International Master of Science in Environmental Technology and Engineering

Transcription

1 Master s dissertation submitted in partial fulfilment of the requirements for the joint degree of International Master of Science in Environmental Technology and Engineering an Erasmus Mundus Master Course jointly organized by UGent (Belgium), ICTP (Prague) and UNESCO IHE (the Netherlands) Academic year Exploring potential of iron and cerium oxide nanoparticles to reduce the uptake of arsenic by paddy rice Host University: Ghent University, Belgium Shilpi Misra Promotor: Prof. dr. ir.gijs Du Laing This thesis was elaborated and defended at Ghent University within the framework of the European Erasmus Mundus Programme Erasmus Mundus International Master of Science in Environmental Technology and Engineering " (Course N ) 2014 Ghent, Belgium, Shilpi Misra, Ghent University, all rights reserved.

2 Copyright The author and the promoter give permission to use this thesis for consultation and to copy parts of it for personal use. Any other use is subject to the Laws of Copyright. Permission to produce any material contained in this work should be obtained from the author. Ghent University, August 2014 The Promoter: Prof. ir. Gijs Du Laing The Author: Shilpi Misra i

3 Acknowledgment I would like to show my gratitude to my promoter, Prof. Gijs Du Laing for giving me the opportunity to work in the Laboratory of Analytical Chemistry and Applied Ecochemistry (Ecochem) as a Master s student, and also for his valuable comments and suggestions during the whole thesis work. I would also like to thank David de Vleesschauwe from the Department of Crop Protection, for providing me with the rice varieties. I would like to acknowledge the kind assistance of the staff members at the Laboratory of Analytical Chemistry and Applied Ecochemistry, Joachim, Roseline and Katty. They helped me a lot in carrying out the experiments especially Joachim who was always there when I needed him. Thanks to my parents for letting me spread my wings and fulfill my dreams. I would also like to express lots of love for my friends who were working me in the lab (Alebel, An Chen, Albana) for all the fun and company and especially to Gilda, Nadya and Mariana who have made my study period in Europe memorable. My Srilankan friends in UNESCO-IHE were there with me like a family because of them I never felt alone especially my friend Nadeeka who was like an elder sister taking care of me. A special thanks to the love of my life, for bearing all my tantrums and still supporting me through thick and thin, without you I won t be here. I also wish to express my sincere thanks to the European Union and the board of the IMETE program for selecting me as a grant student, giving me the chance to study a promising field within an international atmosphere. I also want to thank the staff members of the IMETE program for their unreserved help whenever I needed it, during my whole studying period. ii

4 Abstract In many areas of the world, especially South-east Asia, presence of high concentrations of arsenic in groundwater is very common. Rice, which accumulates arsenic to high levels in the grain, is the main staple diet in these countries, and groundwater is the main source of irrigation especially during the dry season. Accordingly, presence of arsenic in rice poses a health risk to the consumers. Various arsenic mitigation measures have been tested and used before trying to deal with this problem, but the use of metallic nanoparticles, which are successfully used for arsenic removal from wastewater, was never explored. Hence, this study aimed at studying the impact of metallic nanoparticles supplied with arsenic-contaminated irrigation water on the fate of the arsenic in a paddy soil environment. Therefore, a laboratory experiment was conducted during eight weeks in which rice (japonica variety Dongjin cv.) was grown on a soil. Arsenite As (III) and arsenate As (V) contaminated water were used for irrigation at a concentration of 100 μg/week. In different treatments, one kg of soil was treated with bulk soil amendments - 0.1% Fe2O3, 0.1%CeO2, FeCl3 (expressed as 0.1% Fe2O3) - or nanoparticles - Fe2O3 and CeO2 supplied through the irrigation water at two different dosages: 0.05g and 0.5g for the whole experimental period. Arsenic contaminated water and nanoparticles were supplied during the first 4 weeks. During the experiment, availabilities of arsenic, iron and cerium in the soil solution were determined regularly. Plant growth parameters (biomass yield, plant height and root length) and accumulation of arsenic in the different plant parts were also measured at the end of the experiment. The study revealed that in most of the cases Bulk FeCl3, 0.5g Fe2O3 and CeO2 nanoparticle treatments were able to reduce arsenic availability in the soil solution, enhance plant growth and reduce arsenic concentration in the plant parts. However, it should be noted that although the nanoparticle treatments seemed to be effective in reducing arsenic availability in soil solution and concentration in plants often no significant differences were observed when compared to the untreated plants, especially at the lower application dose. Therefore, further research using different arsenic and nanoparticle concentrations is still needed. Keywords: Rice, Fe2O3, CeO2, nanoparticles, plant uptake iii

5 Table of Contents Contents Copyright Acknowledgment Abstract Table of contents List of Tables List of Figures List of Abbreviations Page i ii iii iv vi viii x 1. Introduction Background Objectives of the study 2 2. Literature review Arsenic abundance and its sources Arsenic speciation in groundwater Arsenic in irrigation water, soil and rice crops Arsenic uptake in rice and its toxic effects on plants Arsenic transformation in rice paddies Human exposure to Arsenic Arsenic mitigation measures Soil amendments Application of nanoparticles Material and methods Experimental setup Preparation of pot experiment Growth conditions Experimental treatments Harvest of plants and measurement of plant height, root length, fresh and dry weight Analysis Soil analysis Analysis of pore water for total As, Fe and Ce contents Analysis of rice shoots and roots for arsenic concentration Statistical analysis Results Soil Characterization Arsenic concentrations in pore water Effects of treatments on As concentrations in pore water of soil 25 contaminated with As (III) Effects of treatments on As concentrations in pore water of soil 26 iv

6 contaminated with As (V) 4.3 Iron concentrations in pore water Effects of treatments on Fe concentration in pore water of soil contaminated with As (III) Effects of treatments on Fe concentration in pore water of soil contaminated with As (V) Cerium concentrations in pore water Effects of treatments on Ce concentration in pore water of soil contaminated with As (III) Effects of treatments on Ce concentration in pore water of soil contaminated with As (V) Plant growth Effects of treatments on plant growth for As (III) contaminated soil Effects of treatments on plant growth for As (V) contaminated soil Arsenic concentration and content in plants grown on soil irrigated with As (III) contaminated water Total As in root and shoot Total As in whole plant Arsenic concentration and content in plants grown on soil irrigated with As (V) contaminated water Total As in root and shoot Total As in whole plant Discussion Release of Fe in pore water from soils irrigated with As (III) and As (V) contaminated water Release of Ce in pore water from soils irrigated with As (III) and As (V) contaminated water Effects of treatments on As concentrations in soil solution for soils irrigated with As (III) and As (V) contaminated water Effect of treatments in reducing impact of As on the plant growth parameters Effect of treatments in reducing As accumulation in rice plants Arsenic concentration (mg/kg) and content (μg/pot) in roots Arsenic concentration (mg/kg) and content (μg/pot) in shoot Arsenic concentration (mg/kg) and content (μg/pot) in whole plant Arsenic translocation from root to shoot Correlation analysis Relation between As/Fe and As/Ce ratio in soil solution and rice shoot Economic feasibility of using bulk versus nanoparticle treatment options Conclusions and Recommendations 62 References 64 v

7 List of Tables Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Physicochemical properties of the soil (Mean ± SD, n=3) Page As concentrations in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) 28 As concentrations in the soil solutions of soil irrigated with As (V) contaminated water during the experimental period (Mean ± S.D., n=3) 28 Fe concentrations in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) 31 Fe concentrations in the soil solutions of soil irrigated with As (V) contaminated water during the experimental period. (Mean ± S.D., n=3) Table 4.6 Table 4.7 Ce concentrations (μg/l) in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) (D.L. detection limit = 0.5 µg/l) Ce concentrations (μg/l) in the soil solutions of soils irrigated with As (V) contaminated water during the experimental period (Mean ± S.D., n=3) (D.L. detection limit = 0.5 μg/l) Table 4.8 Table 4.9 Table 5.1 Total arsenic concentration and content in the roots, shoots and whole plant grown in As (III) contaminated water (Mean ± S.D., n=3) 37 Total arsenic concentration and content in the roots, shoots and whole plant grown in As (V) contaminated water (Mean ± S.D., n=3) 38 Correlation coefficients between As concentration in soil solution and As in plant for soil irrigated with As (III) contaminated water 53 Table 5.2 Correlation coefficients between As concentration in soil solution and As in plant for soil irrigated with As (V) contaminated water 54 vi

8 Table 5.3 Table 5.4 Table 5.5 Table 5.6 Page Correlation coefficients between As concentration in soil solution and plant growth parameters for soil irrigated with As (III) contaminated water 55 Correlation coefficients between As concentration in soil solution and plant growth parameters for soil irrigated with As (V) contaminated water 55 Correlation coefficients between As in plant and plant growth parameters for soil irrigated with As (III) contaminated water 57 Correlation coefficients between As in plant and plant growth parameters for soil irrigated with As (V) contaminated water 58 Table 5.7 Economic feasibility of different treatment options 61 vii

9 List of Figures Figure 2.1 Different Arsenic (As) species commonly occurring in the environment 4 Figure 2.2 Eh-pH diagram of aqueous arsenic species in the system As-O2-H2O at 25 C and at 1 bar total pressure (Smedley and Kinniburgh, 2005) Figure 2.3 Biogeochemical cycling of arsenic in the rice paddy 7 Figure 2.4 Dynamics of arsenic species in the rhizosphere of rice. Green arrows show adsorption to soil particulates. Red arrows show active plant transport processes. The blue circle illustrates the zone of oxygenation 9 around the growing rice root. (Meharg 2004) Figure 3.1 Experimental set up in the laboratory 16 Figure 3.2 Overview of the amendments used in the experimental setup 18 Figure 4.1 Root, shoot and total dry matter yield of rice plants irrigated with As (III) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant difference between treatments based on Tukey s test at P < Figure 4.2 Shoot height and root length of rice irrigated with As (III) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant 33 different between treatments based on Tukey s test at P < 0.05 Figure 4.3 Root, shoot and total Dry matter yield of rice irrigated with As (V) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant different between treatments based on Tukey s test at P < Figure 4.4 Shoot height and root length of rice irrigated with As (V) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant 35 different between treatments based on Tukey s test at P < 0.05 Figure 5.1 Fe2O3 nanoparticles forming flocks in the water layer above soil in the pots. 39 Figure 5.2 Brownish red deposition of Fe-plaque on the rice roots and iron oxide deposition on the oxic soil surface and sides of the pots 41 Figure 5.3 Average As concentrations (μg/l) in the soil solution for the different treatments during the whole experimental period. 46 Figure 5.4 Comparison of root, shoot and total dry weights in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p < Page 5 viii

