in the LIFE10 ENV/GR/000601 project: CHARM - Chromium in Asopos Groundwater System: Remediation Technologies and Measures.

<figure><img src="image_109.png" /></figure>
# MONITORING OF IRON ABSORPTION USING A CONSTRUCTED WETLAND PILOT PLANT WITH AN HYBRID LAYOUT

**Elena Comino**, Adriano Fiorucci, Stefania Menegatti, Vincenzo Riggio

Engineering Department of Environment, Territory and Infrastructure Politecnico di Torino, Torino, 10129, Italy

Corresponding author email: elena.comino@polito.it

## ABSTRACT

The main purpose of this study was strictly focused on the treatment of iron pollution present in an artificial wastewater (obtained starting from iron sulphate heptahydrate) by the use of constructed wetland system.

The effect of different tanks technologies (vertical and horizontal subsurface flow), bedding material, and plant species was also investigated. A pilot-scale study was conducted on an hybrid layout CW system operated in vertical and horizontal mode. Two vertical systems differed from each other with their bedding material and planted with different species (Juncus and Typha Latifolia). Also one horizontal system, filled with different substrate and planted with Papyrus, was used after the two vertical tanks.

The CW was designed to work as an hybrid system where the flow, of 50 l/day, crossed alternately from the two vertical tanks and then to the horizontal one. The hydraulic retention time was set equal to about 4 days and the Iron concentration in the inlet was 50mg/l. The test was designed to use batch feeding inside the system as a tank of 1000l was initially filled with 490l of water and 25g of iron sulphate diluted in 10l of water. After ten days of operation the inlet tank was refill again to restart another feeding phase.

The test was conducted during two years of efficiency monitoring. Both physical and chemical parameters were collected with a multiparameters probe three times a week at three different sites of the system, in order to had sufficient and representative data. To determine the abatement of the initial concentration of iron in the CW system were done different samplings at the exit of each tanks (VF1, VF2 abd HF).

The target to the CW system was provided from the Italian legislation (Legislative Decree 152/2006) for wastewater discharges into surface water bodies and drains. Concentration based on the best removal efficiencies for VF1, VF2 and HF were Iron 87,9%, 91%, 12,2% and a total removal efficiency of 90,7%.

<figure><img src="image_110.png" /></figure>
# INSIGHTS ON THE ROLE OF VEGETATION ON NITROGEN REMOVAL IN SUBSURFACE FLOW CONSTRUCTED WETLANDS

**Myrto Tsiknia**, Niovi Stasinou, Nikolaos Paranychianakis, Nicolas Kalogerakis

School of Environmental Engineering, Technical University of Crete, Chania, Greece

Corresponding author email: niko.paranychianakis@enveng.tuc.gr

## ABSTRACT

In this study we investigate the role of vegetation on nitrogen cycling in Constructed Wetlands (CWs) subsurface flow fed with synthetic municipal wastewater. Six pilot CWs were set, duplicates of the following treatments, without vegetation and planted with *Typha latifolia* and *Arundo donax*. Analyses of COD, TKN, NO₃⁻-N, and NH₄⁺-N were carried out at the inlet and the outlet of CWs in a weekly basis to investigate their treatment efficiency in terms of N removal. We also monitored the abundance of ammonia oxidizing organisms and denitrifiers through qPCR to provide insights on the pathways operating. Our findings revealed a seasonal impact of the vegetation on N removal rates between 67th and the 178th day of the experiment, when CWs planted with *T. latifolia* showed higher removal rates for TNK and NH₄⁺-N compared to the unplanted or planted with *A. donax* CWs. Before and after this interval there was no effect of vegetation on the treatment performance of CWs. Analysis of variance revealed that there was not any significant effect of vegetation on the abundance of gene copies of ammonia oxidizers and dentrifiers. Ammonia oxidizing bacteria (AOB) dominated over ammonia oxidizing archaea throughout the study period implying that they had a higher contribution on nitrification. This hypothesis is strengthened by the significant correlation between AOB and NH₄⁺-N removal rate and denitrification genes. Finally, denitrification genes found to be highly abundant indicating denitrification as the major pathway of N removal.

**Acknowledgements:** The financial support by FP-7 project WATER4CROPS Grant No. 311933 is greatly appreciated.

<figure><img src="image_111.png" /></figure>
# EVALUATION OF A PILOT-SCALE CONSTRUCTED WETLAND WITH JUNCUS ACUTUS L. FOR THE REMOVAL OF BISPHENOL A FROM SECONDARY-TREATED WASTEWATER

Stavros Christofilopoulos, Stella Voutsadaki, Nikolaos Nikolaidis, Nicolas Kalogerakis

School of Environmental Engineering, Technical University of Crete, Greece

Corresponding author email: nicolas.kalogerakis@enveng.tuc.gr

## ABSTRACT

Previous studies have shown the ability of halophytes to grow in soil polluted by heavy metals and toxic ions and also the tolerance of *J. acutus* endophytic bacteria to heavy metals and bisphenol A [BPA, 4-[1-(4-hydroxyphenyl)-1-methylethyl]phenol). Moreover, several endophytic strains of *J. acutus* have the potential to enhance not only bioremediation of bisphenol A but also plant growth. Thus *J. acutus* was demonstrated as an appropriate species for implementing phytoremediation strategies in areas with contaminated groundwater. Application of the phytoremediation strategy in secondary treated municipal wastewater is now investigated.

The aim of this study was to evaluate the removal of the phenolic estrogen BPA, in a small pilot-scale constructed wetland system fed with secondary-treated municipal wastewater, spiked with known amounts of BPA. For that reason a constructed wetland system, planted with *J. acutus*, was designed and operated in the WWTP of city of Chania, Crete, Greece. The unit was running in a horizontal flow with a surface area of 1m². In the evaluation of the CW, removal efficiencies of BPA was monitored and several parameters were measured; Chemical Oxygen Demand (COD), Biochemical oxygen demand (BOD), pH, Total Suspended Solids (TSS), Total Dissolved Solids (TDS), Total Organic Carbon (TOC), Total Phosphorus (TP) and Dissolved Nitrogen. Experiments were conducted at different hydraulic residence times (HRTs) and robustness of the system was also tested by changing the BPA concentration of the influent stream from 20ppb to 200ppb. The results indicated significant removals of BPA, while plants did not show any toxicity symptoms.

***Acknowledgements:*** This work was co-funded by the European Union in the Work Programme 2012 "COOPERATION" FP7-KBBE-2012-6 project - "Integrating biotreated wastewater reuse and valorization with enhanced water use efficiency to support the Green Economy in EU and India" (WATER4CROPS, Grant No. 311933).

<figure><img src="image_112.png" /></figure>
CONSTRUCTED WETLAND WITH HALOPHYTES FOR THE DEGRADATION OF BISPHENOL A IN PRIMARY-TREATED MUNICIPAL WASTEWATER

**Stavros Christofilopoulos**<fnref n="1" />, **Michalis Fountoulakis**<fnref n="2" />, **Eleni Michalodimitraki**<fnref n="1" />, **Thrassyvoulos Manios**<fnref n="2" />, **Nicolas Kalogerakis**<fnref n="1" />

Corresponding author email: nicolas.kalogerakis@enveng.tuc.gr

ABSTRACT

Evaluation of the removal of the phenolic estrogen bisphenol A (BPA, 4-[1-(4-hydroxyphenyl)-1-methylethyl]phenol) in a constructed wetland system fed with primary-treated municipal wastewater and spiked with known amounts of BPA, is investigated in pilot-scale. The influents of the CW are artificially spiked, primary-treated municipal wastewater from the wastewater treatment plant of the city of Heraklion, Crete. The halophyte-based rhizo-degradation CW, planted with four Mediterranean wetland halophytic species (*Tamarix parviflora*, *Limoniastrum monopetalum*, *Sarcocornia perennis* and *Juncus inflexus*), is operated as a Horizontal Subsurface Flow system (HSF). In the evaluation of the CW several process parameters were measured; namely, Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD), pH, Electrical Conductivity (EC), Total Nitrogen (TN), Total Phosphorus (TP) and Total Boron (Bo). Three different hydraulic residence times (HRTs) were applied to the system in order to evaluate the dependency of treatment efficiency on HRT with and without bioaugmentation with BPA degrading strain *Sphingomonas* sp. strain TTNP3.

The study was conducted during a 7 month period. The average removal efficiency for BPA was 98%, under nominal conditions during summer period. However significant sensitivity in the system was observed by changing HRT. Degradation rate decreased when HRT was switched to half and started to increase again, after increasing HRT, as expected from the literature. Bioaugmentation with *Sphingomonas* sp. strain TTNP3 showed a slight increase on the removal of BPA. For almost all the other examined parameters, low removal efficiencies were observed.

<figure><img src="image_113.png" /></figure>

<footnote n="1">¹School of Environmental Engineering, Technical University of Crete, Greece</footnote>

<footnote n="2">²School of Agricultural Technology, Technological Educational Institute of Crete, Heraklion, Greece</footnote>
# THE PLANTS THAT PURIFY THE WATER
(LE PIANTE CHE DEPURANO L'ACQUA)

Maria Cristina Grandi<fnref n="1" />, Angelo Massacci<fnref n="2" />, Laura Passatore<fnref n="2" />, Floriana Romagnolli<fnref n="3" />

## ABSTRACT

The book LE PIANTE CHE DEPURANO L'ACQUA, published by IL CAMPO, BOLOGNA (ITALY) in February 2014, with a preface of Paola Rossi Pisa (Department of Environmental Science and Technology, University of Bologna, Italy), describes the botany and the ecology of native and non-native aquatic plants and several hygrophytes that can be used in applications of constructed wetlands (HF, VF, FWS) for wastewater treatments, phytoremediation and natural swimming pools. It is the very first book in Italy that illuminates and joins scientific aspects and technical applications of aquatic plants. In-depth knowledge of aquatic macrophytes and hygrophytes is offered in this book as a foundation for their appropriate and informed application in constructed wetlands with the purpose of water purification.

<figure><img src="image_114.png" /></figure>

The first chapter of the book contains information about ecology, cultivation and maintenance of aquatic plants; the following three chapters describe their applications. The fifth chapter is organized in 60 plant profiles, regarding the botanical, ecological and phenological aspects of native aquatic plants, non-natives and hygrophytes. Each profile provides descriptive photos of the main parts of the individual plant. The description of every species is completed by its possible application in constructed wetlands, in phytoremediation and in regeneration areas of a natural pools. The aim was to create a work-book that provides hands-on experiences and tested knowledge from different fields of phytoremediation and water purification: the goal is to investigate and to encourage a wide range of possibilities in the use of aquatic species and hygrophytes, emphasizing that each plant must be seen and respected in its ecological characteristics, in order to ensure a long life in an demanding environment. Preference is given to native species rather than non-natives, as they seem to proof more resistant and naturally adapted to the climatic parameters of the environment in which they will live. This is illustrated in graphs with Ellenberg indicators and other tables. Another goal is to mimic nature in its biodiversity, so the authors are going to suggest plant communities that create ecosystems as close to natural example as possible, plant communities that can successfully thrive in semi-natural environments. In addition, the book describes in detail the cultivation and maintenance of all plants, as we wanted to show how to manage them and how to keep them healthy for many years, once the installation has come to life and begun to fulfill its purpose. The book is also supported by a glossary and a complete plant list.

<figure><img src="image_115.png" /></figure>

<footnote n="1">¹NINFEA Bologna, Italy www.ninfeawaterplants.com</footnote>

<footnote n="2">²IBAF - CNR Roma, Italy www.ibaf.cnr.it</footnote>

<footnote n="3">³STUDIO VIS Reggio Emilia, Italy www.fitodepurazionevis.com</footnote>
# PELLICE RIVER BASIN: ECO-RESTORATION WITH A SMALL RIVERINE WETLAND AND OLD FRUIT SEED BANK CREATION

## Elena Comino, Maurizio Rosso

Engineering Department of Environment, Territory and Infrastructure Politecnico di Torino, Torino, 10129, Italy
*Corresponding author email: elena.comino@polito.it*

## ABSTRACT

From the IPS website the "Phytotechnology is the strategic use of plant ... and by restoring ecosystem services in managed landscapes". The aim of this abstract is to describe a case study of eco-restoration and habitat recreation with a small riverine wetland. The result is the valorisation of the entire river basin, with the population, local authorities and stakeholder's involvement. The river basin is an ecosystem service and for this reason, our goal is to increase its value.

The paper carried out in the frame an ALCOTRA Project between Italy and France still on going TT:Co:Co focused on river management.

The River basin is characterized by an unstable balance between conflicting aspects which represents two sides of the same system. On one side, the fluvial ecosystem constitutes an great ecological and recreational value to be conserved in order to protect the autochthone flora and fauna; on the other side the maintenance of the quantity and the quality of the water needs to be improved to make better use of this natural resource for human activities.

The study area is located in Pellice Valley, which belongs to the Piedmont Region in North-western Italy. The alpine valley coincides with the catchment of the Pellice River, which is the first left tributary of the Po River the major Italian river. This area is characterized by an important vegetation coverage which makes the Pellice valley one of the most wooded Italian valleys. The presence of a rich forest landscape and of two Sites of European Community Importance (SCI) (European Commission, 2001) gives the territory a particular natural and environmental value. Furthermore, presence of the river gives the area a great ecological value, because it represents an ecological corridor characterized by linearity and organized into complex networks, that need to be protected (European Commission, 2001).

The paper presents the restoration project of a polluted area in the community of Villar Pellice. Old train wagons used as "caravan" in a non-authorized campsite will be removed and the area will be returned to the population. A new designed small wetland will be the natural link between two existing artificial ponds, while another part of the area will be planted with ancient autochthons fruit trees in order to create a seed bank for the entire valley. The creation of a specific didactical structure will improve the two important described ecological services.

**Acknowledgements:** The authors thanks the French partners of the TT:CoCo. Project and the financial support by ALCOTRA

<figure><img src="image_116.png" /></figure>
<figure><img src="image_117.png" /></figure>
<figure><img src="image_118.png" /></figure>

METALS
<figure><img src="image_119.png" /></figure>
# EFFECT OF HYPERACCUMULATOR PLANTS AND ASSOCIATED RHIZOBACTERIA ON THE EFFICIENCY OF NICKEL EXTRACTION

Marie Rue, Guillaume Echevarria, Emile Benizri

UMR 1120 Sols et Environnement - Université de Lorraine – INRA, 2 avenue de la Forêt de Haye, TSA 40602, 54518 Vandoeuvre les Nancy Cedex, France

Corresponding author email: emile.benizri@univ-lorraine.fr

## ABSTRACT

Many works have attempted to relate the association of different plants on the efficiency of inorganic pollutants extraction, with the hypothesis that these multi-species covers promoted the development and the activity of rhizosphere microorganisms, such as PGPR. Up to now, the focus has been on crop associations (maize, tobacco, Brassica species). Only few studies have concerned the effect of the combination of metal hyperaccumulator plants with other species non-hyperaccumulators. These experiments showed that co-cropping with nonhyper accumulator plants could enhance the growth of the hyperaccumulator and metal accumulation.

The objective of this work was to study the effect of species richness in vegetation cover (mono- vs co-cropping), which only consisted of four hyperaccumulator plant species (Brassicaceae), on the efficiency of Nickel (Ni) extraction from an ultramafic soil containing significant nickel concentrations (Ni = 1480 mg.kg⁻¹). The effects on some soil physicochemical properties and on microbial communities colonizing the rhizosphere were also evaluated.

An experiment was set up with four hyperaccumulator species (Leptoplax emarginata, Noccaea tymphaea, Alyssum murale and Bornmuellera tymphaea). Six treatments had been realised (one mixed cover, four monospecies covers and unplanted soil) with 7 replicates for each. After four months of culture in controlled conditions, the estimation of plant biomass and Ni concentrations in shoots and roots were evaluated. In the meanwhile, microbial biomass carbon, size of cultivable rhizosphere bacterial community (UFC), as well as the potential production of auxin compounds (AIA), were evaluated for each treatment. Bacterial communities were also characterized by genetic (SSCP) and metabolic (Biolog Ecoplate™) structures. Moreover, different microbial enzymes (ACCD - 1-Aminocyclopropane-1-carboxylate deaminase, urease, acid phosphatase, Í-glucosidase, arylsulphatase and FDA-fluorescein diacetate hydrolysis) were measured in rhizosphere soil samples.

The presence of a cover, whether single or multi-species, caused a reduction in the concentration of extractable Ni from the soil. In our case, this effect was most pronounced in the presence of mesocosms planted with *B. tymphaea* and *N. tymphaea*. Similarly, the Ni bioconcentration factor showed a good correlation with shoot biomass and Ni concentration in shoot, especially for *B. tymphaea* and *N. tymphaea* and in a less extend for cocropping species. Moreover, the treatment with *N. tymphaea* showed the lowest pH value – which favours Ni solubility in soil. A strong correlation between pH, microbial enzyme activities and the size of the bacterial community was also observed. No significant change in enzyme activities was observed with covers, except in the case of arylsulfatase. The characterization of bacterial communities from soil samples by genetic (SSCP) and metabolic (Biolog Ecoplate™) structures revealed differences between mesocosms.

The co-cropping of the four hyperaccumulator species did not significantly improve the process of phytoextraction. However, the biomass produced by *B. tymphaea* was in the same range as that of *N. tymphaea* and *L. emarginata* but *B. tymphaea* bioconcentration factor was the highest among all four species (i.e. more than 1% Ni in its dried shoots). Therefore, *B. tymphaea*, and to a lesser extent *N. tymphaea*, were the two species with the greatest potential of phytoextraction of Ni in co-cropping systems. A combination of different hyperaccumulator plants appears promising in phytoremediation practices, but further research is needed to unravel the links between aboveground hyperaccumulating plants, the belowground rhizosphere microbial communities in metaliferous soils and the implication of these microbial communities in the survival of plants and their ability to extract Ni. In particular, associations of plants should be tested in pairs, to define the plant cover providing the best phytoextraction.

**Acknowledgements:** This work was supported by the French National Research Agency through the national program "Investissements d'avenir" with the reference ANR-10-LABX-21 - RESSOURCES21 and by the project LORVER, financed by the FEDER and the "Région Lorraine".

<figure><img src="image_120.png" /></figure>
# REGULATION OF COPPER DELIVERY TO PLASTOCYANIN VIA THE PAA2/HMA8 TRANSPORTER

## Marinus Pilon

Biology Department, Colorado State University, Fort Collins, CO 80523

Corresponding author email: pilon@lamar.colostate.edu

## ABSTRACT

Copper (Cu) is an essential micronutrient for plants, as it is a cofactor for enzymes that are involved in housekeeping processes such as respiration (cytochrome-c oxidase) and photosynthesis (plastocyanin). Cu is thus essential for plant biomass formation. Under Cu limiting conditions, several mRNAs encoding seemingly non-essential cuproproteins such as Cu/ZnSOD are subject to Cu-microRNA-mediated down-regulation. The concerted expression of these Cu-microRNAs is mediated by the Cu-responsive transcription factor SPL7. We have proposed that the Cu-microRNAs serve in order to economize the available cellular Cu for use in plastocyanin (PC), thus allowing to maintain photosynthesis under Cu deficient conditions (Burkhead et al., 2009, New Phytol. 182, 799-816). Indeed PC is a preferred target for Cu delivery when previously Cu-depleted plants are re-supplied with Cu. Studies in Poplar confirmed and extended this Cu economy model (Ravet et al., 2011, Plant Physiol. 157:1300-1312). How is the prioritization of Cu delivery to PC achieved? PC mRNA is not a target of a microRNA. In order for Cu to reach PC in the thylakoid lumen, cytosolic Cu is first transported over the inner chloroplast envelope and subsequently the thylakoid membrane by two P18-type ATPases, PAA1/HMA6 (inner chloroplast envelope) and PAA2/HMA8 (thylakoid membrane). We confirmed the subcellular localization of these proteins by direct biochemical approaches and analyzed their targeting to the correct chloroplast membrane systems. Since PC seems to be the primary target in Cu deficiency, we investigated if PAA1 or PAA2 regulation are involved in subcellular Cu distribution. We found that PAA2 protein is most stable at low Cu concentrations and its abundance decreases significantly with Cu addition (Tapken et al., 2012, J. Biol. Chem. 287:18544-18550). This regulation occurs post-translationally in an SPL7-independent manner via turnover mediated by the CLP protease system.

**Acknowledgements:** This project was funded by the National Science Foundation, grant nrs IOS-0847442 and MCB 1244142 and USDA-NIFA grant nr 2012-67013-19416.

<figure><img src="image_121.png" /></figure>
# ANALYSIS OF SELENIUM ACCUMULATION, SPECIATION AND TOLERANCE OF POTENTIAL SELENIUM HYPERACCUMULATOR *SYMPHYOTRICHUM ERICOIDES*

Ali F. El Mehdawi, Elizabeth A. H. Pilon-Smits

Biology Department, Colorado State University, Fort Collins, CO USA

## ABSTRACT

Selenium (Se) hyperaccumulator plants can accumulate Se to extraordinary levels (0.1-1.5%) of their dry weight, levels toxic to most other organisms. Selenium promotes hyperaccumulator growth and offers the plant several ecological advantages through negative effects on Se-sensitive partners (for a review see [1]). High tissue Se levels can reduce herbivory, and high-Se litter deposition can inhibit neighboring plants [2]. *Symphyotrichum ericoides* was found to contain hyperaccumulator levels of Se (up to 4,000 mg kg⁻¹ DW) on a seleniferous field site. It often grew next to other Se hyperaccumulators *Astragalus bisulcatus* and *Stanleya pinnata*, where it was on average 2-fold larger and suffered less herbivory than when growing next to non- hyperaccumulators [3]. This raised two questions, whether *S. ericoides* is capable of hyperaccumulation without neighbor assistance, and whether its Se-derived benefit is merely ecological or also physiological. In a comparative greenhouse study, Se accumulation and tolerance of *S. ericoides* were analyzed in parallel with hyperaccumulator *A. bisulcatus*, Se accumulator *Brassica juncea* and related Asteracea *Machaeranthera tanacetifolia* [4]. *Symphyotrichus ericoides* and *M. tanacetifolia* accumulated Se to hyperaccumulator levels: up to 3,000 and 1,500 mg Se kg⁻¹ DW, respectively. They were completely tolerant to these Se levels and even grew 1.5-2.5 fold larger with Se than without. *Symphyotrichus ericoides* showed very high leaf Se/S and shoot/root Se concentration ratios, similar to *A. bisulcatus* and higher than *M. tanacetifolia* and *B. juncea*. Se X-ray absorption near-edge structure spectroscopy showed that *S. ericoides* accumulated Se predominantly (86%) as C-Se-C compounds indistinguishable from methyl-selenocysteine, which may explain its Se tolerance; the remainder was inorganic Se. Thus, in the greenhouse study *S. ericoides* displayed all of the characteristics of a hyperaccumulator; whether it can hyperaccumulate independently in the field requires more investigation. The bigger size displayed by the high-Se *S. ericoides* growing next to hyperaccumulators may be explained by a Se-related physiological benefit, in addition to the ecological benefit demonstrated earlier. These findings are useful for the management of seleniferous areas and cultivation of Se-rich crops for phytoremediation or biofortification.

## References:

1. El-Mehdawi AF, Pilon-Smits EAH (2012) Ecological aspects of plant selenium hyperaccumulation. *Plant Biology* 14, 1-10.

2. El-Mehdawi AF, Quinn CF, Pilon-Smits EAH (2011) Effects of selenium hyperaccumulation on plant-plant interactions: Evidence for elemental allelopathy? *New Phytologist* 191, 120-131.

3. El-Mehdawi AF, Quinn CF, Pilon-Smits EAH (2011) Selenium hyperaccumulators facilitate selenium-tolerant neighbors via phytoenrichment and reduced herbivory. *Current Biology* 21, 1440-1449.

4. El Mehdawi AF, Reynolds RJB, Prins CN, Lindblom SD, Cappa JJ, Fakra SC, Pilon-Smits EAH (2014) Analysis of selenium accumulation, speciation and tolerance of potential selenium hyperaccumulator *Symphyotrichum ericoides*. *Physiologia Plantarum*, in press. doi: 10.1111/ppl.12149. http://onlinelibrary.wiley.com/doi/10.1111/ppl.12149/pdf

**Acknowledgements:** Financial support for these studies was provided by a PhD research scholarship from the Libyan government.

<figure><img src="image_122.png" /></figure>
# TOLERANCE LEVEL OF DOUGLAS FIRS (PSEUDOTSUGA MENZIESII) PLANTLETS TO METALLIC TRACE ELEMENTS

Amandine Bonet<fnref n="1" />, Grégoire Pascaud<fnref n="2" />, Céline Faugeron<fnref n="1" />, Marilyne Soubrand<fnref n="2" />, Emmanuel Joussein<fnref n="2" />, Vincent Gloaguen<fnref n="1" />, Gaëlle Saladin<fnref n="1" />

Corresponding author email: gaelle.saladin@unilim.fr

## ABSTRACT

Tree species can be good tools for phytoaccumulation because of their high biomass and their deep root system. Gymnosperms are not widely used although they can be as efficient as angiosperms [1,2,3]. The aim of this study was to evaluate the tolerance of Douglas firs to metallic trace elements (MTE) when soils are contaminated by several MTE instead of a single one [4]. Indeed, polluted soils are generally the result of multiple contaminants.

Douglas fir seeds were stored for 2 months at 4°C in plastic bags containing moist sterile sand for stratification. They were then transferred in greenhouse in pots with soils contaminated with several MTE. Soil samples (the upper 15-20 cm) were collected on two sites in France: the first is a former mine of lead and silver in Pontgibaud (Puy-De-Dôme, Auvergne) and the second is a former mine of gold in La Petite Faye (Creuse, Limousin). The MTE in soils of Pontgibaud (mean content in mg. kg⁻<fnref n="1" /> dry soil) are Pb (11270), As (910), Zn (460) and Cd (3). The soils of La Petite Faye contained Pb, As, Sb and Cd but were sampled in two zones (Z1 and Z2) due to the gradient of MTE concentrations of soils [4]. Mean concentrations (in mg.kg⁻<fnref n="1" /> dry soil) of Pb, As, Sb and Cd were 119900, 21300, 1400 and 2.2 in Z1 and 14500, 68100, 930 and 1.7 in Z2, respectively.

Preliminary experiments showed that the rate of Douglas fir germination was close to 70% on uncontaminated soil. Thus, approximately 50 seeds per pot were sowed in order to obtain at least 30-35 plantlets per pot. Germination was performed directly on contaminated soils or on a mixture of contaminated soil and horticultural compost / sand (75/25, v/v) with a final content of contaminated soil of 25 or 50%. Germinations on 100% horticultural compost / sand were performed in parallel as a control of optimal growth for Douglas fir. All pots were watered with demineralized water for 10 weeks after seed sowing (the first two weeks corresponded to the time of germination) and plantlets were then harvested. Root system was rinsed in demineralized water and then incubated in 20 mM EDTA to remove metallic cations adsorbed on root surface. Roots, stem and needles were then weighted and divided into several batches for analyses: dry weight, MTE quantification, photosynthetic pigment and soluble carbohydrate contents, oxidoreduction status and thiol-rich oligopeptide content (often involved in Cd and other MTE scavenging). Moreover, MTE concentrations measured in the different organs will be discussed as a function of MTE speciation in soils. Analyses are actually in progress.

## References:

1. Astier C, Gloaguen V, Faugeron C (2014) Phytoremediation of cadmium- contaminated soils by young Douglas fir trees: effects of cadmium exposure on cell wall composition. *International Journal of Phytoremediation*, 16, 790-803.

2. Cho NS, Aoyama M, Seki K, Hayashi N, Doi S (1999) Adsorption by coniferous leaves of chromium ions from effluent. *Journal of Wood Science*, 45, 266-270.

3. Moussavou Moudouma CF, Riou C, Gloaguen V, Saladin G (2013) Hybrid larch (*Larix x eurolepis* Henry): a good candidate for cadmium phytoremediation? *Environmental Science and Pollution Research* 20(3), 1889-1894.

4. Wanat N, Austruy A, Joussein E, Soubrand M, Hitmi A, Gauthier-Moussard C, Lenain JF, Vernay P, Munch JC, Pichon M (2013) Potentials of *Miscanthus x giganteus* grown on highly contaminated Technosols. *Journal of Geochemical Exploration, 126-127*, 78-84.

<figure><img src="image_123.png" /></figure>

<footnote n="1">¹ LCSN EA 1069 (Laboratoire de Chimie des Substances Naturelles / Laboratory of Chemistry of Natural Substances), Faculty of Sciences and Techniques, University of Limoges, France</footnote>

<footnote n="2">² GRESE EA 4330 (Groupement de Recherche Eau Sol Environnement / Research Group Water Soil Environment), Faculty of Sciences and Techniques, University of Limoges, France</footnote>
# ARSENIC PHYTOEXTRACTION BY AN INDIAN ECOTYPE *PTERIS VITTATA*: MOLECULAR EVIDENCE OF ITS GENETIC POTENTIALITY

**Sarita Tiwari, Bijaya Ketan Sarangi**

National Environmental Engineering Research Institute, Council of Scientific and Industrial Research Nehru Marg, Nagpur- 440020

Corresponding author: bk_sarangi@neeri.res.in

## ABSTRACT

Arsenic(As) is increasingly enriched in soil and water due to unsustainable industrial activities such as; mining, coal burning, arsenical pesticide, geogenic formation due to disproportionate underground water harvest, indiscriminate discharge of As waste and natural contamination. Due to its non-degradability, As concentration in the environment is exceeding the safe level, and its acute toxicity is posing a great threat to mankind. Remediation of As pollution through phytoextraction by hyperaccumulator plants is being increasingly investigated to develop an alternative, and sustainable method over the existing conventional *ex situ* methods. The inherent ability of a plant for metal accumulation with high Bioaccumulation Factor (BF) is the primary requisite to make such proposition a reality. In this study, As remediation potentiality of an Indian ecotype *Pteris vittata* is determined and the mechanism underneath its tolerance and accumulation has been investigated. The plant was treated with different concentration of As (25, 50, 100, 150, 200 mg L⁻¹) in hydroponic condition. This ecotype accumulates up to 9000 mg As Kg⁻¹ dry weight of biomass with BF up to 65. There were no visible toxicity symptoms of the plant after treatment with 50 mg L⁻¹ As for one month indicating its high tolerance for As. Molecular study confirmed presence of a orthologs of *arsC* gene of the *ars* operon, which encodes arsenate reductase (AR) that mediates conversion of AsV to AsIII. Arsenate reductase activity was increased by two fold in As treated plant in comparison to control, and more activity was observed in frond to that of root. Occurrence of the molecular determinant for arsenate reduction similar to other hyperaccumulator for detoxification and accumulation in the plant biomass, confirms this plant as As hyperaccumulator. The present study establish this *Pteris vittata* Indian ecotype is a As hyperaccumulator and a suitable plant species for As remediation application.