10 Figure 5.5a Figure 5.5b Figure 5.6 a,b,c,d, Figure 5.6 e, f Comparison of shoot height in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05. Comparison of root length in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05. Comparison of As concentration (mg/kg) and content (μg/pot) in root (a, b) and shoot (c, d) of soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05 Comparison of As concentration (mg/kg) and content (μg/pot) in whole plant of soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05 Page Figure 5.7 Figure 5.8 a,b,c,d Comparison of root to shoot As translocation factor between different treatments for soils irrigated with As (III) and As (V) contaminated water. 52 Bars with same colour and different letter show significant difference between treatments at p < 0.05 Relation between As/Fe and As/Ce ratio in soil solution and rice shoot for soils irrigated with As (III) and As (V) contaminated water. 60 ix

11 List of Abbreviations As As (III) As (V) Ce CEC CeO2 Fe Fe2O3 FeCl3 ICP- OES ICP-MS NP SD Arsenic Arsenite Arsenate Cerium Cation Exchange capacity Cerium oxide Iron Iron oxide Ferric chloride Inductively coupled plasma-optical emission spectrometry Inductively coupled plasma-mass spectrometry Nanoparticles Standard Deviation x

12 1. Introduction 1.1 Background Arsenic is a toxic metalloid, widely distributed in the Earth s crust. It can be found in trace concentrations in water, soil, air and rocks. It is spread through both natural and anthropogenic sources. Natural sources include volcanic eruption, dissolution of minerals, exudates from vegetation and wind-blown dusts. Metal smelting, mining, combustion of fossil fuels, use as wood preservative and pesticides are the main anthropogenic sources of arsenic in the environment. Arsenic is of great environmental concern due to its occurrence in the groundwater and soil. Arsenic compounds are characterized as inorganic or organic and it can exist in the environment in four valency states (-3, 0, +3, +5). Inorganic forms of arsenic As (III) and As (V) are more common than methylated ones such as MMA (monomethylarsinic acid) and DMA (dimethylarsinic acid). Unlike most trace metals, arsenic forms oxyanions which tend to become less strongly sorbed to soil as the ph increases. Most groundwaters have a nearneutral ph but sometimes these anions can be present in solution even at neutral ph values. Accordingly, Arsenic is considered a major groundwater contaminant throughout the world. It is causing most serious health issues such as cancer in Bangladesh, West Bengal, India and China. Groundwater is the main water source for drinking as well as crop irrigation in these areas. The maximum limit of arsenic in drinking water has been changed from 50 μg/l to 10 μg/l by the World Health Organization (WHO) and USEPA, although in most of the South- Asian countries, such as Bangladesh, this limit is still set at 50 μg/l. Problems with arsenic mainly prevail in rice growing areas, where rice acts as the main staple food and rice straw is being used to feed animals. Rice is grown under waterlogged conditions and groundwater which is contaminated with arsenic is used for irrigation of the rice crops. As a result, the rice plants can accumulate arsenic from contaminated irrigation water. Inorganic species of arsenic, As (III) and As (V), are common in those contaminated waters. In the flooded environment, reducing conditions develop which leads to the conversion of As (V) to As (III), which is the more toxic form of arsenic. Arsenic gets easily associated with iron oxides in soil. Various studies previously focused on the use of iron oxides and hydroxides as bulk soil amendments to immobilise arsenic in 1

13 contaminated soil. Moreover, some researchers have reported that cerium oxides (CeO2) are effective for treatment of arsenic-contaminated wastewater. Furthermore, increasing attention goes to the use of nanoparticles for environmental cleanup. Therefore, we studied the potential impact of adding nanoparticles of iron oxide (Fe2O3) and cerium oxide (CeO2) to the irrigation water and their effect on mobility and availability of arsenic in the soil, arsenic accumulation in rice crops growing on the soil, and the translocation of arsenic within the crop. 1.2 Objectives of the study Specific objectives of this study were: Investigate the effectiveness of iron oxide and cerium oxide nanoparticles to reduce the availability and mobility of arsenic supplied to the soil through irrigation water. Study the impact of iron oxide and cerium oxide nanoparticles on uptake of Arsenic by rice and its translocation to the different plant parts. Compare the impact of supplying nanoparticles to the irrigation water with the impact of bulk soil amendments added to the solid soil phase (CeO2, Fe2O3, FeCl3). 2

14 2. Literature review 2.1 Arsenic abundance and its sources Arsenic (As) is an ubiquitous trace element. It is the 20 th most abundant among the naturally occurring elements in the Earth s crust. Often referred to as a heavy metal, it is actually a metalloid with predominant non-metallic character. Arsenopyrite (FeAsS) is the most widespread Arsenic mineral, a major constituent in most rocks, ores and coals (Reimann et al. 2009). Arsenic immediately ends up in the environment due to its widespread use as a pesticide, insecticide, herbicide or wood preservative. According to FAO, Codex Alimentarious (2013), a major share of the world arsenic production, i.e. 70%, is used in timber treatment as copper chrome arsenate (CCA), 22% in agricultural chemicals, and the remainder in glass, pharmaceuticals and non-ferrous alloys. Among the anthropogenic sources mining, metal smelting and burning of fossil fuels are the major contributors. Major natural sources of Arsenic consist of volcanic eruptions, forest fires and wind-blown dust. Most arsenic is released into the environment due to its mobilization from minerals under natural conditions. Problems related to the presence of Arsenic in groundwater are most pronounced in Asia. The countries in Asia with most reported cases of groundwater contamination are Bangladesh, Cambodia, China, India, Nepal, Pakistan, Thailand, and Vietnam. Among them, Bangladesh and the West Bengal region in India have the most severe cases of contamination of groundwater due to the weathering of aquifer rocks (Rahman et al., 2007). Bangladesh and the West Bengal region receive their water mainly from the Gangetic delta plain which has a naturally high concentration of As in groundwater (Siegel 2002). Groundwater is mainly used for irrigation during the dry season and drinking purposes in these areas, resulting in serious health implications for people using it regularly. 2.2 Arsenic speciation and its toxicity in groundwater Arsenic is present at an average concentration of 2 mg/kg in the Earth s crust. Its major valency states are -3, 0, +3 and +5 and it occurs in both inorganic and organic forms. Figure 2.1 shows the different forms of arsenic which commonly occur in the environment. 3

15 Figure 2.1 Different Arsenic (As) species commonly occurring in the environment Inorganic forms of arsenic are more prevalent in soils and groundwater as they exist as oxyanions of trivalent (As 3+ ) arsenite, dominant under reduced conditions, and pentavalent arsenate (As 5+ ), dominant under oxygenated conditions. Arsenate species are AsO4 3, HAsO4 2, H2AsO4 while arsenite species include As (OH)3, As(OH)4, AsO2OH 2 and AsO3 3 (Mohan and Pittman 2007). Generally, it is considered that inorganic species of arsenic are more toxic than the methylated ones. The toxicity of arsenic species follows the order AsH3 > As (III) > As(V) > MMAA (monomethylarsonic acid) > DMAA (dimethylarsinic acid). Toxic properties of arsenic contaminated water depend largely on its chemical speciation. Arsenate comprises 50% of the total arsenic in groundwater (Samanta et al. 1999). The stability of arsenic species under natural water conditions is mainly dependent on ph, redox potential, precipitating metals such as Fe (II/III), organic matter and microbial activity. Arsenic is a unique metalloid as it is more mobile at ph range of , which normally exists in groundwater, and under both reducing and oxidizing conditions (Smedley and Kinniburgh, 2005). Arsenic speciation is mainly governed by redox potential (Eh) and ph 4

16 variations (Fig. 2.2). At low ph (less than about ph 6.9) and under oxidizing conditions, the dominant species of arsenic is H2AsO4 whereas, HAsO4 2 becomes dominant at higher ph. Highly acidic and alkaline conditions show presence of H3AsO4 0 and AsO4 3 arsenic species, respectively. Uncharged species of arsenic such as H3AsO3 0 predominate under reducing conditions at a ph less than about 9.2. Fig. 2.2 Eh-pH diagram of aqueous arsenic species in the system As-O2-H2O at 25 C and at 1 bar total pressure (Smedley and Kinniburgh, 2005) 2.3 Arsenic in irrigation water, soil and rice crops Due to shortage of water supply during dry seasons, crops are often irrigated with groundwater. Long term use of As contaminated groundwater for irrigation might elevate the As concentrations in the soils and also in the crops irrigated with this water (Rahman et al., 2007). 5

17 If irrigation is performed with As contaminated water, the soil arsenic concentrations can go up to 83 mg/kg whereas the soils irrigated with non-polluted water contain 4-8 mg/kg of Arsenic (Ullah, 1998). High arsenic concentrations in soil and the use of irrigation water with high arsenic may ultimately lead to elevated concentrations of arsenic in cereals, vegetables, and other agricultural products of arsenic-affected areas (Abedin et al., 2002b). According to Meharg and Rahman (2003), in many arsenic affected areas of Bangladesh where irrigation is done using water from shallow tube wells, arsenic levels of 1.8 mg/kg have been recorded in the rice plants. However, depending on the rice cultivar, arsenic uptake and accumulation from the irrigation water and contaminated soil might vary. In Bangladesh, 75% of the total cropped area and 83% of the total irrigated area are used for growing rice (Dey et al., 1996, Smith et al., 2008). In the major arsenic contaminated areas, rice is the staple food and rice straw is used for feeding cattle. Abedin et al. (2002b) observed that if irrigation is done with As-contaminated groundwater, rice straw used for feeding to cattle can accumulate levels of As up to 92 mg/kg, whereas the legal limit for straw fed to cattle is 0.2 mg/kg in many countries (Nicholson et al., 1999). 2.4 Arsenic uptake in rice and its toxic effects on plants Rice plants are natural arsenic accumulators in comparison to other crops. Under normal condition rice plants take up a lot of silicon from soil which is used to strengthen their stems and the husks that protect the grain against pest attack and many other abiotic and biotic stresses. Rice accumulates up to 10% silicon in its shoot, which is very high amount (Ma et al., 2007). Chemical similarity between arsenic and silicon under soil conditions in flooded rice paddies results in arsenic transport to the plant parts via the silicon transporters. Thus arsenic is integrated into the plant as it grows and finds its way into the grain which we consume. Abedin et al. (2002b) conducted a greenhouse study using As contaminated irrigation water to determine the effects of As concentration in the water on growth of rice, and uptake and speciation of arsenic in the rice. Two phosphate doses (14.3 mg P/kg and 28.6 mg P/kg) and five As (V) doses (0-8 mg/l) were applied. With increasing concentration of arsenate in the irrigation water, a decrease in plant height, grain yield, the number of filled grains, grain weight, and root biomass was observed. At the same time an increase in arsenic concentrations in root, straw, and rice husk was noticed. It was concluded that arsenic 6