<figure><img src="image_124.png" /><figcaption>**Figure:** *arsC* amplification using gene specific primers in *Pteris vittata* genomic DNA</figcaption></figure>

Legends: 1: 2-7: 3A & 3B amplicon, 8: negative control
1 : 100bp ladder

<figure><img src="image_125.png" /></figure>

Legends: 1: 100bp ladder, 2: negative control, 3: ACR2 amplicon,
4: ACR3 amplicon;

<figure><img src="image_126.png" /><figcaption>**Figure:** Arsenic accumulation in plant biomass</figcaption></figure>

**Acknowledgements:** The financial support by CSIR under the XIth Five Year Plan Project is acknowledged. The authors are thankful to Director, NEERI and providing research facility to carry out the work.

<figure><img src="image_127.png" /></figure>
# THE STUDY OF THE PROCESS OF METALS ACCUMULATION BY FLOATING MACROTHYTES UNDER WATER POLLUTION CONDITIONS

**Olga Shuvaeva<fnref n="1" />,<fnref n="2" />**, Ludmila Belchenko<fnref n="2" />, Tamara Romanova<fnref n="1" />,<fnref n="2" />, Maria Denisyuk<fnref n="1" />,<fnref n="2" />, Alla Bryanskaya<fnref n="3" />

*Corresponding author E-mail: olga@niic.nsc.ru*

## ABSTRACT

Phytoremediation technology is an effective sustainable, energy and cost economic method of environment clearing due to the plants ability to hyper-accumulation of the pollutants. The floating macrophytes water hyacinth, *Eichhornia crassipes* (EC) and *Pistia Stratiotes* (PS) are applied most often to sewage treatment of the industrial enterprises and may consider as an efficient tool for contaminated waters cleaning up.

Unfortunately in contemporary literature the studies are mainly focused on the actual metals uptake by plants but sufficiently less is known about the process of their transport and transformation inside the plant's tissue and practically nothing is known about the role of microorganisms in the process of metals bioaccumulation. At the moment there is a great need in strengthening of such knowledge as well as those of the mechanism of the process which would help to manage the pollutants extraction technology for its practical application.

In frames of present investigation the following questions were considered on the base of natural modelling approach with cadmium as a model element:

- an estimation of the contribution of the different ways of pollutants removal from contaminated water;

- the study of the mechanism of penetration of metals within the plant and its fate during accumulation act.

As it is known there are at least two channels of metals removal from contaminated natural water: the sedimentation process and accumulation by plankton organisms. We found that these channels provide water purification at the level of about 40%, however, in the presence of the floating plants the plankton channel doesn't work. As a result of microorganisms species diversity investigation in water and in plant's rhizosphere the microorganisms-destructors resistant to pollutant were identified. It was shown that the efficiency of metal extraction by microorganisms may reach about 50% of the total value of those accumulated by plant. Herewith the rhizosphere microorganisms demonstrate the highest accumulative capacity. It has been assumed that their possible role consists in the destruction of dying plankton and heavy metals transformation into the forms which may be accumulated by the plants more effectively.

It was also shown that at the first stage the pollutant sorption on the surface of the roots takes place at that metal is mainly localized in rhizodermis, then the pollutant penetrates into the tissues of the stem according to its translocation factor with the formation of the complexes rich in cysteine.

An approach for identifying of the binding forms of the element the methodology based on HPLC separation of the plant extract with UV and ICP-OES detection to investigate peptide compounds formed in plants during bioaccumulation process was developed. The obtained results allow assuming that in water-soluble fraction isolated from hyacinth cadmium is bound mainly with the peptides similar to phytochelatins. It has been assumed that flavonoids perform an intermediate role in the accumulation process, taking part in the transport and combat an oxidative stress.

In contrast to the traditional black-box approach, investigations of contaminant speciation in plant tissues have given sound understanding of the phytoremediation phenomenon. Such advancements could provide a basis for future improving the efficacy of the biological remediation processes.

<figure><img src="image_128.png" /></figure>

<footnote n="1">¹ Institute of Inorganic Chemistry, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia</footnote>

<footnote n="2">² Novosibirsk State University, Novosibirsk, Russia</footnote>

<footnote n="3">³ Institute of Cytology &Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia</footnote>
# POSSIBILITIES OF AIR POLLUTANTS REMOVAL: HEAVY METALS, PARTICULATE MATTER AND PAH

Stanislaw Waldemar Gawronski, Helena Gawronska

Laboratory of Basic Research in Horticulture, Faculty of Horticulture, Biotechnology and Landscape Architecture, Poland

Corresponding author email: stanislaw_gawronski@sggw.pl

## ABSTRACT

The air pollutants became in recent years on the top of harmful factors to man wellbeing and health. The ease of air movement together with pollutants makes difficult to clean it up. For purifying air the use of plants can be employed, what to some extend is possible to control by man via phytoremediation. Plants due to their large leaf area index – LAI (from 2 for grasses up to 14 in some trees), morphological characteristics and some metabolic properties are very efficient in air pollutants accumulation but they vary in this regard depending on species. Additionally an important role in cleaning up air plays rain which washes down pollutants suspended in air and these accumulated/deposited on surfaces including plants leaves. However, part of contaminants retained in the plants wax. Also wind participates in blowing up pollutants from surfaces but this part might be and often is re-suspended in air.

For years in our Lab we estimated accumulation of PM on leaves of over 60 trees, shrubs and climbers species.

In this work we attempt to evaluate the role of rain in washing off PM from surface *Tilia cordata* trees, For evaluation were chosen only slow rainfalls (not exceeding 20 mm). After every episode of the rainfall, amount of rain collected in plastic containers placed under tree was measured, the liquid was filtrated and after drying PM were determined gravimetrically. Results showed that for evaluation were chosen three, slow rainfalls (not exceeding 20 mm). After every episode of the rainfall, collected in plastic containers placed under tree, the amount of rain was measured and the liquid was filtrated and after drying PM were determined gravimetrically. Results showed that the amount of rainwater measured under the trees was the smallest near the trunk and it was increasing up to the last 60 cm to the edge of the crown when achieve the same amount as the rainfall outside the trees. However, in the water collected under the trees 7.9% higher content of PM it was noted.

We also determined amount of Pb in soil samples collected from underneath *Tilia cordata* trees planted in 1936 using XRF spectrometer Alpha 4000. In this experiment confirmed the role of rain in washing Pb off leaves to the soil under the crown. Lead is washed from the leaves but in very little quantities is transported to leaves therefore it become a good marker for the distribution of Pb under the tree. The results of this experiment on many years growing trees shows lower amount of Pb near the trunk increasing along with distance from trunk and again lower at the edge of the crown where number of the leaves is much lower.

The aim of other experiments were to assess the role of the plants in removing PAHs from the air, their flow from leaves to the soil, PAH degradation in soil and role of plants in the intensification of this process.

Plants of *Canna x generalis* were grown in containers with soil spiked with phenanthrene, chrysene, benzo(k)fluoranthene, benzo(g,h,i)perylene and in non spiked soil (control). The amount of PAHs was by 21.6% higher in combination with *Canna X generalis* plants compared to the combination without plants. Higher PAHs content in the soil with *Canna X generalis* plants is due to uptake of these pollutants from air, their accumulation on leaves and washing off the leaf surface to the soil.

**Acknowledgements:** This study was supported by the Warsaw Plant Health Initiative FP7-REGPOT 2011-1-286093.

<figure><img src="image_129.png" /></figure>
# MITIGATION MEASURES FOR GEOGENIC CHROMIUM RICH SOILS

Maria Lilli<fnref n="1" />, Nikolaos Nikolaidis<fnref n="1" />, Nicolas Kalogerakis<fnref n="1" />, George Karatzas<fnref n="1" />, Mike Mueller<fnref n="2" />

Corresponding author email: marialilli02@gmail.com

## ABSTRACT

Heavy metals at high concentrations in soils cause toxicity to biota, toxic effects to plants, lead to losses in agricultural productivity as well as potential human health effects. Therefore, it is of great importance to reduce the mobility and bioavailability of these metals from the soil. Within the framework of the LIFE+ project CHARM, the Technical University of Crete has used mitigation technologies that can reduce the mobility of chromium (by converting Cr(VI) to Cr(III) and/or by sequestration), so that the overall amount of chromium uptaken by the cultivated crops or leached to the groundwater is significantly reduced. The objective of this study was to evaluate the efficacy of zero valent iron products and compost to stabilize chromium in the soil and reduce its bioavailability.

The overall experimental design was conducted in two phases. In the first phase, a sampling campaign was conducted in order to collect soil and plant (carrots) samples from cultivated sites in the Schimatari area of Asopos river basin. Asopos river basin has agricultural soils containing geogenic origin chromium and other heavy metals that have the potential to pose a threat to human health and environment. Groundwater samples from wells irrigating the carrots were also collected. Samples were analyzed for base cations and heavy metals and specifically chromium, according to EPA 3051A method and ICP-MS (Agilent- CX). Regarding carrots, the leaves, the peel and the inside of carrot were analyzed separately. In the second phase, a field scale experiment demonstration was conducted where carrots were cultivated in treatment plots with amendments of Zero Valent Iron, DARAMEND® and compost as soil stabilization agents. It is hypothesized that ZVI and DARAMEND® (which is a ZVI based product) will reduce Cr(VI) into Cr(III) in situ which in turn will precipitate, whereas the amendment of compost in the soil will stimulate the formation of water stable aggregates which will incorporate the silt-clay fractions into micro and macro aggregate, reducing in this way the mobility and bioavailability of Cr(VI) in soil solution.

The results of the initial sampling (before the application of the mitigation measures) showed that the average dry weight chromium concentration in carrots was 0.35 ppm for the inside of carrot, 3.07 ppm for the peel and 2.76 ppm for the leaves (Figure 1). After wash, the average dry weight chromium concentration was 0.54 ppm for the inside of carrot and 0.91 ppm for

the peel. The results suggest that chromium is found on the peel of the carrot and washing can reduce its concentration by 70%. Amendments of Zero Valent Iron, DARAMEND® and compost all reduced the bioavailability and stabilize chromium in the soil.

Acknowledgements: This research was co-funded by the LIFE+ CHARM project (LIFE10 ENV/GR/000601).

<figure><img src="image_130.png" /></figure>

<figure><img src="image_131.png" /></figure>

<footnote n="1">¹School of Environmental Engineering, Technical University of Crete, Greece</footnote>

<footnote n="2">²FMC Soil and Groundwater Remediation Technologies, Brussels, Belgium</footnote>
# CITRIC ACID-ASSISTED PHYTOEXTRACTION OF LEAD: A FIELD EXPERIMENT

Eriberto Freitas, Clístenes Nascimento, Fernando Bruno Silva, Wildson Silva

Federal Rural University of Pernambuco, Department of Agronomy, R. Dom Manuel de Medeiros, s/n – Dois Irmãos, 52171-900 Recife, PE, Brazil

Corresponding author email: clistenes@depa.ufrpe.br

## ABSTRACT

Lead is one of the most common inorganic pollutants in soils. This metal has a high persistence in the soil and is potentially toxic, even at low concentrations. In the United States, the Environmental Protection Agency (EPA) considers levels of Pb in soil at between 400 and 500 mg kg⁻¹ as high risk to human health. Conventional techniques, such as excavation, backfilling, fixation and leaching are remediation strategies available for soils contaminated by heavy metals. Alternatively, emerging technologies, such as phytoremediation, which is especially suitable for moderately polluted areas, can also be used. Phytoextraction which removes metals from the soil through absorption and subsequent translocation from the plant's roots to its aerial parts is often proposed for the remediation of soils contaminated with metals due to lower cost, public acceptance and reduced environmental impact. This study was carried out to assess the performance of Zea mays and Chrysopogon zizanoides in the phytoextraction of Pb from a soil contaminated by battery recycling activities. The experiment was conducted in an area of slag deposition in the vicinity of a company operating in the recycling of automotive electric batteries, located in Rio Tinto, PB, Brazil (06°48'11" S latitude, 35°04'50" W longitude). The species were planted with different spacings between rows (0.80, 0.65 and 0.50 m). Citric acid (40 mmol kg⁻¹) was applied on each experimental plot on the 61st day of cultivation in order to solubilize the Pb and assist the phytoextraction. The results showed that the chelating agent promoted a 14-fold increase in the Pb concentration in maize shoots as compared to the control, which accumulated only 111 mg kg⁻¹ of the metal. The citric acid induced a Pb concentration in vetiver shoots that was 7.2-6.7 fold higher than the control at both the 0.65 and 0.50 m plant spacing, respectively. The use of citric acid increased substantially the uptake and translocation of Pb to the shoots, regardless of plant spacing. The use of citric acid at 40 mmol kg⁻¹ was effective in the Pb solubilization of the soil and in the induction of Pb absorption by the studied species. Commercial citric acid is suitable for use in the area, due to low cost and high biodegradability. Due to the natural low solubility of Pb and to the long time required (more than 150 yr), phytoextraction without chelating agents is not feasible to remedy the studied area. Conventional spacings used for both species (0.80 m) and also 0.65 m for maize coupled with the application of the chelating agent is the recommended phytoextraction strategy for the remediation of areas with moderate Pb contamination. For higher concentrations of this metal, other remediation approaches should be adopted to make the process practical and economically viable.

**Acknowledgements:** The financial support by CNPq (Brazilian National Council for Scientific and Technological Development) is greatly appreciated.

<figure><img src="image_132.png" /></figure>
# CHELANT-ENHANCED HEAVY METAL UPTAKE BY *EUCALYPTUS* SP. UNDER CONTROLLED DEFICIT IRRIGATION (CDI)

Pinchas Fine<fnref n="1" />, Rathod Paresh<fnref n="1" />,<fnref n="2" />, Anna Beriozkin<fnref n="1" />, Oz Ein-Gal<fnref n="1" />, Amir Hass<fnref n="3" />

*Corresponding author email: finep@volcani.agri.gov.il*

## ABSTRACT

Enhancement of phytoremediation of heavy metal polluted soils employs organic ligands, aimed to solubilize, phytoextract and translocate metals into the canopy. The use of EDTA, an efficient yet environmentally persistent chelants is phasing out. We tested the hypothesis that CDI of fast growing, salinity resistant *Eucalyptus* coupled with timely EDTA application/cessation enhances sediment phytoremediation while minimizing leaching of metal complexes below the root-zone by allowing for their biodegradation prior to uncontrolled winter leaching.

Two experimental setups were examined: (i) 24 lysimeters (220-L) packed with a mixture of metal polluted sludge and quartz sand (planting in 2006), and (ii) two 60 m<fnref n="3" />, drainage-controlled basins filled with dredged, polluted, saline river sediment. Both were planted (in 2006 and in 2010, respectively) with *Eucalyptus* saplings (1 and 5 species, respectively). The trees were matured for a year before applying the CDI and chelants. In the next 2 summers the lysimeters were irrigated with tap or RO water amended with Na₃EDTA, Na₃EDDS and citric acid (each at 0.05-2 mM) or not amended (control). In late 2009 the trees were cut (for analyses) and then allowed to recover for 2 years before EDTA application restarted. This time EDTA was as either 4Na or 4NH₄ salt (by H₄EDTA titration with NH₄OH). The basins were irrigated with secondary wastewater effluent with intermittent additions of 10 mM EDTA during the summers of 2011 & 2012. In 2011 it was applied once a week from May through mid-September and in 2012 it was applied in every irrigation in May and then in July. Leaching was prescribed yet frequent (overall LF ≈2%) for soil solution monitoring. Tree leaves were analysed 2-3 times a year.