18 translocated almost equally to both root and shoot parts since the concentrations of arsenic in rice straw were similar to those in the roots for the highest As treatment (i.e. up to 91.8 mg/kg and mg/kg respectively). The effects of phosphate supply on the arsenic concentrations in rice plant parts and on plant growth were almost negligible. Abedin and Meharg (2002) conducted a short term study on the toxic effects of arsenite and arsenate on rice seedlings at different dosages (0-8 mg/l). They found that arsenite was more toxic for rice seed germination in comparison to arsenate. Also the shoot height and root tolerance index for rice seedlings decreased with increasing concentrations of arsenite and arsenate. These findings clearly indicate that inorganic As species may have a negative impact on plant growth parameters. 2.5 Arsenic transformation in rice paddies Arsenic in paddy rice mainly originates from geogenic weathering, due to soil formation from local bedrock or sediments carried from upstream (Meharg and Zhao 2012). Only in areas where rice is grown near metal smelters, atmospheric deposition is also a possible source of arsenic. Figure 2.3 Biogeochemical cycling of arsenic in the rice paddy Paddy rice is mainly grown under lowland (flooded) conditions, but it can also be cultivated in upland conditions (non-flooded). Due to the flooded conditions in paddy fields, anaerobic conditions are prevalent which leads to a reduction in redox potential (Eh). Cycles of 7

19 flooding and non-flooding result in a variation of the redox potential. Takahashi et al. (2004) reported that the concentrations of Fe, Mn and As during the flooded period were higher in the soil pore water. Under anaerobic conditions, As (III) is present, whereas As (V) is present under aerobic conditions (Fig 2.3). As was presented in Section 2, these species differ in mobility. In groundwater As (V) is more common but when this water is used for irrigation of paddy soil under anaerobic condition it transforms to As (III). Arsenic solubility and transformation to As (III) increases under flooded conditions thus leading to exposure of rice crops to Arsenic. However, the fact that Arsenic solubility increases under flooded conditions should mainly be attributed to the reductive dissolution of iron oxides/hydroxides. Together with this process, arsenic mobilization also occurs since iron oxide minerals are an important host phase for arsenic in soil. Before flooding, arsenic is mainly sorbed to soil particles in the form of arsenates, but after flooding arsenite can be found for 60-80% in the solution phase (Yamaguchi et al., 2011). Arsenic present in the soil pore water can be readily absorbed by the rice roots but the absorption mechanisms differ between arsenate and arsenite. As (V) is taken up via the high affinity phosphate uptake system (Meharg, 2004). Arsenite is more mobile than arsenate and it is transported into rice roots via water channels (aquaporins) (Meharg and Jardine 2003). Arsenite is uncharged and behaves as a water analogue under neutral ph with respect to plasma membrane transport and therefore, it is easily transported through the aquaporins (Meharg 2004). Figure 2.4 shows the complex chemistry of arsenic in the rhizosphere of rice plants. Even under flooded conditions, the rhizosphere region of the rice plants may still be aerobic due to the release of O2 into the rhizosphere by transport of oxygen from the leaves to the roots. Under these conditions precipitation of iron hydroxides (FeOOH) around the roots takes place which leads to the formation of iron plaque. Iron plaque acts as scavenger of As (V), ultimately forming highly insoluble iron arsenate. It should be noted that there is always an interconversion between species of arsenic taking place under paddy field conditions. Some microbes are capable of methylating inorganic arsenic to MMA and DMA. Therefore, though arsenite dominates in anaerobic conditions, As (V), MMA and DMA are also present. 8

20 2.6 Human exposure to arsenic Carcinogenic properties of arsenic were discovered in early 1879 due to increasing rates of lung cancer in miners exposed to As (Smith et al., 2002). Inorganic species of arsenic are more toxic than methylated ones. Among inorganic arsenic, trivalent (As 3+ ) compounds are usually more toxic than As 5+ compounds. The mode of exposure to arsenic is either oral ingestion or inhalation. Long term exposure to inorganic arsenic species can lead to arsenicosis. Effects of arsenic poisoning can take several years to develop and they also include skin lesions, peripheral neuropathy, gastrointestinal symptoms, diabetes, renal system effects, cardiovascular disease and cancer depending on the level of exposure. Figure 2.4 Dynamics of arsenic species in the rhizosphere of rice. Green arrows show adsorption to soil particulates. Red arrows show active plant transport processes. The blue circle illustrates the zone of oxygenation around the growing rice root. (Meharg 2004) Human exposure to As is mainly due to consumption of contaminated groundwater for drinking and cooking purposes. According to the WHO guideline the level of arsenic in drinking water should not exceed 10 μg/l but this threshold is often exceeded in Arsenic contaminated areas. The 9

21 tolerable intake level of arsenic in food is 3.0 μg/kg body weight per day (2 7 μg/kg body weight per day based on the range of estimated total dietary exposure) (WHO, 2010). In accordance to Chinese food safety standard, the inorganic As concentration in rice should not exceed 0.15 mg/kg. In the arsenic affected areas in South East Asia the main staple compound of the diet is rice which is grown under flooded conditions with contaminated groundwater. Approximately, 0.45 kg rice is consumed on daily basis in some villages in Bangladesh and West Bengal, India, and As contaminated rice contributes for between 11 to 32% to the dietary intake (Correll et al., 2006). Similarly, Meharg (2004) projected that consumption of rice containing 0.05 mg As /kg and drinking water with As concentrations of 0.01 mg/l contribute for approximately 60% to the human dietary As exposure. With the increase in concentration of arsenic in rice roots the concentration in straw also increases (Lin et al., 2013). Depending on the concentration of As in irrigation water the mean As concentration in rice grain grown in a greenhouse can range from 0.26 mg/kg to 0.74 mg/kg (Abedin et al., 2002b). Land degradation, loss in productivity due to effects on plant growth and contamination of the food chain can occur due to increased levels of arsenic in irrigation water (Brammer, 2005; Duxbury and Zavala, 2005). Thus, it becomes essential to find some mitigation measures to reduce the effects of arsenic pollution. 2.7 Arsenic mitigation measures Scientists have been working on the problems associated with Arsenic pollution for a long time. Various agricultural measures have been tested to reduce the translocation of Arsenic from the soil or contaminated water into the rice plants. These measures mainly include remediation of the soil with soil amendments. Soil amendments reduce the effects of arsenic by changing its speciation, reducing its uptake by the rice plants and/or affecting its solubility Soil amendments Iron containing materials have been often recommended to reduce the impact of arsenic pollution. Under natural soil conditions as well, Arsenic is generally bound to Iron hydroxides (FeOOH). It has been observed that Iron oxides suppress negative impacts of 10

22 arsenic pollution by either creating a high redox potential in the soil (Yoshiba, et al., 1996), by sorption of arsenic in/on the iron oxides (Ultra et al., 2009) and by precipitating low solubility arsenic compounds. Application of iron oxides to soils slows down the decrease in Eh under flooded conditions. This suppresses the reduction of arsenate to arsenite and dissolution of arsenic from soil solids (Yoshiba, M., et al., 1996). Also, arsenite has a lower affinity for most soil minerals such as aluminosilicate clay minerals and manganese oxides etc., except for iron oxides/hydroxides. Xie and Huang (1998) conducted an experiment in flooded paddies and found that application of FeCl3 H2O (25 mg Fe kg -1 soil or % Fe2O3) strikingly decreased the arsenic concentrations in the soils and husked rice by 25% and 9.2%, respectively. Moreover, the plants receiving the iron treatment showed better growth. A pot experiment conducted by Hossain et al. (2009) under flooded conditions confirmed that the addition of bivalent ionized iron (Fe 2+ ) leads to the formation of iron oxide plaque on the root surfaces, which in turn reduces the arsenic toxicity and its effects on rice growth. It was also observed that the arsenic concentration in rice grain and straw reduced and there was an increase in grain yield. Ultra et al. (2009) added 0.1% and 0.5% amorphous iron oxides/hydroxides to soil in a pot experiment using an arsenic-contaminated water supply containing 5 mg /l arsenate. Fe-oxide dosage of 0.1% enhanced the formation of iron plaque around the root surfaces leading to a decreased As concentration in the rice plants and an improved plant growth by increasing arsenic concentration on the root surface. Characteristics of iron plaque accumulation on mature rice plants and its impact on As (V) accumulation and speciation in the plants was studied by Liu et al. (2006) using a compartmented soil-glass bead culture system. The distribution of arsenic in the plant parts was in the order iron plaque > root > straw > husk > grain and there was a significant difference in iron plaque formation and arsenic accumulation in grain between genotypes. The main species of arsenic in the rice grain were inorganic arsenic and dimethyl arsinic acid (DMA). Liu et al. (2004) conducted an experiment on rice seedlings of different genotypes. They have grown the seedlings in water having different Fe concentrations ranging from 20 to 100 mg Fe 2+ /l to induce Fe- plaque and then transferred them into nutrient solution having an arsenate 11

23 concentration of 0.5 mg/l. It was observed that there was no significant effect on rice growth due to Fe 2+ concentrations in the pretreatment solution and 0.5 mg As/l in the treatment solutions. Iron plaque on the roots accumulated up to 75-89% of total As, and there was no difference between the genotypes. However, arsenic concentration differed in shoots of the different genotypes. Adsorption of arsenite and arsenate on iron oxides/hydroxides is decreased markedly by application of phosphates. Phosphate (PO4 - ) is an analogue of arsenate; thus, it increases desorption of arsenate from soil, enhancing the chance for reduction to arsenite under reducing conditions (Reynolds et al., 1999). A pot experiment using rice plants under flooded conditions was conducted by Hossain et al. (2009) in which it was found that addition of phosphate fertilizers increases arsenic accumulation in rice. Due to the addition of phosphates (0-50 mg P/kg-soil) arsenic adsorption on the iron oxide plaque formed on root reduced by 21%. Moreover, it was observed that addition of higher doses of arsenic (15-30 mg As/kg-soil) in combination with a high dose of phosphate (50 mg P/kg-soil) had a negative effect on grain yield. Though, it should be noted that once arsenate is displaced by phosphates it may be converted into arsenite under reducing conditions, thus reducing the competitive suppression of arsenic uptake by phosphate Application of nanoparticles Particles with at least one dimension in the range of nm are named nanoparticles (Morose, 2010). They should have a specific surface area per unit volume greater than 60 m 2 /cm 3 (European Commission, 2011). Nanoparticles have remarkable physical properties and chemical reactivity, e.g. a high specific surface area, and they exhibit a high sorption capacity for inorganic and organic compounds. This is the reason why they have received considerable attention recently in the fields of science and engineering. Use of nanoparticles for pollutant removal from water/wastewater is becoming very common and gaining popularity. Different types of nanoparticles can be used for water treatment, e.g., metalcontaining nanoparticles, carbonaceous nanomaterials, dendrimers and zeolites (Savage, 2005). In the section below, we will focus mainly on metal-based nanoparticles with special attention to iron and cerium based nanoparticles. 12