**Lysimeters:** EDTA was the only effective chelant in solubilizing and translocating metals into the canopy, and the growth of *E. camaldulensis* seemed unaffected by the salination of the soil solution (up to 35 dS m⁻<fnref n="1" />) and the build-up of EDTA concentrations in the soil solution (up to 100 mM). EDTA slowly degraded, with a half-life of ≥27-d at 10 mM solution concentration. Comparably, the EDDS half-life was 5-11 days. The peak average (6-18 replicates) metal concentrations in the lysimeter soil solution and in the *Eucalyptus* leaves, in the EDTA vs. control treatments were (all respective values): Cd: 200 mg L⁻<fnref n="1" /> vs. 1.0, and 67 vs. 21 mg kg⁻<fnref n="1" />; Cu: 90 vs. 1.5 mg L⁻<fnref n="1" />, and 17 vs. 3.0 mg kg⁻<fnref n="1" />; Cr: 4.0 vs. 1.4 mg L⁻<fnref n="1" />, and 3.0 vs. 1.0 mg kg⁻<fnref n="1" />; Ni: 60 mg L⁻<fnref n="1" /> vs. 14, and 20 vs. 6.0 mg kg⁻<fnref n="1" />; Pb: >44 vs. 0.1 mg L⁻<fnref n="1" />, and 9.0 vs. 1.0 mg kg⁻<fnref n="1" />; and Zn: 650 vs. 4.0 mg L⁻<fnref n="1" /> and 200 vs. 70 mg kg⁻<fnref n="1" />. In 2013, ≈7 years after planting, metal's concentrations in the upper (15-30 cm) soil layer of the EDTA and control treatments decreased as follows: Cd (53 mg kg⁻<fnref n="1" />): -67% and -20% (*p*<0.05); Zn (206 mg kg⁻<fnref n="1" />): -58 and -6% (*p*<0.01); Pb (23 mg kg⁻<fnref n="1" />) (0-15 cm): -60% and -28%. Decreases in the concentrations of other metals (e.g., Cu (37 mg kg⁻<fnref n="1" />): -55 & -28%; Ni (14 mg kg⁻<fnref n="1" />): -29%; Mn (50 mg kg⁻<fnref n="1" />): -75%; Fe (4580 mg kg⁻<fnref n="1" />): -27%) were not significantly different between the treatments.

**Sediment basins:** The concept worked similarly well, with respect to high growth rate of the selected species under high salinity and ensuing salination at the CDI (EC >20 ds m⁻<fnref n="1" /> in the summer time). Average weights of 3.5 years old *E. camaldulensis* and *E. occidentalis* were 55 and 45 kg/tree. EDTA degraded fast in the sediment with peak soil solution concentrations not exceeding 17 mM immediately after addition. The concentration quickly decrease to 0-2 mM, and in the winter leachate water they were 0-0.1 mM. EDTA addition increased metal concentration by >2 orders of

<figure><img src="image_133.png" /></figure>

<footnote n="1">¹Inst. of Soil, Water and Environmental Sciences, Volcani Center, ARO, PO Box 6, Bet-Dagan 50250, Israel</footnote>

<footnote n="2">² currently at the Department of Earth System Analysis, Faculty of Geo-information Science & Earth Observation, University of Twente, Hengelosestraat 99, 7514 AE, Enschede, The Netherlands</footnote>

<footnote n="3">³West Virginia State University</footnote>
magnitude. In 2011 they returned to near background concentration 2-3 months after the secession
of EDTA application and in 2012 this return to background occurred within a month. Thus the
uncontrolled winter leaching was not affected by the summer applied EDTA. Leaves metal
concentrations followed EDTA addition in 2011 but not in 2012. Cd concentration in the above two
species and in *E. Spatulate* were usually >15 mg kg⁻¹.

To conclude, the study suggests that sustainable phytostabilization and phytoextraction of heavy
metals are achievable under CDI with EDTA fertigation at low dose, continuous or intermittent. The
biodegradable EDDS and citric acid were completely ineffective. RO water Irrigation with (NH₄)₃-
EDTA removes all salinity components, and further widened the scope of this CDI-based
remediation technique.

***Acknowledgements:*** We greatly appreciate the financial support by the Italian Ministry of the Environment, Territory and Sea, within the Italian-Israeli Cooperation on Environmental Technologies – Project 5, and by the European Commission in the framework of the Eco-Innovation Programme, the AGRIPORT project.
# CITRIC ACID- AND TWEEN® 80- ASSISTED PHYTOREMEDIATION OF MULTI-CONTAMINATED SOILS VEGETATED WITH ALFALFA

**Ana Carolina Agnello**<fnref n="1" />,<fnref n="2" />, **David Huguenot**<fnref n="2" />, **Eric van Hullebusch**<fnref n="2" />, **Giovanni Esposito**<fnref n="1" />

*Corresponding author email: david.huguenot@univ-mlv.fr*

## ABSTRACT

The combination of phytoextraction and rhizodegradation phytotechnologies can be employed together with the aim to remediate soils multi-contaminated by inorganic and organic pollutants [1]. However, low bioavailability of contaminants represents a significant limitation in phytoremediation [2]. To overcome this constraint, biodegradable amendments such as low molecular weight organic acids with metal chelating ability and surfactants, which enhance the solubility of organics, can be added to soils [3].

The present study assessed the phytoremediation potential of alfalfa (Medicago sativa L.) in a multi-contaminated soil as well as the effects of citric acid and Tween® 80 (polyethylene glycol sorbitan monooleate), applied individually and in combination, on the phytoremediation process.

The experimental design included a pot experiment in the growth chamber. One month old alfalfa seedlings were transplanted to a soil contaminated by heavy metals (Cu, Pb, Ni and Zn) and petroleum hydrocarbons. Pots were fortnightly treated with citric acid (15 mmol kg⁻<fnref n="1" /> dry soil), Tween® 80 (0.036 mmol kg⁻<fnref n="1" /> dry soil), or the combination of both compounds. Vegetated and non-vegetated controls received distilled water instead of amendments. Each condition was repeated in triplicates. Plants were harvested after 30, 60 and 90 days, every time three days after amendment application. Soil samples (from the rhizosphere in the case of vegetated pots) were taken at the same times. Plant parts were dried, weighed, mineralized and analyzed for their content in Cu, Ni, Pb and Zn by ICP-OES. The number of aliphatic hydrocarbon degraders and the activity of lipase enzyme were determined in soil samples by the most-probable-number method and a colorimetric assay, respectively [4,5].

The results showed that alfalfa plants could tolerate and grow in the multi-contaminated soils. Over the 90-day experimental time, shoot and root biomass increased and negligible plant mortality arose. Heavy metals were uptaken by alfalfa to a limited extent, and mostly by plant roots. Heavy metal concentration in plant tissues were in the following order: Zn > Cu > Pb > Ni. Alfalfa rhizosphere effect was manifest, enhancing both microbial population (alkane degraders) and activity (lipase enzyme), with rhizosphere effects of 28.11 and 2.04, respectively, after 90 days. Soil amendments did not significantly enhance plant metal concentration or total uptake. By contrast, the combination of citric acid and Tween® 80 significantly improved alkane degraders (5.3-fold increase) and lipase activity (1.0-fold increase) in the rhizosphere of amended plants, after 30 days of experiment.

This evidence supports the phytoremediation potential of alfalfa species to facilitate the remediation of multi-contaminated soils and the possibility to enhance the phytoremediation process through the joint application of citric acid and Tween® 80.

## References:

1. Salt, D.E., Smith, R.D., Raskin, I., (1998) Phytoremediation. *Annual Review of Plant Physiology and Plant Molecular Biology* 49, 643-668.

2. National Research Council (2003) Bioavailability of Contaminants in Soils and Sediments: Processes, Tools, and Applications. The National Academies Press, Washington, DC.

3. Agnello, A.C., Huguenot, D., van Hullebusch, E.D., Esposito, G., (2013) Enhanced phytoremediation: a review of low molecular weight organic acids and surfactants used as amendments. *Critical Reviews in Environmental Science and Technology*. DOI:10.1080/10643389.2013.829764

4. Wrenn, B.A., Venosa, A.D., (1996) Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. *Canadian Journal of Microbiology* 42, 252-258.

5. Margesin, R., Feller, G., Hämmerle, M., Stegner, U., Schinner, F., (2002) A colorimetric method for the determination of lipase activity in soil. *Biotechnology Letters* 24, 27-33.

**Acknowledgements:** The financial support by the Erasmus Mundus Joint Doctorate Programme ETeCoS³ (Environmental Technologies for Contaminated Solids, Soils and Sediments) under the grant agreement FPA n°2010-0009 as well as the MOY labelled programme n°2010/038 are greatly appreciated.

<figure><img src="image_134.png" /></figure>

<footnote n="1">¹Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di Cassino e del Lazio Meridionale, Italia</footnote>

<footnote n="2">²Laboratoire Géomatériaux et Environnement, Université Paris-Est Marne-la-Vallée, France</footnote>
# LEAD TOLERANCE AND ACCUMULATION IN METALLICOLOUS AND NON-METALLICOLOUS POPULATIONS OF *HIRSCHFELDIA INCANA*

Mouna Fahr<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Laurent Laplaze<fnref n="3" />, Mohammed El Mzibri<fnref n="2" />, Najib Bendaou<fnref n="1" />, Valérie Hocher<fnref n="3" />, Didier Bogusz<fnref n="3" />, Abdelaziz Smouni<fnref n="1" />,<fnref n="2" />

Corresponding author email: azizsmouni@yahoo.com

## ABSTRACT

Morocco is known for its mining activities that are sources of pollution by heavy metals. In the eastern region, the soil index pollution is considered very high because of the simultaneous presence of different metallic trace elements with a predominance of Pb. This makes the region toxic to flora, fauna and local populations.

*Hirshfeldia incana* is a member of the Brassicaceae family, that growth well in these contaminated sites. This plant, with a significant biomass, tolerates and accumulates high concentrations of Pb [1]. *H. incana* is a pseudometallophyte that grows also in unpolluted sites in the north of the country. In the context of phytoremediation program, we compared the growth, Pb accumulation, some physiological characteristics and genes expression in a metalicolous (MP) and a non-metalicolous population (NMP) of *H. incana*.

In soil culture, MP appears more tolerant than NMP, while both populations accumulate Pb at the same level. In hydroponic culture, Pb accumulations in the aboveground parts of the NMP were 2.5 times higher than the MP when plants were supplied with 100 µM of Pb. However, MP was more tolerant to Pb than the NMP. The MP had significantly lower concentrations of anthocyanins and higher concentrations of chlorophyll a than NMP.

An heterologous hybridization of *H. incana* transcripts on *A. thaliana* microarrays showed that several genes are regulated by lead. QPCR analysis for *HMA4*, *GCN2* and *MRP14* genes revealed that all of them are regulated by Pb in leaves of MP and NMP. This can suggest a functional role of the 3 genes in Pb accumulation. Interestingly, *MRP14* and *GCN2* genes were expressed constitutively higher in NMP. These results can explain the difference in accumulation between the two populations and suggest a possible adaptation of the MP by a regulation of Pb accumulation. Our results demonstrate that the MP has adapted to tolerate and accumulate Pb and thus has a good potential for phytoremediation of Pb contaminated soil.

## References:

1. Auguy F., Fahr M., Moulin P., Brugel A., Laplaze L., Mzibri M., Filali-Maltouf A., Doumas P., Smouni A. (2013) Lead tolerance and accumulation in *Hirschfeldia incana*, a Mediterranean Brassicaceae from metalliferous mine spoils. PLoS ONE, 8, e61932.

<figure><img src="image_135.png" /></figure>

<footnote n="1">¹ Laboratoire de Physiologie et Biotechnologie Végétale, Faculté des Sciences, Université Mohammed V-Agdal. Rabat, Maroc</footnote>

<footnote n="2">² Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Laboratoire de Biotechnologie des Plantes, UBRM-DSV. Rabat, Maroc</footnote>

<footnote n="3">³ Institut de Recherche pour le Développement (IRD), UMR DIADE (IRD/UM2), Equipe Rhizogenèse. Montpellier- France</footnote>
# CADMIUM UPTAKE AND REPARTITION OVER THE VEGETATIVE STAGE IN HELIANTHUS ANNUUS GROWN AT Cd 2 AND 20 nM

Jean-Yves Cornu<fnref n="1" />,<fnref n="2" />, Rémi Bakoto<fnref n="1" />,<fnref n="2" />, Christophe Nguyen<fnref n="1" />,<fnref n="2" />

Corresponding author email: jycornu@bordeaux.inra.fr

## ABSTRACT

Although processes governing the fate of Cd in plant tissues are well described at high level of Cd exposure (vacuolar sequestration, xylem loading, xylem-to-phloem transfer), the literature is lacking of a formalization of this knowledge in a simulation model. There are some indications showing that the concentration of metals in a plant organ can depend on the growth of this organ due to a dilution effect (Chen et al., 2008; Ekvall and Greger, 2003). These observations can be summarized by the following model: $C = a * Biom^{-b}$ where C is the concentration of trace elements in the plant compartment (leaves, stems, seeds), Biom is the biomass of the plant compartment, a and b are parameters. The parameter b describes the dilution intensity when the parameter a synthesizes the effect of the bioavailability and that of the plant species. The main objective of this work is to test this model on Helianthus annuus, for variations in biomass resulting from the phenology. We worked at a level of Cd exposure that commonly occur in agricultural soils (nM range) and for which metals repartition between plant organs has rarely been described.

One cultivar of sunflower (ES BIBA) commonly cultivated in France was grown in greenhouse in a ¼ Hoagland nutrient solution with 2 or 20 nM Cd(NO₃)₂. Plant sampling was performed at five phenological stages (6, 9, 14, 19 and 31 expanded leaves). Plants were divided into roots, stems, leaves (excluding the petiole) and flower bud (when present). All plant tissues were freeze-dried, weighed and milled. Their total N content was measured by the combustion method of Dumas and their content in Cd, Cu and Zn by ICP-MS after a microwave digestion in a mix HNO₃-H₂O₂. Xylem sap was collected on every plant by the "root pressure" method and analysed for its concentration in metals (ICP-MS), organic acids and amino acids (LC-DAD).

The figure shows the evolution of [a] plant dry weight (g), [b] plant uptake of Cd (µg) and [c] partitioning coefficient of Cd to shoots (unitless) over thermal time (° days) and the relationships between [d] Cu content, [e] Zn content, [f] Cd content (µg g⁻<fnref n="1" />) and biomass (g) in plant shoots, at Cd 2 and 20 nM.

Plant growth was not affected by the level of Cd exposure. The 10-fold rise in the concentration of Cd in nutrient solution induced an almost 10-fold increase in the plant uptake of Cd. The partitioning coefficient of Cd to shoots varied over time, contrary to that of Zn, and was fairly similar at Cd 2 and

20 nM. All these results suggest that at low level of Cd exposure, which characterize agricultural soils, the uptake and the root-to-shoot translocation of Cd in sunflower are not much regulated. Investigations are in progress to check whether the molecular processes involved in Cd homeostasis (e.g. PCs synthesis) are induced at this low concentration.

A decrease in Cu and Zn content was observed with plant growth in stems, leaves (so shoots) and roots. This result confirms that the dilution of Cu and Zn in plant biomass has a physiological origin. The "dilution model" presented above described nicely the experimental data for Cu and Zn. A comparison of the b value with the one obtained for N is necessary to know whether the dilution of Cu and Zn can be explained in the same way as the one of N. For Cd, the re-concentration of the metal in aerial tissues observed at the two last stages of vegetative growth still needs to be figured out. Interestingly, a similar trend was observed in the concentrations of Cd and of some amino acids and organic acids measured in xylem sap.

**Acknowledgements:** The financial support by the funding ANR 2011 CESA 008 01 is greatly appreciated.

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /></figure>

<footnote n="1">¹INRA, UMR ISPA 1391, CS 20032, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²Univ. Bordeaux, UMR ISPA 1391, CS 40201, F-33175 Gradignan, France</footnote>
# SELECTION OF PHYTO- AND/OR PHYCO-REMEDIATION BASED SYSTEMS TO APPLY ON NUCLEAR CONTAMINATION

Nathalie Vanhoudt<fnref n="1" />, Paul Janssen<fnref n="2" />, Natalie Leys<fnref n="2" />, Chrystelle Verhoest<fnref n="3" />, Hildegarde Vandenhove<fnref n="1" />

Corresponding author email: nathalie.vanhoudt@sckcen.be

## ABSTRACT

Although there are various studies dealing with the application of terrestrial plants, aquatic plants and eukaryotic or prokaryotic algae for the decontamination of certain radionuclides from aqueous solutions, no review exists that compares these different approaches to identify the most promising decontamination scheme for scenarios related to nuclear installations. In addition, these studies are conducted under specific environmental conditions with little information onto what extent the extraction capacity depends on the experimental conditions (e.g. pH, exposure time, amount of biomass used, etc.). In order to evaluate the applicability and feasibility of plant/algae based remediation systems for scenarios related to nuclear installations, a more in depth evaluation and testing of existing rhizofiltration/biosorption-driven remediation systems is much needed.

The goal of this research is to assess the potential of using algae (pro- or eukaryotic) and higher plants for the remediation of radioactively contaminated water within different scenarios related to nuclear installations.

A review is made of the existing literature on plant and algae based water decontamination systems for radionuclides relevant for contamination scenarios related to nuclear installations (e.g. ¹³¹I, ¹³⁷Cs, ⁹⁰Sr, ⁵⁸Co, ⁶⁰Co, ¹¹⁰mAg, ⁵⁴Mn, ⁵⁹Fe, ⁵¹Cr, ⁵⁶Zn, etc.). An important aspect of this literature review relates to the conditions in which the rhizofiltration/biosorption tests were performed as this determines the removal efficiency of the system (e.g. radionuclide loading, contact time, volume brought in contact with the contaminated water mass, the batch/flow-through setup, environmental conditions, etc.). Growth and nutrition of biomass and age can also affect rhizofiltration or biosorption due to changes in plant size/cell size, cell wall composition and extracellular product formation. For all organisms studied for bioaccumulation/biosorption, the minimum and optimal conditions of growth are identified. Environmental parameters such as pH, salt content, water hardness, temperature, etc. are considered. It is for example known that the biosorption of Cs and Sr by the Laminaria sp. (eukaryotic algae) is highly dependent on the pH of the solution causing up to 50 % variation in removal efficiency [1]. In addition the influence of competing ions on the uptake of radionuclides by plants is investigated. Sr<fnref n="2" />⁺ uptake is for example reduced in the presence of Ca<fnref n="2" />⁺ while Cs⁺ uptake is restricted in the presence of K⁺ [2-4].

The following step within this research is to perform a SWOT analysis (strengths, weaknesses, opportunities and threats) in a qualitative and descriptive way. Criteria that are considered are efficiency of radionuclide removal, robustness of the system to changing environmental conditions, efficiency of the system to remove a mixture of contaminants vs. specificity of decontamination, ease to grow or harvest biomass, ease to process the contaminated biomass, ease to apply the system in an industrial context, etc. Based on the results of the literature review and the preliminary SWOT analysis, the species and/or strains that are identified as most promising for the removal of radioactive pollutants from water systems will be selected as prime candidates for further research.

Although this study is still on-going, preliminary results based on 81 research studies indicate that phyto- and phyco-remediation techniques have a high potential for radionuclide removal in scenarios related to nuclear installations. Available data also point to rhizofiltration, using aquatic plants, and biosorption, using algae, as the most promising lines of techniques to decontaminate radionuclides from aqueous solutions. Because the accidental release of radionuclides will be mostly in a mixed form, it will be necessary to find out which combination of bioremediation schemes would be best for specific scenarios.

## References:

1. Pohl and Schimmack (2006) J Appl Phycol 18, 135-143

2. Singh et al. (2008) Ecotox Environ Safe 69, 306-311

3. Soudek et al. (2006) J Environ Radioactiv 88, 236-250

4. Zhu (2001) J Environ Radioactiv 55, 303-314

<figure><img src="image_138.png" /></figure>

<footnote n="1">¹Biosphere Impact Studies, Belgian Nuclear Research Centre (SCK•CEN), Belgium</footnote>

<footnote n="2">²Microbiology, Belgian Nuclear Research Centre (SCK•CEN), Belgium</footnote>

<footnote n="3">³Sustainable Process Technology, Laborelec - GDF Suez, Belgium</footnote>
# CHROMIUM BEHAVIOUR IN SILENE VULGARIS

Ana E. Pradas del Real<fnref n="1" />, David H. McNear Jr<fnref n="2" />, Pilar García<fnref n="1" />, Mª Carmen Lobo<fnref n="1" />, Araceli Pérez-Sanz<fnref n="1" />

Corresponding author email: araceli.perez@madrid.org

## ABSTRACT

Chromium is a transition metal which represents the seventh most abundant element on earth. Due to its corrosion-resistant properties it is used in many industrial activities. Its world production is in the order of 10,000,000 tons per year. The wide and increasing use of Cr in industry has led to the disposal and dispersal of large amounts of chromium into soils and waters making it a metal of important environmental concern. Phytomanagement could be a viable alternative in areas polluted with wastes from chromium-using industries. More important than the total chromium concentration is its speciation which determines its solubility, bioavailability and toxicity. Cr(III) and Cr(VI) are the two most common oxidation states of Cr but Cr(VI) is much more available and toxic to living organisms than Cr(III). *Silene vulgaris* (Moench) is a perennial dicotyledonous facultative methallophytewidely distributed throughout Europe, North America, Asia and North Africa. The tolerance of this species to a diversity of metals make it very useful in the initial stages of revegetation and soil remediation. This study investigated the ability of *S. vulgaris* to take up Cr(III) and Cr(VI).

Three experiments were performed. The first experiment was made with a population of *S. vulgaris* grown semi-hydroponically with different concentrations of either Cr(III) or Cr(VI). A combination of synchrotron X-ray spectroscopic techniques, scanning electron and light microscopy and infrared spectroscopy were used to determine the distribution and speciation of Cr and in *S. vulgaris*. A second experiment was made also in semi-hydroponic with different concentrations of either Cr(III) or Cr(VI) to determinate the influence of metal speciation in root exudation of two genotpes of *S. vulgaris*. The exudation rate, the elemental analysis and the concentration of organic acids were determined in the root exudates. And finally, a third experiment was made in soil conditions, artificially polluted with Cr(VI), to assess of rhizosphere bacterial communities in two genotypes of *S. vulgaris* grown on chromium polluted soil. The diversity of the microbial community was analyzed though the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and profiles of 16S rRNA gene fragments of culturable bacteria from bulk soil and rhizosphere.

The results showed that *S. vulgaris* accumulated more Cr when grown with Cr(VI), resulting in an overall reduction in biomass. Starch accumulation in leaves of *S. vulgaris* at higher Cr(VI) doses may be attributed to an impartment between carbon utilization and assimilation resulting in stunted plant growth but not the complete inhibition of photosynthesis indicating that *S. vulgaris* possess tolerance mechanisms that allows it to survive in Cr(VI) rich environments. One of these potential mechanisms is related to root exudates. Hexavalent Chromium expositions increased the root exudate release in *S. vulgaris*. Chromium accumulation and levels of lipid peroxidation in *S. vulgaris* genotypes were positive correlated to root exudates releases rates. The concentration of C, N and H, and specially S, clearly changes as consequence of hexavalent Chromium. The general banding profile corresponding to soil showed the largest number of bands in rhizospheric soil; similar pattern of bands among repetitions; different pattern of bands between the two studied genotypes; loss of bands and increased band intensity after chromium treatment.

The primary mechanisms implicated in Cr(VI) tolerance are a) the total reduction of Cr(VI) to Cr(III) in the rhizosphere or just after uptake in the fine lateral root tips and b) chelation of Cr(III) to the cell wall both of which reduce metal interference with critical cell functions and c) the rate of root exudates and the diversity of the microbial community analyzed. These mechanisms make *S. vulgaris* suitable for in situ remediation of Cr polluted soils.

**Acknowledgements:** This work has been supported by FP13-APL (IMIDRA), EIADES PROGRAM S2009/AMB-1478.

<figure><img src="image_139.png" /></figure>

<footnote n="1">¹Dept of Agro-environmental Research, IMIDRA, Spain</footnote>

<footnote n="2">²Rhizosphere Science Laboratory, University of Kentucky, USA</footnote>
# ROLE OF PHRAGMITES AUSTRALIS (COMMON REED) FOR HEAVY METALS PHYTOREMEDIATION OF ESTUARINE SEDIMENTS

Diego Cicero Fernández<fnref n="1" />, Manuel Peña Fernández<fnref n="1" />, Jose A. Expósito Camargo<fnref n="1" />, Blanca Antizar-Ladislao<fnref n="1" />

Corresponding author email: asocria@gmail.com

## ABSTRACT

According to a recent study conducted by the European Commission, out of 1.17×10⁶ identified contaminated sites reported in 33 countries in Europe, only 58 have been identified remediated. The main contaminant category is heavy metals contributing 30-35% to soil and groundwater contamination respectively. There is a clear need for sustainable and appropriate sediment remediation technologies, which will depend on the site characteristics, concentration, types of pollutants to be removed, and the end use of the contaminated medium.

The focus of this study is on the investigation of phytoremediation of estuarine sediments contaminated with heavy metals. Therefore, for the purpose of this study, the ability of *Phragmites australis* to take up heavy metals (i.e., Co, Ni, As, Mo, Cd, Pb, Se, Ba, Cr, Cu, Fe, Mn, Zn and Hg) from estuarine sediments on the northern Atlantic Spanish coast was investigated using a pilot plant experimental approach. The aim of this study was to elucidate the role of *Phragmites australis* in the restoration of sediments. Translocation factors were calculated for two ecotypes of *P. australis*, one adapted to contaminated estuarine sediments (M) and one adapted to non-contaminated estuarine sediments (G), both growing in estuarine contaminated sediment (R) saturated with freshwater during the length of the experimental research. Plants were harvested in October and December 2012, coinciding with the maximum annual vegetative growth and senescence periods respectively.

Results indicated that both ecotypes successfully grew in the contaminated sediment. For a period of four months ecotypes of *P. australis* adapted to sediment M increased their rhizosphere surface in a 1254%, while ecotypes of *P. australis* adapted to sediment G registered an increase of 219%, which suggests acclimatization in terms of vegetative development for ecotypes of *P. australis* adapted to contaminated sediment.

Results of heavy metals analyses indicated that a relative higher concentration of Fe, Mn and Zn, as compared to the rest of elements under investigation, was present in the sediments used as substrate for the experiment (R), as well as in the sediments where both ecotypes of *P. australis* were taken from in the field. All sediments presented a different profile in concentration levels. All elements analysed were present in all sediments. Hg was not detected in plant tissue samples. Translocation factors indicated that ecotypes of *P. australis* adapted to sediment M were able to upward translocate Ni, Mo, Se, Ba, Cr, Mn and Zn, and ecotypes of *P. australis* adapted to sediment G were able to upward translocate Ni, Mo, Se, Ba, Cr, Zn. Results also indicated that for both ecotypes most of the absorbed Co, As, Cd, Cu and Fe were retained belowground, phytostabilizing the metals and reducing their concentration in the sediments. These results suggested that *P. australis* can be used to immobilize some metals (Co, As, Cd, Pb, Cu, Fe) and store them belowground. The accumulation of some other metals in the aboveground biomass (Ni, Mo, Se, Ba, Cr, Mn, Zn) by *P. australis* showed a temporal variation with the highest values in the pre-senescence and senescence for ecotypes from G and M, respectively, which suggested the best time of the year to harvest within the first annual cycle.

A comparative analysis of results between ecotypes indicated that in pre-senescence period seven metals (Fe, Co, Se, Mo, Ni, Cr and Cu) were accumulated belowground in a major concentration by the ecotype M, while six (Cd, As, Pb, Zn, Mn and Ba) were accumulated in a major concentration by G. For the same period all the metals, with the exception of Cu, were accumulated aboveground in a major concentration by G. In senescence period all the metals were accumulated belowground in a major concentration in G, while aboveground five metals (Co, Mo, Ni, Cr and Ba) were in a higher concentration in G, and seven (Fe, Se, Pb, As, Zn, Mn and Cu) were highly accumulated by M, and Cd was found in the same concentration in both ecotypes. There is not a clear influence on the phytoremediation potential of *P. australis* associated to ecotypes adaptation to contaminated sediments.

**Acknowledgements:** The financial support by Ayuntamiento de Camargo, Cantabria, Spain.

<figure><img src="image_140.png" /></figure>

<footnote n="1">¹Asociación RIA, C/ José Barros, 39600 Muriedas, Cantabria, Spain</footnote>
# ZINC/CADMIUM ACCUMULATION AND CELLULAR LOCALIZATION IN A NEW HYPERACCUMULATOR SEDUM PLUMBIZINCICOLA GROWN IN MINE AREA

Zhu Li<fnref n="1" />, Yaodong Wang<fnref n="2" />, Jolanta Mesjasz-Przybylowicz<fnref n="3" />, Wojciech Jozef Przybylowicz<fnref n="3" />, Pengjie Hu<fnref n="1" />,
**Longhua Wu**<fnref n="1" />, **Yongming Luo**<fnref n="1" />,<fnref n="4" />

*Corresponding author email: lhwu@issas.ac.cn*

## ABSTRACT

During the last decade there has been a growing interest in metal-hyperaccumulating plants, due to their unique physiology of metal tolerance and accumulation as well as their potentials for phytoremediation applications. Quantitative studies on the distribution of metals within plant tissues are important for understanding plant metabolisms, and the mechanisms of metal tolerance and hyperaccumulation. *Sedum plumbizincicola*, a new Cd/Zn hyper accumulator has the potential phytoextraction for Cd/Zn polluted soils. But the accumulation ability and mechanisms of plant grown in mine area are still unknown. In this study, *Sedum plumbizincicola* plants and the corresponding rhizosphere soils were collected from four different plant growing places of mine area, and the localization of Zn/Cd within root, stem and leaf frozen-hydrated cross-sections of *Sedum plumbizincicola* was quantitatively investigated using Cryo-micro-Proton-Induced X-ray Emission (cryo-µ-PIXE). The results showed that *Sedum plumbizincicola* showed Cd/Zn hyperaccumulating ability with high Cd/Zn (1.47/14.6 g kg⁻<fnref n="1" />) in mine area, but the variation of Zn/Cd accumulation in plant was much more largely depended on the available metals than total metals in the soils. In root, 96.6% of Zn in frozen-hydrated root tissue was distributed in the cortex with an

average concentration of 3111 mg kg⁻<fnref n="1" />, but Zn was low localized in central cylinder with an average concentration of 562 mg kg⁻<fnref n="1" />. In stem, Zn and Cd were preferentially accumulated in the epidermis with the average concentration of about 21375 and 1211 mg kg⁻<fnref n="1" />, respectively. Zn was also enriched in vascular bundle and its surrounding parenchyma. More interestingly, some of the vascular bundles were absent from Zn distribution. In leaf, the average concentration of Zn in the whole cross section of frozen-hydrated leaf was 1571 mg kg⁻<fnref n="1" />. Zn highly concentrated in the upper and lower epidermis with the concentration 5155 and 5660 mg kg⁻<fnref n="1" /> respectively, while Zn concentrations in palisade

<figure><img src="image_141.png" /><figcaption>Figure 1 Optical image and elements mapping of *S. plumbizincicola* growth in mine soil (g kg⁻<fnref n="1" /> FHW).</figcaption></figure>

and spongy mesophyll were 1201 and 1173 mg kg⁻<fnref n="1" />, respectively. While Cd extend relative evenly distributed in leaf with the highest concentration of 280 mg kg⁻<fnref n="1" /> in vascular and the lowest concentration of 128 mg kg⁻<fnref n="1" /> in spongy mesophyll, and 85.5 % of total leaf Cd was distributed in mesophyll when taking the tissue area into account. In conclusion, preferential sequestration of heavy metals especially Zn to epidermis in stem and leaf is one of the important mechanisms, and different mechanisms might operate Zn and Cd storage and detoxification in hyperaccmulator *Sedum plumbizincicola*.