24 Iron based nanoparticles Nanoparticles are increasingly being used for environmental cleanup and for arsenic removal and most of the attention goes to iron nanoparticles (FeNP) (Shipley et al. 2009). Shipley et al., (2011) conducted column tests using iron oxide nanoparticles (19.3 nm magnetite and 37.0 nm hematite) to study the removal of arsenate and arsenite from soil. The columns contained 1.5 or 15 wt% iron oxide nanoparticles and soil. Aqueous solutions of arsenate (100 μg/l or 500 μg/l) or arsenite (100 μg/l) were pumped through the iron oxide nanoparticles and soil column, and effluents were collected. The concentration of arsenite or arsenate was determined in the effluent. Furthermore, an arsenic desorption experiment was also conducted by flushing arsenic free solution through the column and measuring arsenite and arsenate concentrations in the effluent. It was finally concluded that magnetite and hematite nanoparticles can be used to remove arsenic in sandy soils due to their strong adsorption, large retardation factor, and resistant desorption capacity. Zerovalent iron Fe (0) is considered an effective method to remove heavy metals. Therefore, Bang et al. (2005) conducted batch experiments to study their reaction with As (III) and As (V) in water. It was observed that when iron filings ( mesh) were mixed with arsenic solutions purged with nitrogen gas, the As (III) removal rate was higher than that for As(V) in the ph range of 4 7. Under anaerobic conditions, electrochemical reduction of As (III) to sparsely soluble As (0) and adsorption of As (III) and As (V) to iron hydroxides formed on the Fe(0) surface were observed. Removal rates increased under aerobic conditions and As (V) removal was faster than As (III) removal. Tang et al. (2011) used ultrafine iron oxide (α- Fe2O3) nanoparticles of 5 nm diameter to remove arsenic species (III and V) from labprepared and natural water samples. It was found that these particles were effective in removing arsenic species. The adsorption capacities of the α-fe2o3 nanoparticles for As (III) and As (V) in the lab-prepared water samples were found to be no less than 95 mg/g and 47 mg/g, respectively, at near neutral ph. Mayo et al. (2007) focused on the effect of Fe3O4 nanoparticle size on the adsorption and desorption behavior of As (III) and As (V). With a decrease in particle size from 300 to 12 nm, an increase in adsorption up to 200 times was observed for both As (III) and As (V). 13

25 Zhang et al., (2010) studied the effectiveness of zero-valent iron (ZVI), iron sulfide (FeS), and magnetite (Fe3O4) nanoparticles for immobilization of arsenic in soils. They found that Fe3O4 nanoparticles appeared to be more effective (5% or more) than other nanoparticles for immobilizing arsenic. Cerium oxide based nanoparticles Cerium is one of the most abundant elements among the rare earth elements. Use of cerium oxide in production of nanoparticles is becoming very prevalent now. Some studies have confirmed their effectiveness for removal of arsenic. It has been reported by Tabelin et al. (2013) that by the formation of inner sphere complexes, cerium oxides can adsorb As (III) and As(V) by making Ce-O-As bonds. Combining Cerium oxide with other metals is also very common to enhance their properties. Zhang et al. (2003) evaluated the capacity of a Ce (IV)-Fe doped iron oxide adsorbent for As (V) removal. The adsorbent was found to be very effective. Li et al., (2012) used hydrous cerium oxide (HCO) nanoparticles with a specific surface area of 198 m 2 /g and demonstrated exceptional adsorption capacity for both As (III) and As (V). At neutral ph, the adsorption capacity reached over 170 mg/g for As (III) and 107 mg/g for As (V). Over a wide ph range from 3 to 11, the HCO nanoparticles were able to remove As (III) by adsorption, which was not observed previously for other arsenic adsorbents. Ce Ti hybrid oxide adsorbent ( nm) with high sorption capacities for As (V) and As (III) were prepared by Li et al. (2010). They observed that the powdered adsorbent had a high sorption capacity, up to 7.5 mg/g for As (V) and 6.8 mg/g for As (III) at the equilibrium arsenic concentration of 10 μg/l. Also Deng et al. (2003) previously reported that the sorption capacity of As (V) increases when Ce Ti oxide adsorbents are combined by hydrolysis-precipitation rather than both used separately. Other metal based nanoparticles Calcium peroxide nanoparticles (15-25 nm) were synthesized and their potential to remove As (III) from contaminated water samples was tested by Olyaie et al. (2012). A dosage of 40 mg/l was able to remove up to 88% of arsenic in 30 minutes and at ph 7.5. CaO2 nanoparticles could effectively remove total As at natural ph conditions (between 6.5 and 8.5). Therefore, they have also been suggested as promising As removal method. 14

26 Martinson and Reddy in (2009) evaluated the efficiency of cupric oxide (CuO) nanoparticles (surface area 85 m 2 /g and diameter nm) to adsorb As (III) and As (V) from groundwater. The CuO nanoparticles were effective in removing As (III) and As (V) between ph 6 and 10 and the maximum adsorption capacity was 26.9 mg/g for As(III) and 22.6 mg/g for As(V). The CuO nanoparticles were also able to remove arsenic to less than 3 μg/l from the groundwater samples, which proves their capability as an effective adsorbent. All above studies prove that soil amendments can be used to reduce the mobility of Arsenic in soils, whereas nanoparticles have been proven to be able to remove Arsenic from water. However, until now nanoparticles have rarely been tested as potential adsorbent for Arsenic in soil-water-plant systems. Therefore, we studied whether metal oxide (Fe2O3 and CeO2) nanoparticles supplied to rice fields together with the Arsenic contaminated irrigation water could affect the Arsenic mobility and availability in the soil, as well as plant uptake and translocation within the rice plant. We compared the impact of these nanoparticles with the impact of bulk soil amendments. 15

27 3. Material and Methods 3.1 Experimental Set-up Preparation for pot experiment The soil used in the pot experiment was obtained from Ooike (Kruishoutem, Belgium), and completely mixed and homogenized. Paddy rice seeds (Oryza sativa L.), a Japonica variety (Dongjin cv.), was obtained from the Department of Crop Protection of Ghent University. In order to sterilize the seeds two tablets of Sodium hypochlorite (NaClO) were dissolved in 250 ml deionized water and seeds were kept in it for half an hour followed by thorough washing with deionized water. The seeds were then kept for germination at 28 C in the dark in an oven on a moist tissue paper placed in glass dishes. Plastic pots of two liter capacity (closed at the bottom) were filled with 1 kg of soil. After 30 days of germination, uniform seedlings were selected and transplanted to the pots (Figure 1). One seedling was transplanted to each pot. Nutrient solution, containing urea CO(NH2)2 and potassium dihydrogen phosphate KH2PO4, was added to the soil to reach an application rate of 50 mg N/kg, 50 mg P/kg and 63 mg K/kg. Fig Experimental set up in the laboratory 16

28 3.1.2 Growth conditions All experiments were carried out in a controlled environment under laboratory conditions. Daylight was supplied with three lamps (36 watt each) (4FT 36W T8 GROLUX) at a day light period of 16 hours/day. The temperature fluctuated during the whole experimental period between 25 and 30 C. The relative humidity was 60 % Experimental treatments The total experimental period was 8 weeks during which Arsenic was supplied with irrigation water to all pots. Arsenic was supplied as arsenite As (III) or arsenate As (V), at a same concentration of 100 μg week -1 to each pot containing one kg soil for a period of 4 weeks. Arsenite was supplied as a solution of Sodium Meta arsenite (NaAsO2, MW: g/mol, Fluka, Switzerland) and arsenate as disodium hydrogen arsenate heptahydrate (Na2HAsO4.7H2O, MW: g/mol, MERCK, Darmstadt, Germany). These solutions were mixed with distilled water to prepare As (III) and As (V) stock solutions of 100 ppm. For each As species, 7 different amendments were tested. Three treatments were bulk soil amendments, i.e., powdered forms of Iron (III) oxide (Fe2O3 0.1%), Cerium (IV) oxide (CeO2 0.1%) and FeCl3 (0.1% expressed as Fe2O3). The other 4 treatments consisted of two different types of nanoparticles, i.e. Fe2O3 and CeO2 both added in two different concentrations 0.05 g/kg soil and 0.5 g/kg of soil for the entire period of experiment. Fe2O3 and CeO2 nanoparticles were supplied with irrigation water (dosage divided equally within the 4 weeks along with As contaminated water) hereas, the bulk amendments were mixed thoroughly with the soil before starting the experiment. The nanoparticles were obtained from PlasmaChem GmbH (Berlin, Germany). They were produced by chemical synthesis and supplied as 5% aqueous solution, having a Fe2O3 and CeO2 concentration of 50,000 mg/l. The average particle sizes of the purchased CeO2 and Fe2O3 ENPs were 4 nm and 4-8 nm, respectively. An overview of the different amendments is given in Figure 3.2. The whole experiment was conducted in triplicate. The rice plants were grown under paddy field conditions, i.e. with the water level kept at 3 to 4 cm above the surface of the soil, up to vegetative stage. The water level was maintained by adding deionized water twice a week. 17

29 Total 48 pots Soils irrigated with As (III) contaminated water One control + 7 treatments = 8 pots X 3 replicates each Control Fe 2O 3 NP 0.05g/kg soil Fe 2O 3 NP 0.5g/kg soil CeO 2 NP 0.05g/kg soil CeO 2 NP 0.5g/kg soil 0.1%Bulk CeO 2 0.1%Bulk Fe 2O 3 Bulk FeCl 3 = 24 pots Soils irrigated with As (V) contaminated water One control + 7 treatments = 8 pots X 3 replicates each Control Fe 2O 3 NP 0.05g/kg soil Fe 2O 3 NP 0.5g/kg soil CeO 2 NP 0.05g/kg soil CeO 2 NP 0.5g/kg soil 0.1%Bulk CeO 2 0.1%Bulk Fe 2O 3 Bulk FeCl 3 = 24 pot Fig Overview of the amendments used in the experimental setup. 18

30 Rhizon soil moisture samplers (length 10 cm) were used to regularly extract small volumes of pore water from the soil. Dissolved compounds in the pore water of the soil can be measured in these extracts without any further filtration since the samplers contain filter material with a pore size of µm. Samplers were placed vertically in all the pots for sampling of the pore water with a time interval of 7 days. Total Arsenic, iron and cerium were measured in each pore water sample Harvest of the plants and measurement of plant height, root length, fresh and dry weight After 8 weeks of growth, the plant height was measured before harvesting. Subsequently, the shoots were harvested carefully by cutting them 4 cm above the soil to avoid any contamination with arsenic containing water. Plant roots were also carefully removed from the soil, washed thoroughly with tap water several times, followed by washing with deionized water, and dried using tissue paper to remove excess water. Afterwards the root length was measured. The fresh weights of both root and shoot parts were measured using an analytical balance. Subsequently, the shoot and roots were dried at 70 C for 3 days in an oven. The dried shoot and root samples were weighed again and cut into small pieces for further analysis. 3.2 Analysis Soil analysis Before the experiment started, soil properties were measured in triplicate according to Van Ranst et al. (1999). Field capacity The field capacity is the amount of soil moisture or water content held in soil after excess water has drained away and the rate of downward movement has materially decreased. This usually takes place within 2-3 days after a rain or irrigation in pervious soils of uniform structure and texture. A white band filter was put at the bottom of an empty perforated pot, then 400 g of soil were added to it and the pot was weighed. After that, 1 liter of water was added to the pot and the water was allowed to leach by gravity. The pot was weighed next day after leaching and the remaining moisture content in the soil was determined. This was 19

31 done in order to determine how much Arsenic contaminated irrigation water along with the nanoparticles can be added to the pots each week. ph Soil ph was measured using two methods: Actual acidity (ph-h2o): the acidity of the free H + -ions in the soil. 10 g of soil were weighed in a 100 ml beaker and diluted with 50 ml of distilled water. After the solution had been equilibrated for 16 hours, it was again brought to suspension with a glass rod and the ph of the supernatant was measured using an Orion 520A ph meter. Potential acidity (ph-kcl): ph-measurement after exchange of the adsorbed H + -ions with K + -ions 1 M KCl solution was prepared by dissolving g KCl in a 250 ml volumetric flask. 10 g of soil were weighed in a 50 ml beaker and 25 ml of 1 M KCl were added to it. The solution was shaken for 10 min and ph of the suspension was measured using an Orion 520A ph meter. Electrical Conductivity (EC) 20 g of soil were weighed in a 300 ml Erlenmeyer flask and 100 ml of distilled water were added to it. The flask was put on the shaker for 30 min; afterwards, the suspension was filtered using the filter paper and electrical conductivity (EC) of the filtrate was measured using a WTW LF537 conductivity meter. Organic matter content (Walkley&Black method) One gram of dry soil was weighed into 500 ml Erlenmeyer flasks. Two blanks were also prepared without soil. 10 ml of Potassium dichromate solution was added into each flask containing soil and mixed. Then, 20 ml of concentrated Sulfuric Acid was added to each flask and mixed gently under the fume hood. The flasks were kept for 30 minutes to allow complete oxidation of the organic matter. Then 150 ml deionized water, 10 ml phosphoric acid (H3PO4) and 1 ml indicator was added and the contents were titrated with ferrous sulphate until the colour changes to brilliant green. 20

32 Cation exchange capacity (CEC) In order to assess the capacity of soil to exchange cations, a saturation of the adsorption sites with ammonium ions (NH4 + ) and subsequent release of these ions with 1 M KCl solution was performed. Two grams of soil mixed with 12.5 g sand was weighed and added to percolation tubes. A layer of 0.5 cm sand was added above and below the soil and sand mixture. After percolation of 75 ml 1 M ammonium acetate (NH4OAc), 150 ml denatured ethanol (C2H5OH) 95% was added to wash away the excess NH4 + ions. Afterwards, 250 ml KCl (1 M) solution was added to remove the exchangeable NH4 + ions. The final percolate was then captured in a 250 ml volumetric flask. Afterwards, 50 ml of the KCl extract was transferred to a distillation flask and 0.1 mg MgO was added. The NH3 formed was captured by steam distillation as NH4 + in an Erlenmeyer flask that contained 20 ml of 2% boric acid. This final solution was titrated with 0.01 N HCl using a Methrohm 718 STAT Titrino system until the colour shifted to pink. Soil texture analysis via (Bouyoucos) Hydrometer Method Fifty grams of dry soil were weighed into a 1 l beaker and 15 ml of deionized water and 15 ml H2O2 (30%) were added. The beaker was covered with a watch glass. In case of frothing a few drops of ethanol were added and the beaker was left overnight. Afterwards, the beaker was heated in warm water bath at 80 C and 5-10 ml of 30% H2O2 was added regularly in order to decompose the organic matter. After 1 h, 300 ml of deionized water was added and the beaker was kept on a hot plate to boil for 1 h. Subsequently, the beaker was allowed to cool down and soil to be settled, and the supernatent was decanted. The beaker was kept for drying in an oven at 105 C for 24 h. Forty grams of the dried soil was put into a 1 L plastic bottle with 100 ml dispersing agent (40 g sodium hexametaphosphate and 10 g of soda in 1 L of water) and was kept for overnight shaking. The contents were transferred into a sedimentation cylinder of 1 l along with 900 ml deionized water. A blank without soil was also used. The cylinders were kept in warm water bath at 24 C. The cylinder was shaken thoroughly and a hydrometer (i.e. standard hydrometer, ASTM nr. 152H with bouyoucos scale in g/l) was lowered into the solution. Readings on the hydrometer were taken in triplicate at a time interval of 30s, 60s, 90, 120, 960 and 1440 min. 21

33 Total Nitrogen One gram of dry soil was transferred into a digestion flask and 7 ml of a mixture of sulphuric acid and salicylic acid were added. The contents were allowed to react for 30 min. Then 0.5 g sodium thiosulfate was added and the mixture was allowed to react for 15 min. After this, 5 ml of concentrated H2SO4, 0.2 g catalyst and 4 ml H2O2 were added. The tubes were kept for digestion at 380 C for 1 h and 30 ml deionized water was added after cooling. The contents were distilled with 40% NaOH in a distillation assembly and ammonia (NH3) was absorbed in 20 ml boric acid (2%). Subsequently, the contents were back-titrated with 0.01 N HCl using a Methrohm 718 STAT Titrino system until the colour turns slight pink. Phosphorus determination 1 ml of soil solution was transferred into a test tube. Successively 5 ml milli-q water, 1 ml Scheel (I) solution, and 1 ml Scheel (II) solution were added and this mixture was shaken for a perfect homogenization. It was allowed to react for 15 min. Afterwards, 2 ml Scheel (III) solution was transferred to the tube, after which the tube was shaken again and allowed to react for another 15 min. The absorbance was read at 700 nm using the spectrophotometer. Determination of trace elements in the soil For the determination of trace metal concentrations, the soil samples were first digested using aqua regia. One gram of soil was weighed into a 100 ml Erlenmeyer flask and 7.5 ml of concentrated hydrochloric acid (HCl) and 2.5 ml of concentrated nitric acid (HNO3) were added. The flasks were covered with a watch glass. After keeping the flasks for overnight digestion, 3 ml of deionized water was added to each flask and the samples were digested for 2 hours on a heating plate (150 C & 50 Watt). After cooling, each extract was filtrated through an acid-resistant filter into a 100 ml volumetric flask. Each flask was rinsed 5 times with 1% HNO3 by transferring the liquid over the filter. The extracts were diluted to 100 ml with deionized water. The flasks were covered with parafilm and shaken. Each diluted extract analysed for total Iron (Fe), Cerium (Ce) and manganese (Mn) using ICP-OES (Vista- MPX CCD Simultaneous ICP-OES) and for Arsenic (As) using ICP-MS with Rh as Internal Standard (Perkin Elmer SCIEX ELAN DRC-e). 22

34 3.2.2 Analysis of pore water for total As, Fe and Ce contents Pore water samples were collected using the Rhizon samplers and acidified with one drop of concentrated HNO3. The samples were analysed for As, Fe and Ce concentrations. The concentration of Fe was determined using ICP-OES (Vista-MPX CCD Simultaneous ICP- OES) and the concentrations of As and Ce were analysed using ICP-MS with Rh and Ga as Internal Standard (Perkin Elmer SCIEX ELAN DRC-e) Analysis of rice shoots and roots for total Arsenic concentration For shoot 0.25 g and for root 0.1 g oven-dried sample were weighed into 100 ml Teflon vials. Ten ml of concentrated HNO3 was added and vials were kept for sonication (Bandelin Sonorex super RK103H) at a frequency of 35 KHz for 15 min. The samples were then digested in a Microwave (Mars 5 CEM, BRS) at 1200 W and 600 psi for 25 min until the temperature reaches 190 C, holding this temperature for 15 min. After cooling down, the digested samples were filtered and transferred to 50 ml volumetric flask with continuous washing with Milli-Q water. Total Arsenic, iron and cerium were measured in the plant samples using ICP-MS (Perkin Elmer SCIEX ELAN DRC-e) with Rh and Ga as Internal Standards and ICP-OES (Vista-MPX CCD Simultaneous ICP-OES). Necessary reagent blanks and sample replicates were also included to check the accuracy of the analytical procedure. 3.3 Statistical analysis In order to assess whether any significant differences were present between various groups, statistical tests were performed in the software package IBM SPSS Statistics 22. The tests used were One-way ANOVA, Welch-ANOVA for groups that failed to fulfil all assumptions and Tukey test to conduct multiple comparisons. Correlation analysis was also done in order to study the relationships between Arsenic concentration in soil solution, plants and plant growth parameters. 23

35 4. Results 4.1 Soil Characterization The major physicochemical properties of the soil used for the experiment are presented in Table 4.1. The ph of the soil was near-neutral and its electrical conductivity was ± 0.07 μs/cm. The cation exchange capacity (CEC) and organic matter content (%) were 11.4 ± 0.33 meq/100 g soil and 2.9 ± 0.18 %, respectively. According to soil texture analysis it was found that the soil type is silt sandy loam. The total Fe, As and Ce concentration of the soil were also analyzed. Table 4.1 Physicochemical properties of the soil (Mean ± S.D., n=3) Soil properties Average values ph (in H2O) 6.7 ± 0.01 ph (in KCl) 6.4 ± 0.03 Electrical conductivity (μs/cm) ± 0.07 Organic matter (%) 2.9 ± 0.18 CEC (meq/100g) 11.4 ± 0.33 % Clay 14.1 ± 0.21 % Sand 33.4 ± 1.77 % Silt 52.7 ± 1.63 Soil texture Silt sandy loam Total nitrogen (%) 0.12 ± Total iron (%) 1.3 ± 0.04 Total arsenic (mg/kg) 7.25 ± 0.12 Total cerium (mg/kg) ± 5.95 Available phosphorus (mg/kg) ± 3.8 Total iron and arsenic in the soil were 1.3 ± 0.04 % and 7.25 ± 0.12 mg/kg, but the total cerium concentration was quite high, i.e ± 5.95 mg/kg. The nitrogen and available phosphorus content in the soil were 0.12 ± % and ± 3.8 mg/kg, respectively. 24