**Acknowledgements:** The financial support by the National Natural Science Foundation of China, project No. 41325003 is greatly appreciated.

<figure><img src="image_142.png" /></figure>

<footnote n="1">¹Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China</footnote>

<footnote n="2">²School of Botany, The University of Melbourne, Parkville, VIC 3010, Australia</footnote>

<footnote n="3">³iThemba LABS, PO Box 722, Somerset West 7129, South Africa</footnote>

<footnote n="4">⁴Yantai Institute of Coastal Zone Research, Yantai 264003, China</footnote>
# NATURALLY SELECTED BIO-ACCUMULATORS OF HEAVY METALS IN A BATTERY WASTE DUMPSITE IN IBADAN, SOUTH-WESTERN NIGERIA

**Comfort Modupeore Adeoye<fnref n="1" />, Gideon Olajire Adeoye<fnref n="1" />, Oladele Osibanjo<fnref n="1" />, Jose Morenike Adeoye-Isijola<fnref n="2" />**

*Corresponding author email: kutulolaola@yahoo.com*

## ABSTRACT

A post impact assessment of a battery waste dumpsite was made in a local community acquired as a housing estate. Despite the devastation caused by the battery waste, certain group of plants grew luxuriantly. Soil and plant samples were therefore taken to evaluate the content of heavy metals in the soil and plants. Analysis was carried out in the Analytical Services Laboratory of International Institutes of Tropical Agriculture, (IITA), in Ibadan, Nigeria. Arrays of naturally selected bioaccumulation of heavy metals were identified by the level of Lead (Pb) in the roots and shoot/leaves of plants. The crops include: *Sporobolus pyramidalis* with (6,700 and 24,200 mgkg⁻<fnref n="1" />) lead in the leaf/stem and root respectively. In the same environment, *Panicum maximum* has 3,480 mgkg⁻<fnref n="1" /> Pb in the leaf/stem and 21,000 mgkg⁻<fnref n="1" /> in the root; *Imperata cylindrical* had 3,080 and 6,010 mgkg⁻<fnref n="1" /> Pb in the /stem and root respectively. *Agaratum conizoides* contains 4,950 mgkg⁻<fnref n="1" /> Pb in leaf/stem and 19,800 mgkg⁻<fnref n="1" /> in the root. *Gomphrena celosoides* has 20,200 mgkg⁻<fnref n="1" /> Pb in the leaf/stem and 23,610 mgkg⁻<fnref n="1" /> Pb in the root. It was observed that there is generally more accumulation of Lead in the roots than the shoot. The above plants should be screened as phyto-mop of Pb and further investigation is being carried out to determine the efficiency of each plant for further use. Judging from allowable concentration of Pb in plants by WHO (0.02 – 0.05 mgkg⁻<fnref n="1" />), these plants are hyper accumulators and should be investigated further as phyto remediators.

<figure><img src="image_143.png" /></figure>

<footnote n="1">¹ University of Ibadan, Ibadan, Nigeria</footnote>

<footnote n="2">² BABCOK University, Nigeria</footnote>
# CADMIUM BIOAVAILABILITY IN AN INTEGRATED BIOSYSTEM, AND THE USE OF AGARICUS SUBRUFESCENS AS A REMEDIATION AGENT

Ketil Stoknes<fnref n="1" />, Ewelina Wojciechowska<fnref n="2" />, Agnieszka Jasińska<fnref n="2" />, Ales Hanc<fnref n="3" />

Corresponding author email: ketil.stoknes@bio.uio.no

## ABSTRACT

Introduction: Cadmium in food stuffs receives considerable public and scientific attention due to this heavy metals toxicity and tendency to be taken up in crops. The bioavailability of Cd varies strongly in different materials, such as natural soils, organic wastes and inorganic fertilizers.

Integrated biosystems are connected food production systems where wastes are recycled into new crops in a sustainable way, often comprising industrial rather than traditional agricultural processes. We present here a case including anaerobic digestion of food waste connected to a closed greenhouse system in which mushroom and plant crops were grown using the digester residue (digestate) as well as some paper, straw and green waste. Cd enters with the raw waste; its mobility in the internal processes such as digestion, digestate dewatering, composting and crops growing was investigated. Some cultivated *Agaricus* mushroom species are known to bioconcentrate Cd. We were interested in testing if edible mushroom culture could be used to control Cd fate in the system.

Methods: Digestate was separated by filtration into a solid and liquid fraction. The solid fraction was composted 10 days with paper and straw for making mushroom compost [1]. *Agaricus subrufescens*, *A. arvensis* and *A. bitorquis* were grown in the mushroom compost. Some of the solid fraction was also vermicomposted using *Eisenia foetida*. Green wastes were composted thermophilically into a stable product. Lettuce, various herbs, tomatoes, beans and *Physalis* were grown in substrate mixes of digestate solids, vermicompost, spent mushroom compost and green waste compost. The liquid fraction of digestate was used for fertigation. Cd was measured by GF-AAS, ICP-AES and ICP-OES. Organic materials were also analyzed by a sequential extraction procedure for assessment of Cd bound to individual fractions: exchangeable, reducible, oxidizable and residual.

Results: Preliminary results from the overall system show that materials had total Cd concentrations between 0.1 and 0.9 mg kg⁻<fnref n="1" /> dry matter (DM), thus within a “normal” range.

*A. subrufescens* had a strong effect on Cd mobility: Mushrooms from the first crop contained 2 to 30 mg kg⁻<fnref n="1" /> DM, some far above the EU limit of ~8 mg kg⁻<fnref n="1" /> DM (corresponding to the limit of 1 mg/kg fresh weight). However, concentration more than halved in the second crop and continued decreasing in consecutive crops. In digestate rich composts, the mushrooms extracted more than 80% of the cadmium present at start, mostly during the first crop, while mushrooms from the second and later crops in fact complied with the EU legislation for consumption.

Mushroom cadmium content was negatively related to mushroom yield and positively related to substrate total cadmium concentration. Within composts of similar cadmium content, but with varying digestate content, a higher digestate content lead to higher mushroom cadmium content. This will be more closely analysed using the sequential Cd extraction results when ready.

We are currently analysing the complete set of crops and materials for Cd and other heavy metals. The mass flow of the integrated biosystem will be presented.

Conclusion: When mushroom culture is appropriate in integrated biosystems or other waste-based cropping systems, *A. subrufescens* can be an efficient Cd remediation agent. The species has a strong ability to take up and translocate Cd to the fruitbodies. This should be of particular interest when working with more polluted wastes.

## References:

1. Stoknes, K., Beyer, D. and Norgaard, E. (2013) Anaerobically digested food waste in compost for *Agaricus bisporus* and *Agaricus subrufescens* and its effect on mushroom productivity. J Sci Food Agric Jul 93(9), 2188-200

Acknowledgements: The financial support by the EU, EcoInnovation (project Food2Waste2Food ECO/11/304388) and Lindum AS, Drammen, Norway, is greatly appreciated.

<figure><img src="image_144.png" /></figure>

<footnote n="1">¹University of Oslo, Department of Biosciences, IBV, Oslo, Norway</footnote>

<footnote n="2">²Poznan University of Life Sciences, Department of Vegetable Crops, Poznań, Poland</footnote>

<footnote n="3">³Department of Agro-Environmental Chemistry and Plant Nutrition, Czech University of Life Sciences, Prague, Czech Republic</footnote>
# PLANT UPTAKE AND TRANSLOCATION OF 223-Ra AS A POTENTIAL RADIOPHARMACEUTICAL NUCLIDE

Stanislav Smrček<fnref n="1" />, Ján Kozempel<fnref n="2" />, Martin Vlk<fnref n="2" />, Šárka Pšondrová<fnref n="1" />

Corresponding author email: stanislav.smrcek@natur.cuni.cz

## ABSTRACT

The radiopharmaceutical therapy and diagnosis play important role in the treatment and imaging of cancer diseases and often make possible healing or life prolongation at malignant prognosis. Together with the use of classical radiopharmaceutical nuclides such as 99m-Tc, 51-Cr, or 67-Ga, the new radioisotopes with better characteristics of emitted particles are continuously investigated. One of new radionuclide for bone therapy is the 223-Ra. Radium-223 is an alpha emitter with a half-life of 11.4 days and after emission of 4 alpha and 2 beta particles decays finally to a stable 207-Pb. The general problem at radiopharmaceutical therapy and/or diagnosis is the impossibility of total recovery of a radioactive material into the radioactive waste. Thus part of the activity is excreted, preferably by urine into municipal or hospital wastewaters and can cause problems in the contamination of the ecosystem. For these reasons it is needed to investigate the possibilities of the removal of these radionuclides. The effective technology should be phytoextraction as additional step to conventional sewage treatment plants. Simultaneously the uptake and translocation of these radionuclides in agriculturally interesting plants can be investigated.

The phytoextraction of 223-Ra was investigated using in vitro cultures of common cultivated plants (Zea mays, Helianthus annuus, Brassica napus and corn plants). 223-Ra was eluted from 227-Ac/223-Ra generator as nitrate and added to cultivation solution. Murashige-Skoog or tap water was used as cultivation medium. To the 2-4 weeks old cultures (depending of plant species) the 223-Ra contaminated medium was added and cultures were cultivated at common conditions. After one or two weeks the plants were removed, root system intensively washed and the radioactivity distribution in plants was evaluated using electronic autoradiography.

The results show that 223-Ra is uptaken and absorbed by the root system, the uptake is markedly plant dependent. Moreover the uptake is strongly influenced by cultivation medium. The significant differences were found at cultivation in medium and tap water. Although the total uptake was below 10 % of inserted radioactivity, it was clearly documented, that the studied radionuclide is not only adsorbed on root surface, but it is effectively absorbed into plant tissue and translocated to shoot parts of plants.

It can be concluded, that the hypothesis about the possibility of food chain contamination by radiopharmaceutical therapy is true and the investigation of the methods for hospital sewage water is needed. The use of contaminant chelators or plant water transport accelerators will be used in further investigations to obtain more effective extraction of studied contaminant.

**Acknowledgements:** This project was funded by the Technology Agency of Czech Republic under contract No.: TA03010027, and its support is greatly appreciated.

<figure><img src="image_145.png" /></figure>

<footnote n="1">¹Charles University in Prague, Faculty of Science, Albertov 6, 12000 Prague, Czech Republic</footnote>

<footnote n="2">²Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Břehová 7, 11519 Prague, Czech Republic</footnote>
# RECOVERING METALS FROM SEWAGE SLUDGE, WASTE INCINERATION RESIDUES AND SIMILAR SUBSTANCES WITH HYPERACCUMULATIVE PLANTS

Johannes Kisser<fnref n="1" />, Heinz Gattringer<fnref n="1" />, Monika Iordanopoulos-Kisser<fnref n="2" />

Corresponding author email: jk@achemia-nova.net

## ABSTRACT

Sewage sludges as well as ashes and slags from waste incineration plants are known accumulation sinks of many elements that are either important nutrients for biological organisms (phosphorus, potassium, magnesium, etc.) or valuable metals when considered on their own in pure form (nickel, chrome, zinc, etc.); they are also serious pollutants when they occur at localized anthropogenic end-of-stream points. Technologies to recover these metals in a reasonable and economically relevant manner from very diffuse sources are practically non-existent or require large amounts of energy and chemicals, which pose environmental risks. Often more than 90% of these same metals have to be imported from abroad for technological use. These primary resources are becoming more expensive as they become more scarce and remaining deposits more difficult to mine, which is a serious concern for industrialized nations. Basic economic and strategic reasoning demands an increase in recycling activities and waste minimization.

On the other hand agriculture uses large volumes of mineral fertilizers, which are often sourced from mines as well, and thus are also subject to the same principle of finiteness and potential shortage in supply. These converted biological nutrients are taken up by crops and through the food chain and human consumption end up in sewage systems and in wastewater treatment plants in great quantities. The metabolized nutrients mostly do not return to agriculture, but due to contamination with heavy metals are diverted to be used as construction aggregates or are thermally treated and end up rather uselessly in landfills.

The project BIO-ORE aims to explore new pathways to concentrate metals from diluted sources such as sewage sludge and wastewater by using highly efficient biological absorption and transport mechanisms. These enzymatic systems from plants work with very little energy input. The process is called bioaccumulation and can be most effectively observed in hyperaccumulating metalophytes, which are studied for its suitability to be incorporated in metal recovery processes.

In a systematic series of tests under laboratory conditions the accumulation behaviour for antimony, chrome, cobalt, manganese, molybdenum nickel, zinc and rare earth metals of a selection of candidate plants growing on sewage sludge is assessed (quantitatively and qualitatively). Growth performance of these plants as well as the most suitable substrate properties were evaluated. Furthermore, an overview of the locations in Austria will be compiled where wastewater streams with comparatively high concentrations of the selected metals occur in interesting quantities. Lastly, stakeholders from all the process steps, from the sewage sludge treatment until final metal refinement, were involved and consulted early on in order to develop the best metal recovery strategies.

The results of this project provide the groundwork for further research and development steps that may bring to practical implementation a technological option with potentially huge benefits:

* The recovery of valuable metal resources from sewage sludge, incineration ashes and metal rich waste waters by environmentally friendly and low energy means

* Simultaneous decontamination of the input substrates from heavy metals, opening the possibility for these nutrient streams to be redirected to biological regeneration processes (for example use as fertilizers in agriculture) without fear of polluting soils with heavy metal loads

<figure><img src="image_146.png" /></figure>

<footnote n="1">¹alchemia-nova, Institute of innovative phytochemistry & closed loop processes, Vienna, Austria</footnote>

<footnote n="2">²Technical office MJKISSER, Mauerbach, Austria</footnote>
* Simultaneous generation of biomass on contaminated substrates, which can yield usable energy surplus through incineration during processing

Metal accumulation occurred in all used plants under certain conditions. Nitrogen seems to play an important role for the plants and their decision, which metals to (hyper)accumulate. Unfortunately *Pteris cretica* showed the worst metal accumulation in comparison to the other plants. *Alyssum murale* showed interesting accumulations for molybdenum in nutrient rich conditions of sewage sludge and good accumulation for nickel, rubidium and manganese under poor nutrient conditions. The accumulator plant *Dryopteris filix-mas* could be interesting for further investigation about vanadium and molybdenum. Two varieties of sunflower were tested, where rubidium and manganese showed promising results. *Eichhornia crassipes* was grown on watered industrial sludge and showed high accumulation for cadmium, nickel, rubidium and zinc. *Phytolacca americana* will also be tested in further trials, as they are said to be one of the rare earth accumulators, which could be partly proven by this first trials.

All in all, the results look very promising to keep up the work and stretch it to other waste streams as well. Therefore a national follow-up research project started this March 2014, where two years of field trials with hyperaccumulative plants and further phytomining strategies (recovering of metals) will be developed.

**Acknowledgements:** The financial support by FFG project "BIO-OREs" contract No. 84363 is greatly appreciated.

<figure><img src="image_147.png" /></figure>
# POTENTIAL OF INDIAN MUSTARD FOR RHENIUM PHYTOMINING

Luís A. B. Novo<fnref n="1" />, Andrea Pereira<fnref n="2" />, Alba Casanova<fnref n="2" />, Claudio F. Mahler<fnref n="1" />, Luís González<fnref n="2" />

Corresponding author email: novo@coc.ufrj.br

## ABSTRACT

Rhenium (Re) is one of the rarest elements found on Earth, with an estimate concentration of 0.4 ng g⁻<fnref n="1" /> in the upper crust, 2.2 pg mL⁻<fnref n="1" /> in freshwater, and 7-8 pg mL⁻<fnref n="1" /> in seawater. Due to its scarcity, Re is also one of the most expensive metals in the world market, presenting a high demand in the aerospace industry where it is used as a high-temperature superalloy. Bioavailable ReO₄ ions are widely dispersed in areas surrounding copper-molybdenum mines, and their accumulation in plants of these regions has been reported. This opens a window of opportunity for phytomining, a cost effective and environment friendly plant based technique to extract precious metals from low grade surface ores or mineralized soils. The objective of this study was to evaluate the potential of Indian mustard (Brassica juncea (L.) Czern), a species well known for its aptitude in the field of phytoremediation, to phytomine Re.

Commercial organic substrate was amended with KReO₄ (potassium perrhenate) to attain 5 different treatments according to the substrate Re concentration: 5, 10, 20, 40, and 80 mg kg⁻<fnref n="1" />. Healthy 14-day-old Indian mustard seedlings were then transferred to plastic pots containing the spiked substrate, at the rate of 5 replicates per treatment/harvest. The plants were allowed to grow under greenhouse conditions, and soil moisture was kept at 35 ± 5% with deionized water, according to field capacity. The plants were harvested for analysis 50 and 80 days after sowing. Fresh and dry biomass weights of the roots and shoots were determined, and the Re content of both plant parts was assessed through inductively coupled plasma optical emission spectrometry (ICP-OES). Analysis of variance (ANOVA) and homogeneity of variance tests were performed (p ≤ 0.05). Tukey and Dunnett's T3 tests were carried out for post-hoc comparisons between groups.

At the end of the experiment, the Indian mustard plants developed in the 5 mg kg⁻<fnref n="1" /> treatment presented

significantly higher shoot dry weight values. The aboveground dry biomass reductions of the plants grown in the 10, 20, 40 and 80 mg kg⁻<fnref n="1" /> treatments in relation to those of the 5 mg kg⁻<fnref n="1" /> treatment were of 29.46%, 37.87%, 43.26%, and 58.72%, respectively. The concentration of Re in shoots at the final harvesting period was strikingly higher in the plants subjected to the 80 mg kg⁻<fnref n="1" /> treatment, registering a mean of 23396.2 mg kg⁻<fnref n="1" /> (Figure 1). In comparison to the results of the 5, 10, 20 and 40 mg kg⁻<fnref n="1" /> treatments, this implied an increment of 1635.88%, 769.88%, 253.43%, and 200.38%, respectively. Nevertheless, the content of Re in the shoots of all treatments were well above the threshold levels of hyperaccumulation for this metal.

<figure><img src="image_148.png" /><figcaption>Figure 1. Concentration of Re in the shoots of Indian mustard 80 days after sowing. Data are means ± SD. Values followed by different letters differ significantly at p ≤ 0.05, n = 5.</figcaption></figure>

Accordingly, the bioconcentration factor for the plants of all treatments was two orders of magnitude higher than 1.

The findings of this study confirm the suitability of Indian mustard for Re phytomining. Although further research is required to assess the profitability of the process, the results suggest that potential earnings from Re phytomining with Indian mustard could be used to at least finance the phytoremediation of copper-molybdenum mining areas.

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

<figure><img src="image_149.png" /></figure>

<footnote n="1">¹Department of Civil Engineering, COPPE, Federal University of Rio de Janeiro, Brazil</footnote>

<footnote n="2">²Department of Plant Biology and Soil Science, University of Vigo, Spain</footnote>
# LOCALISED TRACE METAL SOLUBILISATION AND OXYGEN DEPLETION IN THE RHIZOSPHERE OF SALIX SMITHIANA UPON ADDITION OF ELEMENTAL SULFUR

Christoph Hoefer, Jakob Santner, Markus Puschenreiter, Walter W. Wenzel

Department of Forest and Soil Sciences, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Strasse 24, Tulln, A-3430 Austria

Corresponding author email: christoph.hoefer@boku.ac.at

## ABSTRACT

Phytoextraction by metal-accumulating willows such as *Salix smithiana* has shown promising results for Zn and Cd. However, even on moderately polluted soils, the applicability of phytoextraction is often hindered by presumably long remediation times. Enhanced contaminant solubility and, in consequence, increased contaminant accumulation in plants (i.e. induced phytoextraction), may contribute to higher phytoextraction efficiency, lower remediation times and thus enhance the practical applicability. However, induced phytoextraction may also increase the risk of contaminant leaching to groundwater. This risk might be low when elemental sulfur (S⁰) is used as soil amendment for a controlled and steady mobilization of trace metals.

Here we present data from a rhizobox experiment where *S. smithiana* was grown on a slightly acidic, moderately metal polluted soil with and without application of S⁰. We collected soil pore water from rooted and bulk soil compartments using Rhizon samplers and measured pH, SO₄²⁻, NO₃⁻ (IC) and metal concentrations (ICP-MS). After 61 days, plants were harvested and analysed for biomass production and metal concentrations. Additionally, willows were grown in rhizotrons for monitoring potential localised changes in metal solubility and soil respiration in the willow rhizosphere in response to S⁰. Chemical images of the distribution of metals and O₂ in the root zone were obtained using diffusive gradients in thin film (DGT) gels and planar optode (PO) sensors. We analyzed the DGT gels for the metal distribution in the rhizospere using laser ablation-ICP-MS, whereas O₂ concentrations (PO) in the rhizosphere were assessed using a colour-ratiometric approach.

Our results from the rhizobox trial showed significantly increased Mn, Zn and Cd concentrations in the sulfur treatments compared to the control due to soil acidification caused by the oxidation of S⁰. Enhanced metal mobilization was particularly observed in the S⁰ amended rhizosphere compartments. The corresponding large concentrations of Mn along with O₂ depletion in the rhizosphere indicated a reduction of Mn (oxy)hydroxides, serving as electron acceptors during sulfur oxidation in O₂-depleted zones. Enhanced metal solubility in the S⁰ treatments was generally associated with substantially increased uptake of Cd and Zn in willow tissues. Chemical images of DGT-available Zn and Cd show clear, root associated depletion zones in both, control and S⁰ treatments. Higher bulk soil metal concentrations and root associated solubilisation hot spots were observed in the S⁰ treatment. Increases of dissolved Mn were associated to both, sulfur application and patches of higher soil compaction. PO mapping results showed patchy, root associated, oxygen depletion in the S⁰ treatment, while O₂ depletion was almost absent in the control, supporting the hypothesis of reduction of Mn (oxy)hydroxides upon oxygen depletion due to the oxidation of elemental sulphur.

We conclude that the observed magnification of metal mobilization in the partially anaerobic willow rhizosphere, i.e. at the location of plant uptake, upon addition of S⁰ has a good potential for enhancing phytoextraction efficiency while keeping the risk of metal leaching from the bulk soil relatively low.

**Acknowledgements:** We acknowledge the financial support by the project "Chemical imaging of phosphorus dynamics in the rhizosphere" (P23798-B16) by the Austrian Science Fund (FWF) and the provision of the planar optodes by Dr. Morten Larsen (University of Southern Denmark).

<figure><img src="image_150.png" /></figure>
# HOW DO LOW DOSES OF DFOB AND EDTA AFFECT THE PHYTOEXTRACTION OF Cd, Cu, Pb AND Zn BY HELIANTHUS ANNUUS?

Jean-Yves Cornu<fnref n="1" />,<fnref n="2" />, Clément Dépernet<fnref n="1" />,<fnref n="2" />, Armelle Braud<fnref n="3" />, Thierry Lebeau<fnref n="3" />

Corresponding author email: jycornu@bordeaux.inra.fr

## ABSTRACT

Phytoextraction is the only way for the *in situ* cleaning-up of metal-contaminated soils. This technology is environmental friendly and cost-effective compared to physico-chemical clean-up process but its effectiveness must be improved to cope with the requirements of planners and users. The main weakness of phytoextraction is the long cleaning-up duration (Baker et al. 2000) primarily caused by the low phytoavailability of metals in soil. Inoculation of siderophore-producing bacteria (SPB) has been recently proposed as a strategy to optimize metal phytoextraction (Rajkumar et al. 2010). The localized and continuous production of siderophore in the close vicinity of plant roots, where most of the SPB are established, would help in promoting metal phytoextraction with minimizing the risk of metal leaching. However, there is a need to dissect how siderophores interact with metals at the soil-root interface to better assess the potential of coupling phytoextraction with the inoculation of SPB.

The present study focuses on desferrioxamine B (DFOB), the main siderophore produced by the actinobacteria *Streptomyces pilosus*. DFOB is characterized by a high selectivity for Fe(III) (10³<fnref n="1" /> M⁻<fnref n="1" />) but also by a good affinity for divalent metals including Cd(II) (10⁷.⁹ M⁻<fnref n="1" />), Cu(II) (10¹⁴.<fnref n="1" /> M⁻<fnref n="1" />) and Zn(II) (10¹<fnref n="1" />.<fnref n="1" /> M⁻<fnref n="1" />). The purpose of this work was to compare the efficiency of a low dose of DFOB vs. EDTA on the phytoextraction of Cd, Cu, Pb and Zn by sunflower. The main goal was to dissect the impact both chelators have on (1) the metals mobilisation from the solid phase, (2) their speciation in soil solution, (3) their uptake by plant roots and (4) their translocation from roots to shoots, for two cultivars of sunflower grown on an agricultural poly-contaminated soil.

Two cultivars of sunflower (ES RICA, KAPPLAN) with contrasted pattern of metal repartition (Laporte et al., 2014) were grown for 28 days on a calcareous soil (pH 7.3) contaminated in Cu, Cd, Pb and Zn (334, 5.2, 1191 and 561 mg kg⁻<fnref n="1" /> soil, respectively). Four days after transplanting, DFOB was supplied at the concentration of 200 µmol kg⁻<fnref n="1" /> soil to mimic bacterial production. The same procedure was performed for EDTA. Soil solution was extracted every week (on days 9, 16 and 23) using Rhizon soil moisture samplers. At harvest, xylem sap was collected on every plant by the "root pressure" method. Then, plants were divided into roots and shoots, and all plant tissues were freeze-dried, weighed, milled and digested in a mix HNO₃-H₂O₂. The concentrations of metals (Fe, Cu, Cd, Pb, Zn) in soil solution, xylem sap and plant tissues were assayed by ICP-MS. Investigations

are in progress to quantify in soil solution (i) the concentrations of DFOB and EDTA using the Fe-CAS complex, and (ii) the labile fraction of metals by anodic stripping voltammetry.

The results will provide new insights on the mechanisms by which DFOB alters the soil-plant transfer of metals and, thus, on the efficiency of coupling phytoextraction with the inoculation of siderophore-producing-bacteria.

<figure><img src="image_151.png" /></figure>

**Acknowledgements:** The financial support by the funding ANR 2011 CESA 008 01 is greatly appreciated.

<figure><img src="image_152.png" /></figure>

<footnote n="1">¹INRA, UMR ISPA 1391, CS 20032, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²Univ. Bordeaux, UMR ISPA 1391, CS 40201, F-33175 Gradignan, France</footnote>

<footnote n="3">³Univ. Nantes, UMR LPGN 6112, BP 92208, 44322 Nantes cedex3, France</footnote>
NEW INFORMATION FOR PHYTOEXTRACTION AND PHYTOMINING

X. Zhang<sup>1,2</sup>, B. Laubie<sup>1</sup> V. Houzelot<sup>1</sup>, E. Plasari<sup>1</sup>, A. Bani<sup>3</sup>, J.L. Morel<sup>2</sup>, G. Echevarria<sup>2</sup>, M.O. Simonnot<sup>1</sup>

Corresponding author email: Marie-Odile.Simonnot@univ-lorraine.fr

ABSTRACT

Ni-hyperaccumulators have the ability to store more than 10,000 µg Ni g⁻<fnref n="1" /> of dry matter (DM) in their tissues without heavy metal toxicity symptoms [2]. Nickel content in the organs of 14 hyperaccumulators from three genera: *Alyssum*, *Leptoplax* and *Bornmuellera* collected in Greece and Albania were analyzed [4]. Moreover, two different growing systems aimed to improve the yields of nickel phytoextraction were investigated, and the better cropping system was chosen for the sustainable development of *A. murale*. Results showed *Alyssum murale* (*A.murale*) grown in the Balkans (Albania) had yields of at least 100 kg Ni per ha.

For the huge biomass treatment, an optimized process to obtain a high value of ammonium nickel sulfate hexahydrate (ANSH) was studied in detail [1,3]. Once harvested and dried, *A. murale* biomass is incinerated to obtain concentrated Ni ashes to ensure the success of phytomining. This process is based on acid leaching for Ni extraction, purification of other elements in the leachate and different crystallisation steps to prepare the product at a high purity (≥99%). The main features of this process are presented as well as the newest results obtained at the pilot scale. We have clearly demonstrated the feasibility of the synthesis of this nickel double salt after phytoextraction and a start-up project has been launched to develop this activity.

To complete this approach, other extraction methods and synthesis are currently investigated to obtain various Ni products and will be presented.

References:

1. Barbaroux R, Plasari E, Mercier G, Simonnot MO, Morel JL, Blais JF. A new process for nickel ammonium disulfate production from ash of the hyperaccumulating plant *Alyssum murale*. *Science of the Total Environment* 2012; 423: 111-119.

2. Brooks RR, Chambers MF, Nicks LJ, Robinson BH. Phytomining. *Trends in Plant Science* 1998; 3: 359-362.

3. Mercier G, Barbaroux R, Plasari E, Blais J-F, Simonnot M-O, Morel J-L. Production of a crystallized nickel salt from hyperaccumulor plants. In: 2012.

4. Zhang X, Houzelot V, Bani A, Morel JL, Echevarria G, Simonnot M-O. Selection and combustion of Ni-hyperaccumulators for the phytomining process. *Int J Phytoremediat* 2014; 16: *1058-1072*.