36 4.2 Arsenic concentrations in pore water Effects of treatments on As concentrations in pore water of soil contaminated with As (III) Arsenic concentrations measured in the soil solution during the experimental period of eight weeks are presented in the Table 4.2. In the first week, arsenic concentrations in the soil solution were higher in most treatments compared to the untreated soils (179.4 ± 55.5 μg/l), except for the bulk FeCl3 treatment (73.2 ± 22.6 μg/l). Statistical analysis confirmed that the concentration in the bulk FeCl3 treatment was significantly different (p <0.05) from the concentrations in the untreated soils and the other treatments. In the second week, the bulk FeCl3 (202.2 ± 20.8 μg/l) and the nanoparticles treatments 0.05 g Fe2O3 (209.0 ±16.8 μg/l), 0.5 g Fe2O3 (202.7 ±25.5 μg/l) and 0.5 g CeO2 (206.6 ± 28.4 μg/l) all resulted in lower arsenic concentrations in the soil solution compared to the untreated soils (221.5 ± 8.5 μg/l). Although differences are observed between the treatments, but ANOVA analysis confirmed that these are not significant at the 95% confidence level (p = 0.288). In the third week all treatments showed an increase in soil solution As concentrations compared to the untreated soils however the differences were not significant (p = 0.461). Nevertheless, a reduction in As concentration from week four is observed for bulk FeCl3, 0.05 g Fe2O3, 0.5 g Fe2O3 and 0.5 g CeO2 nanoparticles treatments. Statistically, bulk FeCl3 (173.3 ± 21.0 μg/l) showed a significant difference (p <0.05) compared to the untreated soils (220.2 ± 16.3 μg/l) and soils treated with 0.1% bulk Fe2O3 (212.7 ± 13.9 μg/l). A noticeable reduction in As concentrations is observed during the sixth week, i.e. to ± 23.1 μg/l, ± 2.0 μg/l, ± 28.1 μg/l and ± 4.6 μg/l for 0.5 g CeO2, 0.5 g Fe2O3, 0.05 g Fe2O3 and bulk FeCl3 treatments, respectively. This decrease was observed after stopping the irrigation with Arsenic (III) contaminated water and nanoparticles. However, As concentrations in the pore water of treated soils did not differ significantly from the concentration in the untreated soils, except for the 0.5 g CeO2 nanoparticle treatment. Furthermore, statistically insignificant, but similar trends can be seen for week seven and eight for 0.5 g CeO2, 0.5 g Fe2O3, 0.05 g Fe2O3 and bulk FeCl3 treatments. In general, the bulk treatments of 0.1% CeO2 and 0.1% Fe2O3 resulted in higher As concentrations in the pore water, which may result in a higher availability of As for plant uptake. 25

37 Weekly differences in As release for the different treatments are presented in Table 4.2. Concentrations of As in pore water of the untreated soils revealed that in the beginning of the experiment no significant changes in As concentrations were observed, but in week six As concentrations decreased compared to those measured in the third week. However, for 0.05 g Fe2O3 nanoparticle treated soils no significant differences in As concentrations were observed between the different weeks (p = 0.056). Soils that received the 0.5 g Fe2O3 nanoparticle treatment showed a significant decrease in As concentration at the end of the experimental period. From week six onwards, after the supply of As (III) contaminated water and nanoparticles was stopped, a decline in As concentration was observed. For 0.05 g and 0.5 g CeO2 nanoparticle treated soils also a decrease in As concentration was seen at the end of the experimental period compared to the beginning of the experiment. In soils treated with 0.1% bulk CeO2 As concentrations decreased significantly in the middle of the experimental period, i.e. in week four, five and six, but an increase was observed again toward the end of the experiment. When 0.1% bulk Fe2O3 was used as treatment it was noticed that As concentrations decreased significantly in week six, after the treatments were stopped, but the concentrations increased again by the end of the experiment. In bulk FeCl3 treated soils As concentrations were significantly lower in week one (73.2 ± 22.6 μg/l) compared to the other weeks. However, they started increasing until week three after which a significant decline was observed until the experiment ended Effects of treatments on As concentration in pore water of soil contaminated with As (V) Arsenic concentrations measured in the soil solution during the experimental period of eight weeks are presented in the Table 4.3. In the first week, all treatments exhibited low As concentrations, ranging from 71.9 ± 31.2 μg/l to ± 14.5 for bulk FeCl3 and 0.05 g Fe2O3 nanoparticles treatments, respectively. These As concentrations were lower compared to the untreated soil (231.9 ± 16.9 μg/l). For week one, As (V) irrigated soils showed a trend similar to As (III) irrigated soils, with bulk FeCl3 having the lowest concentrations (71.9 ± 31.2 μg/l). This result differed significantly from the concentration measured in the untreated soils (231.9 ± 16.9 μg/l) and the other treatments. From the second to the eight week, it was noted that the 0.5 g CeO2 nanoparticle treatment resulted in the lowest arsenic concentration in the pore water. Furthermore, these results are 26

38 persistent even after stopping irrigation with the As (V) contaminated water and nanoparticles. Concentrations of arsenic in pore water of the 0.5 g CeO2 nanoparticle treated soil decreased from ± 8.9 μg/l to ± 25.7 μg/l from week two to week eight, respectively. However, observed concentrations did not significantly differ from those in the untreated soils. Weekly differences in As release for the different treatments are presented in Table 4.3. A significant decrease (p < 0.05) in As concentration was seen for untreated soils between week one and seven. For the soils treated with 0.05 g, 0.5 g Fe2O3, 0.5 g CeO2 nanoparticles and 0.1 % bulk CeO2 an increase in As concentration was observed in week two. However, by the end of the experimental period these concentrations declined significantly. ANOVA analysis showed no significant decline in As concentration for the soils that received the 0.05 g CeO2 nanoparticle treatment (p = 0.055). Arsenic concentrations in 0.1% bulk Fe2O3 treated soils also declined in week six compared to the concentrations in week two; however, by the end of the experiment no significant decrease was observed. Soils treated with bulk FeCl3 showed the lowest As concentrations in the first week, which differed significantly from the concentrations in all other weeks of the whole experimental period. Although As concentrations increased in the following weeks, they declined significantly by the end of the experiment. 4.3 Iron concentrations in pore water Effects of treatments on Fe concentration in pore water of soil contaminated with As (III) The Fe concentrations in pore water of soil irrigated with As (III) contaminated water are presented in Table 4.4. In the first week, Fe concentrations in all treated and untreated soils ranged from 54.5 ± 24.5 to ± 15.4 mg/l, with the highest in bulk FeCl3 treated soil which was significantly different (p <0.05) from all other treatments and the lowest in untreated soils. In the second week release of Fe into the soil solution was observed, probably due to formation of reducing conditions. Most of the treated soils showed an increase in Fe concentration with the highest concentration being observed for 0.05 g CeO2 nanoparticles (126.4 ± 16.6 mg/l) and bulk FeCl3 (459.7 ± 42.5 mg/l) treated soils, and the lowest for untreated soils (117.8 ± 22.7 mg/l). 27

39 Table 4.2 As concentrations in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Control ± 55.5a AB ± 8.5a AB ± 25.2a A ± 16.3a AB ± 28.0a AB ± 10.0bc B ± 12.1a AB ± 22.1a AB Fe2O g nano ± 24.7ac A ±16.8a A ± 7.0a A ± 11.0ab A ± 15.0a A ± 28.1abc A ± 52.0a A ± 32.5a A Fe2O3 0.5 g nano ± 18.2ac A ±25.5a AB ± 12.4a A ± 4.1ab AB ± 23.8a AB ± 2.0ab B ± 54.6a B ± 24.0a B CeO g nano ± 8.9ac ABC ± 6.2a BC ± 18.1a C ± 4.5ab ABC ± 17.6a ABC ± 23.2bc A ± 21.0a ABC ± 22.6a AB CeO2 0.5 g nano ±13.5ac BC ± 28.4a BC ± 32.8a C ± 21.1ab ABC ± 17.5a ABC ± 23.1ac A ± 33.7a AB ± 36.7a AB Bulk CeO2 0.1% ±10.0c B ±13.3a BCD ± 8.8a BD ± 6.9ab CD ± 16.5a D ± 14.0bc ACD ± 11.8a BCD ± 5.7b ABCD Bulk Fe2O3 0.1% ±12.3ac B 233.1± 14.3a B ± 17.0a B ± 13.9a AB ± 14.2a B ± 5.7abc A ± 20.1a B ± 25.9b B Bulk FeCl ± 22.6b A ± 20.8a D ± 5.8a C ± 21.0b BD ± 8.8a BD ± 4.6abc B ± 8.1a BD ± 12.5a BD Capital letters (in superscript) denote weekly differences with time for the treatments and means within the same row followed by the same letter are not significantly different from each other based on Tukey s test at P < Small letter denote the differences between the treatments week wise and means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P < 0.05 Table 4.3 As concentrations in the soil solutions of soil irrigated with As (V) contaminated water during the experimental period (Mean ± S.D., n=3) Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Control ± 16.9b A ± 31.0a AB ± 7.4ab AB ± 45.7ab AB ± 29.0ab AB ± 31.3ab AB ± 23.6c B ± 43.2ab AB Fe2O g nano ± 14.5b AB ±12.3a A ± 6.4b AB ± 2.2b AB ± 10.6bc AB ± 35.9b AB ± 17.5bc B ± 25.6ab B Fe2O3 0.5 g nano 204.1± 8.5b B ±16.0a A ± 2.8b AB ± 6.9b AB ± 10.9c AB ± 10.6b B ± 14.0b AB ± 9.4a B CeO g nano ± 36.1b A ±13.2a A ± 17.7b A ± 11.4ab A ± 7.6bc A ± 19.3b A ± 26.4abc A ± 29.1ab A CeO2 0.5 g nano ± 33.2b AB ± 8.9a A ± 18.6a AB ± 9.0a AB ± 22.1a AB ± 12.9a B ± 1.6ac B ± 25.7ab B Bulk CeO2 0.1% ± 8.8b B ± 9.2a A ± 6.9b AB ± 5.1ab AB ± 7.9bc B ± 16.2b B ± 13.3bc B ± 14.9ab B Bulk Fe2O3 0.1% ± 10.7b AB ± 4.3a A ± 5.4ab B ± 9.2ab AB ± 24.1abc AB ± 4.8b B ± 10.4bc AB ± 75.5ab AB Bulk FeCl ± 31.2a ACD ± 23.3a CD ± 17.0b D ± 22.3ab BCD ± 9.9abc BCD ± 11.1ab BD ± 8.3c BD ± 4.2b B Capital letters (in superscript) denote weekly differences with time for the treatments and means within the same row followed by the same letter are not significantly different from each other based on Tukey s test at P < Small letters denote the differences between the treatments for each week and means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P <

40 Furthermore, from the second to the eight week, a significant decline in Fe concentrations is observed for all treatments. However, the Fe concentrations are still higher in treated compared to untreated soils, except for the 0.05 g Fe2O3, 0.5 g Fe2O3 and 0.5 g CeO2 nanoparticles treatments. The Fe concentration in these treatments ranged from 34.5 to 93.1 ± 6.7 ± 9.5 mg/l. In comparison to all other treated and untreated soil, pore water of bulk FeCl3 treated soil always has higher Fe concentrations in the soil solution, with the difference being statistically significant for the whole experimental period Effects of treatments on Fe concentration in pore water of soil contaminated with As (V) In the first week, a high Fe concentration in the soil solution is observed for bulk FeCl3 treated soils (117.8 ± 28.0 mg/l) (Table 4.5), as was also the case for the As (III) contaminated soils. The Fe concentration in pore water of the bulk FeCl3 treated soil differed significantly from all other treatments. An increase in the Fe concentration is observed in week two compared to week one for all treatments. Bulk FeCl3 treated soils showed the highest increase among all treatments (444.2 ± 42.1 mg/l), while average Fe concentrations (107.6 ± 3.8 mg/l) were observed in pore water of the untreated soil. From the third week onwards, low Fe concentrations ranging from 38.8 ± 5.6 to 95.6 ± 7.1 mg/l and from 31.4 ± 3.1 to 88.8 ± 6.7 mg/l are seen in soils treated with 0.05 g and 0.5 g CeO2 nanoparticles, respectively. The Fe concentration in pore water of soil treated with bulk FeCl3 declined from ± 42.1 mg/l in week two to ± 14.1 mg/l in week eight. The difference between Fe concentrations of pore water in bulk FeCl3 treated soil and those of untreated soil and soil treated with other amendments was statistically significant during the entire experimental period. 4.4 Cerium concentrations in pore water Effects of treatments on Ce concentration in pore water of soil contaminated with As (III) In the untreated soils the Ce concentrations were below the detection limit except for the second week where the mean Ce concentration was 0.86 ± 0.6 μg/l (Table 4.6). The pore water of the 0.1 % bulk CeO2 treated soil had Ce concentrations below the detection limit 29

41 during all the weeks. However, in the soil treated with 0.05 g and 0.5 g CeO2 nanoparticles, detectable Ce concentrations were observed (2.63 ± 1.5 and 2.88 ± 1.8 μg/l, respectively) in the first week. By the end of the experimental period, a decline in concentration was seen in the soils treated with nanoparticles. Eventually, concentrations reached up to 0.75 ± 0.2 μg/l in soil treated with 0.05 g CeO2 and 1.00 ± 0.1 μg/l in soil treated with 0.5 g CeO2. However, these differences were not statistically significant Effects of treatments on Ce concentration in pore water of soil contaminated with As (V) Table 4.7 summarizes the mean Ce concentrations detected in pore water of soils irrigated with As (V) contaminated water. The Ce concentrations were below detection limit for both untreated soils and soils treated with 0.1% bulk CeO2. However, in soils treated with 0.05 and 0.5 g CeO2 high Ce concentrations are detected in the pore water in week one (1.83 ± 0.8 μg/l and 2.13 ± 0.7 μg/l, respectively). In soils treated with 0.05 g CeO2 nanoparticles, Ce concentrations were below detection limit from week two to week six. Interestingly, the concentrations increased to 0.91 ± 0.3 and 0.88 ± 0.2 μg/l in week seven and eight, respectively. The Ce concentrations in soils treated with 0.5 g CeO2 nanoparticles increased continuously, except in week two and three, till the end of experimental period. Concentrations above 7 µg/l were reached in the last week. However, these increases in concentration were not statistically significant. 4.5 Plant growth Effect of As contaminated water on the plant growth may be seen directly by means of reduction in dry matter yield and stunted growth. Therefore, root, shoot and total dry matter, shoot height and root length were recorded at the end of experimental period. A positive effect of the treatments on plant growth was observed, in soils irrigated with As (III) as well as As (V) contaminated water. 30

42 Table 4.4 Fe concentrations in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Control 54.5 ± 24.5b BC ± 22.7b A 81.0 ± 4.1b C 65.3 ± 4.6b BC 50.0 ± 7.0bc BC 46.0 ± 2.1b B 43.2 ± 1.8b B 45.2 ± 4.2b B Fe 2O g nano 62.4 ± 10.5b BCD ± 7.2b A 81.1 ± 2.3b CD 66.7 ± 1.2b D 50.8 ± 1.2bc BDE 45.1 ± 11.3b BDE 35.6 ± 13.4b E 39.4 ± 9.5b BE Fe 2O g nano 59.7 ± 6.3b BC 97.2 ± 8.3b A 79.7 ± 5.3b AB 64.9 ± 5.5b B 44.5 ± 4.7c C 41.0 ± 1.1b D 33.1 ± 10.8b E 36.5 ± 4.2b F CeO g nano 61.9 ± 9.2b BD ± 16.6b A 93.1 ± 6.7b CD 75.1 ± 5.2b D 56.5 ± 4.2b BD 53.9 ± 4.4b BD 47.7 ± 6.1b B 46.1 ± 3.8b B CeO g nano 65.0 ± 6.4b BCD 99.9 ± 13.4b A 77.0 ± 8.4b BCD 62.6 ± 6.6b BCD 50.6 ± 1.9bc CD 39.0 ± 7.1b E 33.7 ± 7.0b E 34.5 ± 9.5b E Bulk CeO 2 0.1% 75.9 ± 6.5b AD ± 2.1b B 93.0 ± 5.9b C 77.2 ± 1.3b AD 61.6 ± 3.9b E 54.6 ± 4.7b EF 48.1 ± 4.1b F 45.1 ± 3.0b F Bulk Fe 2O 3 0.1% 72.0 ± 8.0b A ± 10.5b B 90.7 ± 4.8b C 74.8 ± 2.3b ACD 60.0 ± 2.2b ADE 50.7 ± 6.8b E 46.7 ± 6.2b E 47.7 ± 7.4b E Bulk FeCl ± 15.4a A ± 42.5a B ± 14.7a C ± 14.3a D ± 6.0a E ± 11.9a E ± 7.4a E ± 6.9a E Capital letters (in superscript) denote weekly differences with time for the treatments and means within the same row followed by the same letter are not significantly different from each other based on Tukey s test at P < Small letters denote the differences between the treatments for each week and means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P < 0.05 Table 4.5 Fe concentrations in the soil solutions of soil irrigated with As (V) contaminated water during the experimental period (Mean ± S.D., n=3) Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Control 76.9 ± 1.2b ACD ± 3.8b B 93.1 ± 6.4b AC 66.3 ± 7.5b D 56.9 ± 7.2b DE 55.2 ± 3.2b DE 43.2 ± 5.1b E 39.6 ± 5.3bc EF Fe 2O g nano 75.3 ± 3.4b A 99.6 ± 2.4b B 86.6 ± 2.7b BC 65.6 ± 3.1b AD 56.5 ± 0.5b ED 53.9 ± 2.3b E 41.5 ± 3.2b E 38.9 ± 3.7bc F Fe 2O g nano 82.0 ± 3.6b A ± 4.1b B 92.6 ± 4.4b AC 72.2 ± 4.7b AD 59.7 ± 4.0b E 60.1 ± 2.1b E 51.0 ± 2.0 b EF 49.0 ± 2.6c F CeO g nano 61.1 ± 17.0b A 95.6 ± 7.1b BC 78.9 ± 0.7b BC 63.1 ± 1.4b ACD 53.4 ± 3.7b ADE 51.8 ± 3.9b ADE 41.4 ± 4.7b ADE 38.8 ± 5.6bc AE CeO g nano 60.8 ± 11.3b ACD 88.8 ± 6.7b B 67.7 ± 3.4b C 55.7 ± 2.6b D 38.9 ± 3.4b E 38.2 ± 2.7b E 31.9 ± 1.1b E 31.4 ± 3.1b E Bulk CeO 2 0.1% 79.2 ± 7.5b ACD ± 11.9b B 89.5 ± 1.7b BC 67.4 ± 3.5b CD 54.3 ± 2.9b DE 53.4 ± 3.5b DE 44.1 ± 6.0b E 42.0 ± 1.6bc E Bulk Fe 2O 3 0.1% 66.8 ± 5.5b A ± 13.4b B 84.3 ± 3.1b C 67.3 ± 4.2b AD 53.5 ± 1.9b AE 53.3 ± 1.9b AE 44.0 ± 2.0b E 42.7 ± 4.5bc E Bulk FeCl ± 28.0a A ± 42.1a B ± 87.4a ABC ± 65.9a ABC ± 39.6a BC ± 39.9a BC ± 31.2a AC ± 14.1a C Capital letters (in superscript) denote weekly differences with time for the treatments and means within the same row followed by the same letter are not significantly different from each other based on Tukey s test at P < Small letters denote the differences between the treatments for each week and means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P <