Acknowledgements: This work was funded by Institut Carnot Environnement Energie Lorraine (ICEEL). It was also supported by the French National Research Agency through the national program "Investissements d'avenir" (ANR-10-LABEX-21 - RESSOURCES21). Sincere thanks are extended to China Scholarship Council (CSC), University of Lorraine, and INRA for providing financial assistance.

<figure><img src="image_153.png" /></figure>

<footnote n="1"><sup>1</sup> Laboratoire Réactions et Génie des Procédés, UMR 7274 CNRS – Université de Lorraine, 1, rue Grandville BP20451, 54001 Nancy Cedex, France</footnote>

<footnote n="2"><sup>2</sup> Laboratoire Sols et Environnement, UMR INRA Université de Lorraine, 2 avenue de la Forêt de Haye, TSA 40602, 54618 Vandœuvre-lès-Nancy, France</footnote>

<footnote n="3"><sup>3</sup> Agro-Environmental Department, Agricultural University of Tirana, Kamez, Albania</footnote>
# OPTIMISATION OF AMENDMENT EFFECTIVENESS IN STABILISATION OF METALS IN HEAVILY CONTAMINATED SOILS

Grzegorz Siebielec<fnref n="1" />, Petra Kidd<fnref n="2" />, Michel Mench<fnref n="3" />, Monika Pecio<fnref n="1" />, Rafal Galazka<fnref n="1" />, Vanessa Álvarez-López<fnref n="2" />, Jaco Vangronsveld<fnref n="4" />, Wolfgang Friesl-Hanl<fnref n="5" />, Markus Puschenreiter<fnref n="6" />, Andy Cundy<fnref n="7" />

*Corresponding author email: gs@iung.pulawy.pl*

## ABSTRACT

Metals can be stabilized in soil by amendments increasing metal adsorption or altering their chemical form. Few experiments compare different amendments under similar environmental conditions, or consider whether all soil properties or functions (microbes, soil fauna, plant growth, retention, colloid stability, etc.) are similarly protected. Within the EU FP7 Greenland project (266124) we compared the impact of novel soil amendment combinations and traditional materials on metal solubility and response of plants, soil organisms and microbial activity.

One-year greenhouse pot experiments were established: soil A, less toxic agricultural soil contaminated through long-term Zn/Pb smelter emissions in Poland (pH 7.0); soil B, toxic soil contaminated through smelter dust spill in Poland (pH 6.8). Amendments were tested individually and in novel combinations in planted and unplanted soils: compost (GWDA), drinking water residue (DWR), iron grit (IG), Ca-phosphate (PO4), LD slag (LD), Thomas basic slag (TBS), gravel sludge and siderite (GS/SID) and cyclonic ash (CA). Soil B was planted with grasses, and soil A with lettuce. Plants were periodically harvested, yields recorded and metal content determined. Soil metal extractability and bioaccessibility, pH, EC and enzymatic activity were measured. Soil pore waters were analysed for trace element/nutrient concentrations.

Parallel tests evaluated earthworm behaviour and metal accumulation. Earthworms Eisenia veneta were put into jars – five earthworms (previously weighed to record the initial weight) to each jar. The jars were stored at 20 °C and soil moisture was maintained at field water capacity. Earthworms were removed from the soil after 4 weeks, weighted and analysed for metals content (Figure). The soil was thoroughly mixed and samples were taken for the determination of metal solubility and pH.

The earthworms were removed from soil and kept on moist filter papers in glass vessels for 3 days for full depuration of worms. Then they were washed in deionized water, dried on paper towels, and killed by dry-freezing. The dry weight of the earthworms was recorded and the samples were dissolved in concentrated

nitric acid (Baker Analyzed Instra). Metal concentrations in earthworm extracts were measured with inductively coupled plasma mass spectrometry (ICP-MS, AGILENT 7500CE).

Plant-associated bacteria (rhizospheric or endophytic) can improve plant establishment, growth and survival in contaminated sites. In the next study we tested whether effect of selected amendment combinations can be enhanced by bacterial inoculation of plants (Festuca arundinacea. Eight amendment combinations were tested at 2 rates and with 2 types of inoculants.

We measured plant yield and metal concentration and metal soil extractability as indicators of the inoculation effectiveness.

<figure><img src="image_154.png" /></figure>

**Acknowledgements:** The financial support by FP-7 project GREENLAND contract No. 266124 is greatly appreciated.

<figure><img src="image_155.png" /></figure>

<footnote n="1">¹Institute of Soil Science and Plant Cultivation-State Research Institute, Pulawy, Poland</footnote>

<footnote n="2">²Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Spain</footnote>

<footnote n="3">³INRA - University of Bordeaux, France</footnote>

<footnote n="4">⁴Universiteit Hasselt, Diepenbeek, Belgium</footnote>

<footnote n="5">⁵Austrian Institute of Technology GmbH, Tulln, Austria</footnote>

<footnote n="6">⁶BOKU, University of Natural Resources and Applied Life Sciences, Vienna</footnote>

<footnote n="7">⁷School of Environment and Technology, University of Brighton, UK</footnote>
# AIDED PHYTOSTABILISATION OF A Cr, Mo AND Ni-CONTAMINATED TECHNOSOL

Nadège Oustriere<fnref n="1" />, Michel Mench<fnref n="1" />, Lilian Marchand<fnref n="1" />, Jean Luc Bouchardon<fnref n="2" />, Olivier Faure²b

Corresponding author email: oustriere.nadege@gmail.com

## ABSTRACT

The metal-contaminated technosol of a steel smelter heap at Rive-de-Gier, France, displays high potentially available Cr, Ni, and Mo pools with low organic matter and nutrient contents. At this site, aided phytostabilisation may restore a vegetation cover, reduce labile soil Cr, Ni and Mo concentrations, decrease exposure pathways and thus pollutant linkages, notably metal(loid) transfer through natural agencies in the environmental compartments and into the food chain. This study aimed at assessing in controlled conditions the efficiency of Ni/Cd-tolerant plants in mixed stand and soil conditioners to perform aided phytostabilisation on a metal (Cr, Ni, and Mo)-contaminated technosol, while avoiding to increase herbivore exposure.

Plots were implemented on the technosol of Rive de Gier. Soil treatments included: untreated soil (UNT), soil amended with either ramial chipped wood (RCW) or composted sewage sludge (CSS). Topsoils were collected in these plots and an uncontaminated kitchen garden (CTRL) and placed in pots (in triplicates). Potted Unt soil was also amended to singly test two other conditioners, i.e. vermiculite (5%) with compost (5%) (VOM), and iron grit (1%) with compost (5%) (OMZ). Seeds of Ni/Cd tolerant ecotypes of *Festuca pratensis* Huds., *Holcus lanatus* L., and *Plantago lanceolata* L were planted on all soils. Soil pore water was collected, pH and electrical conductivity were measured. After 15 weeks, shoots were harvested. Shoot ionome and soil pore water were analyzed by ICP-MS.

The pH of the soil pore water decreased from 10.2 (Unt soil) to 8.7 (CSS) and 8.8 (RCW) but did not change for the VOM and OMZ treatments. Soil amendment influenced metal concentrations (in mg L⁻<fnref n="1" />) in the soil pore water: Cr concentration decreased from 1.5 mg (UNT) to 0.01 - 0.3 for all amended soils; Mo concentration fell but to a lower extent from 3.5 mg (UNT) to 1.6 - 2.8 on amended soils; whereas Ni concentrations were similar for all soil treatment, i.e. 0.01 - 0.02. Nutrient concentrations and soluble organic matter (SOM) increased in the CSS soil pore water.

After 15 weeks, *F. pratensis* developed well on all soil treatments while *H. lanatus* and *P. lanceolata* only grew on the CTRL, UNT, CSS and CTRL soils and with a low growth rate. The shoot DW yield of *F. pratensis* for the CSS soil was 2 fold higher than that for the CTRL soil: it reached 375 ± 71 g m⁻<fnref n="2" /> (CSS) compared to 162 ± 5 g m⁻<fnref n="2" /> (CTRL). For the UNT, RCW, VOM and OMZ soils, it varied between 12 and 39 g m⁻<fnref n="2" />.

Shoot Cr, Ni and Mo concentrations of *F. pratensis* significantly decreased for the CSS treatment compared to the Unt one, i.e. from 31 to 0.8 mg Cr Kg⁻<fnref n="1" />, 16 to 1.1 mg Ni Kg⁻<fnref n="1" /> and 308 to 108 mg Mo Kg⁻<fnref n="1" />. The RCW, VOM and OMZ treatments reduced between 3 and 7 times the shoot Cr concentration of *F. pratensis*. However, these treatments did not change significantly its shoot Mo and Ni concentrations, which remained between 186 and 376 mg Mo Kg⁻<fnref n="1" /> and 8 and 16 mg Ni Kg⁻<fnref n="1" />.

The CSS treatment best performed to decrease Cr concentration in the soil pore water, increase shoot DW yield of *F. pratensis* and reduce its shoot Ni and Mo concentration. The increase of nutrient concentration in the CSS soil pore water may stimulate shoot production of this Ni/Cd-tolerant *F. pratensis* ecotype which allowed the dilution of the high Ni and Mo concentrations in *F. pratensis* shoot. Other soil treatments did not improve shoot yield of *F. pratensis* and such high shoot Ni, Cr and Mo concentrations may potentially affect herbivores.

Cultivation of a Ni/Cd-tolerant population of *F. pratensis* on this contaminated technosol amended with CSS is a successful combination to restore a dense grass cover. However the shoot Mo concentration of *F. pratensis* remains elevated. Direct evidences of an aided phytostabilisation of metals or only their in situ stabilization/phytoexclusion need further long-term assessment in plots.

**Acknowledgements:** This work was financially supported by ADEME (Physafimm project) and the European Commission under the 7th Framework Programme for Research (FP7-KBBE-266124, GREENLAND).

<figure><img src="image_156.png" /></figure>

<footnote n="1">¹ UMR BIOGECO INRA 1202, Ecologie des Communautés, Université Bordeaux, Bât. B2, Avenue des Facultés, 33405 Talence, FRANCE, and INRA, 69 Route d'Arcachon, 33610 Cestas, France</footnote>

<footnote n="2">² ENSM-SE centre SPIN, 158 cours Fauriel, 42023 St Etienne/ ²b Université Jean Monnet, 23 rue Dr. Paul Michelon, 42100 St Etienne, France</footnote>
# TRACE METALS IN GROWTH MEDIUMS AND PLANT TISSUE MATERIAL FROM A REMEDIATED GOLD MINE TAILINGS DAM IN SOUTH AFRICA

Pieter W. van Deventer<fnref n="1" />, Michael Seiderer<fnref n="2" />

## ABSTRACT

Remediation and rehabilitation of gold tailings is not necessarily a new science but rather a collection of established technologies that have been adapted to suit a new situation. Environmental aspects fundamental to the remediation or rehabilitation of mine dumps and tailings dams are a) aesthetics – shape, form, colour; b) air quality – dust, radioactivity, fumes; c) water quality – surface and ground water; d) soil quality and functions – sterilisation, pollution, land use potential; e) sustainability; and f) post closure land use after mining. Based on the complex integration of different remediation attributes, the most difficult aspects to achieve are the sustainability of applied rehabilitation and remediation techniques, and the post closure land use of tailings dams and mine dumps.

Geochemical imbalances and ongoing chemical reactions, e.g. oxidation of pyrite, do not favour the physical stability of mine dumps and tailings dams. Restrictive factors include the germination potential and survival rate of established vegetation on a hostile growth medium as well as the uptake of potentially toxic trace metals which is an important factor in the consideration of grazing as a proposed post closure land use option.

The geochemical aspects of a remediated gold tailings dam, together with concentrations of specific trace metals in the plant tissue growing on the tailings dam are discussed in this paper. The concentrations of macronutrients and ten other potentially toxic trace metals from tailings and top soil as well as three grass species used for remediation, are compared with each other in order to identify the species most suitable for remediation purposes.

Different correlations exist between soil pH and the bioavailability of trace metals. At pH < 5.3, bioavailable concentrations are distinct for Cr, Co, Ni, Zn and Cd, moderate for Mn and As, and non-existent for Cu, Pb and U. The highest macronutrient concentrations are found in the grass *Digitaria eriantha*, and concentrations of Mn, Co, Zn and As are highest in *Cynodon dactylon*. *Hyparrhenia hirta* shows a great affinity for Cr, and *Digitaria eriantha* for Pb. Only Cu and U exceed toxic limits in *Hyparrhenia hirta* and *Digitaria eriantha* at pH < 5.5.

In conclusion, it is possible to determine the suitability of different species for grazing as a post closure land use option. Relative to this, pH control is an important factor in the limitation of trace metal bioavailability in growth mediums, i.e. gold tailings.

**Keywords:** remediation, rehabilitation, gold tailings, macronutrients, trace metals, absorption, bioavailability

<figure><img src="image_157.png" /></figure>

<footnote n="1">¹School of Geo- and Spatial Sciences / Soil Science, North-West University, Potchefstroom, South Africa</footnote>

<footnote n="2">²Agreenco Environmental Solutions, Potchefstroom, South Africa</footnote>
# ASSESSMENT OF PHYTOSTABILIZATION OF Pb AND Zn IN A TAILING POND FROM SE SPAIN

Raúl Zornoza, Silvia Martínez-Martínez, Ángel Faz, José A. Acosta, María Dolores Gómez

Sustainable Use, Management, and Reclamation of Soil and Water Research Group, Department of Agrarian Science and Technology, Technical University of Cartagena, Paseo Alfonso XIII, 52, 30203 Cartagena, Murcia, Spain

Corresponding author email: raul.zornoza@upct.es

## ABSTRACT

Unreclaimed mine tailings are a worldwide problem due to tailings piles present a source of contamination for nearby communities. These mining areas are affected by the eolian dispersion and water erosion; moreover the wastes from tailing ponds are characterized by high contents of Fe-oxyhydroxides, sulphides and heavy metals. Aided phytostabilization consists of using both plants and organic/inorganic amendments in order to reduce contaminants mobility and bioavailability and recover soil health. In this study, Santa Antonieta tailing pond, located in Cartagena-La Unión mining district (SE Spain), was selected for applying the aided phytostabilization. Different samplings (before application, 6, 12, 18 and 24 months after application of marble waste, raw pig slurry and the solid phase of pig slurry), were carried out. Soil physicochemical and chemical properties were determined, and the vegetation biomass and the Pb-Zn concentrations in different parts (roots, leaves and stems) of the vegetation were analyzed. Results showed that pH, total nitrogen, cation exchangeable capacity, total organic carbon, aggregate stability, available phosphorous and exchangeable potassium increased while salinity decreased after amendments application. In addition, the available concentrations of Pb were reduced from 2019 to < 1 mg kg⁻¹ and for Zn from 107 to < 5 mg kg⁻¹. Fertility improvement and reduction of metals mobility promoted the development of plants and continuous increase of biomass during 18 months after amendments application. The roots of *Lygeum spartum* accumulated the highest Pb and Zn concentrations (~146 and ~290 mg kg⁻¹, respectively), exceeding the Pb concentrations in the roots of this plant the toxicity values proposed by different authors; while the concentrations in shoots were lower than 10 mg Pb kg⁻¹ and 60 mg Zn kg⁻¹. In addition, *Cynodon dactylon* accumulated high concentrations of Zn in roots and shoots (>250 mg kg⁻¹).

**Acknowledgements:** The financial support by LIFE+ project "Post-Mined Polluted Landscapes Reclamation by Means of Valorization of Different Residues" (LIFE 09 ENV/ES/000439) is greatly appreciated.

<figure><img src="image_158.png" /></figure>