43 Table 4.6 Ce concentrations (μg/l) in the soil solutions of soil irrigated with As (III) contaminated water during the experimental period (Mean ± S.D., n=3) (D.L. detection limit = 0.5 µg/l) Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Control < D.L ± 0.6 < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. CeO g nano 2.63 ± 1.5 < D.L. < D.L ± 0.1 < D.L. < D.L ± ± 0.2 CeO 2 0.5g nano 2.88 ± ± ± ± ± ± ± ± 0.1 Bulk CeO 2 0.1% < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. Table 4.7 Ce concentrations (μg/l) in the soil solutions of soils irrigated with As (V) contaminated water during the experimental period (Mean ± S.D., n=3) (D.L. detection limit = 0.5 μg/l) Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Control < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. CeO g nano 1.83 ± 0.8 < D.L. < D.L. < D.L. < D.L. < D.L ± ± 0.2 CeO 2 0.5g nano 2.13 ± ± ± ± ± ± ± ± 3.8 Bulk CeO 2 0.1% < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L. < D.L Effects of treatments on plant growth for As (III) contaminated soil The root dry weight ranged from 0.18 ± 0.06 g to 0.52 ± 0.12 g for 0.05 g CeO2 nanoparticles treatment and bulk FeCl3 respectively (Fig 4.1). However, between all the treatments only 0.05 g CeO2 nanoparticle and bulk FeCl3 treatments showed statistically significant differences at 95% confidence level. ANOVA analysis indicated that shoot dry weight was not affected by the treatment (p=0.097). However, application of 0.05 g CeO2 nanoparticles resulted in the lowest shoot (0.44 ± 0.11 g) dry matter yield, whereas 0.5 g CeO2 nanoparticles resulted in the highest shoot (0.86 ± 0.34 g) dry matter yield. Plants grown on untreated soils also had a very low shoot dry weight of 0.48 ± 0.05 g. Total dry weight of the plant ranged from 0.61 ± 0.17 to 1.32 ± 0.54 g for 0.05 g CeO2 and 0.5 g CeO2 nanoparticle treatments respectively. Total dry weight of the plants grown on untreated soil was 0.76 ± 0.08 g. A significant difference in total dry matter yield is only observed between plants grown on 0.05 g and 0.5 g CeO2 nanoparticles treated soils. The shoot height increased upon treatment, except when 0.05 g CeO2 nanoparticles and 0.1% bulk CeO2 were applied (Fig. 4.2). The plants grown on bulk FeCl3 treated soil showed the 32

44 highest shoot height (63.4 ± 4.1 cm), whereas plants receiving 0.05 g CeO2 nanoparticles treatment had shortest height (53.9 ± 3.8 cm). However, statistical evaluation indicates no significant differences between the treatments (p=0.182). Root length varied from 15.4 ± 0.5 to 20.4 ± 2.0 cm for bulk FeCl3 and 0.05 g Fe2O3 nanoparticles treatments, respectively. For the plants grown on untreated soils the root length was also very low (15.5 ± 0.4 cm). No significant differences are seen in the root length between the treatments (p=0.057). Figure 4.1 Root, shoot and total dry matter yield of rice plants irrigated with As (III) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant difference between treatments based on Tukey s test at P < 0.05 Figure 4.2 Shoot height and root length of rice irrigated with As (III) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant different between treatments based on Tukey s test at P <

45 4.5.2 Effects of treatments on plant growth for As (V) contaminated soil Effects of using As (V) contaminated irrigation water on the plant growth are presented in Fig 4.3 and 4.4. For root dry weight plants grown on untreated soil have the lowest dry weight 0.14 ± 0.06 g and plants grown on soil treated with 0.05 g CeO2 nanoparticles having the highest 0.38 ± 0.03 g. These results differed statistically from each other at 95% confidence level. Shoot dry weight varied from 0.27 ± 0.10 g to 0.81 ± 0.07 g for plants grown on untreated soils and treated with 0.05 g CeO2 nanoparticles respectively. Plants grown on soil that received CeO2 nanoparticles have a higher shoot dry weight compared to plants grown on soils that received other amendments. Shoot dry weight of plants grown on soils treated with 0.05g CeO2 nanoparticles differed significantly from the plants grown on untreated soil (p < 0.05). Total dry matter yield ranged from 0.42 ± 0.16 to 1.19 ± 0.09 g with plants grown on soil treated with 0.05 g CeO2 nanoparticles having highest total dry matter weight and lowest for untreated ones. Plants grown on soil treated with 0.5 g CeO2 nanoparticles and bulk FeCl3 also had high total dry matter 1.08 ± 0.28 g and 1.03 ± 0.29 g respectively. However, only the plants grown on CeO2 nanoparticles treated soil showed a statistically significant difference from the ones grown on untreated soils. Plants grown on soil treated with bulk FeCl3 have the highest shoot height (61.20 ± 3.7 cm), whereas plants grown on untreated soil have the lowest height (47.90 ± 11.0 cm). Figure 4.3 Root, shoot and total Dry matter yield of rice irrigated with As (V) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant different between treatments based on Tukey s test at P <

46 (cm) Though, there was an increase in shoot height for all the treatments but the ANOVA results were not statistically significant (p=0.096). Plants grown on soil treated with 0.05 g CeO2 nanoparticles have the longest root length (20.40 ± 1.1 cm) and plants grown on untreated soil the shortest (13.50 ± 1.3 cm). The difference in root length between plants grown on soil treated with 0.05 g CeO2 nanoparticles and plants grown on untreated soil was statistically significant at the 95% confidence level a a a ab Control Fe2O g nano a ab Fe2O3 0.5 g nano a b CeO g nano a ab CeO2 0.5 g nano a Bulk CeO2 0.1% a a ab ab ab Bulk Fe2O3 0.1% FeCl3 shoot height root length Figure 4.4 Shoot height and root length of rice irrigated with As (V) contaminated water (Mean ± S.D., n=3). Similar letter on the bar indicates no significant different between treatments based on Tukey s test at P < Arsenic concentration and content in plants grown on soil irrigated with As (III) contaminated water Total As in root and shoot Table 4.8 summarizes the total As concentrations (mg/kg dry weight) and content (μg/pot) in rice plants grown on soil irrigated with As (III) contaminated water. Analysis of the different plant parts individually showed that the overall concentration of As in root was higher than in shoot. The As concentration in root ranged from 78.4 ± 14.7 to ± 15.4 mg/kg for 0.1% bulk Fe2O3 and 0.05 g Fe2O3 nanoparticle treated soils respectively. However, the differences in As concentration in root are not statistically significant (p=0.112). As content (μg/pot) in root was highest in plants grown on 0.05 g Fe2O3 nanoparticle treated soils (134.8 ± 32.0 μg/pot), while the lowest As content was found in roots of plants that were grown on 0.05 g CeO2 nanoparticle treated soils (50.3 ± 12.6 μg/pot). There was no significant difference in As content in roots between different treatments (p=0.125). 35

47 The highest As accumulation in shoot was observed in 0.05 g CeO2 nanoparticle treated (7.6 ± 1.83 mg/kg) and the lowest in bulk FeCl3 treated soils (2.1 ± 0.40 mg/kg). Arsenic concentration in shoot of plants grown on bulk FeCl3 treated soils differed significantly from the other treated and untreated soils. Plants grown on soils that received 0.5 g Fe2O3 nanoparticle treatment also showed a low As concentration in shoot (5.2 ± 0.07 mg/kg). This concentration differed significantly from 0.05 g CeO2 nanoparticle treated soils at 95% confidence level. Shoot As content varied from 2.4 ± 0.7 to 7.7 ± 2.4 μg/pot for bulk FeCl3 and 0.1% Fe2O3 treated soil respectively. A significant difference in shoot As content was seen between these two treatments, however, the other treatments did not differ significantly Total As in whole plant The total As concentration in the whole plant was lowest in case of the 0.1% bulk Fe2O3 treated soils (85.6 ± 14.5 mg/kg), followed by the bulk FeCl3, 0.05 g CeO2 and 0.5 g CeO2 nanoparticles treatments. Arsenic concentrations were relatively higher in plants grown on soil treated with 0.5 g Fe2O3 nanoparticles, 0.1% bulk CeO2 and 0.05 g Fe2O3 nanoparticles (105.3 ± 25.9, ± 22.4 and ± 15.7 mg/kg, respectively). However, ANOVA analysis indicated that there was no effect of treatments on total As concentration in the whole plant (p = 0.125). Arsenic content in the whole plant varied from 54.8 ± 13.3 to ± 33.2 μg/pot for 0.05 g CeO2 and 0.05 g Fe2O3 nanoparticle treated soils. However, no significant difference between the treatments was observed (p = 0.137). 4.7 Arsenic concentration and content in plants grown on soil irrigated with As (V) contaminated water Total As in root and shoot Table 4.9 summarizes the results for total As concentrations (mg/kg dry weight) and content (μg/pot) in plants parts. The As concentration in root varied in the range 84.5 ± 18.0 to ± 11.8 mg/kg in plants grown on 0.1% bulk Fe2O3 treated and untreated soils respectively. However, statistically no significant difference was seen between the treatments for As concentration in roots (p = 0.789). Nevertheless, As content in the roots showed somewhat different results with untreated soils and 0.5 g Fe2O3 nanoparticle treated soils yielding least As (42.6 ± 12.2 and 48.7 ± 8.1 μg/pot). These results differed significantly from the 0.05 g 36

48 CeO2 treated soil resulting in the highest As content (118.8 ± 6.1 μg/pot). No significant differences were observed between the other treatments. The lowest shoot As concentrations were seen in plants grown on bulk FeCl3, 0.5 g CeO2 and 0.5 g Fe2O3 nanoparticles treated soils (3.2 ± 0.9, 4.7 ± 0.7 and 5.4 ± 0.4 mg/kg, respectively). In terms of As accumulation in shoot, plants grown on bulk FeCl3 treated soils differed significantly (p <0.05) from 0.05 g Fe2O3, 0.05 g CeO2 nanoparticle, 0.1% bulk Fe2O3 and 0.1% bulk CeO2 treated soils. Arsenic content in the shoot was lowest for plants grown on untreated soils (2.5 ± 1.0 μg/pot) and highest for 0.05 g CeO2 nanoparticle treated soils (7.9 ± 0.5 μg/pot). As content in shoot of plants grown on 0.05 g CeO2 nanoparticle treated soils differs significantly from untreated, 0.5g Fe2O3 nanoparticle treated and bulk FeCl 3 treated soils. Table 4.8 Total arsenic concentration and content in the roots, shoots and whole plant grown on As (III) contaminated soil (Mean ± S.D., n=3) As in root (mg/kg) As in shoot (mg/kg) Total As in plant (mg/kg) As in root (μg/pot) As in shoot (μg/pot) Total As (μg/pot) Control 98.0 ± 11.5a 6.6 ± 0.32bc ± 11.5a 74.2 ± 13.8a 5.0 ± 0.3ab 79.2 ± 14.1a 0.05 g Fe 2O 3 nano 0.5 g Fe 2O 3 nano 0.05 g CeO 2 nano ± 15.4a 6.4 ± 0.30bc ± 15.7a ± 32.0a 7.4 ± 1.3ab ± 33.2a ± 25.8a 5.2 ± 0.07b ± 25.9a ± 47.1a 5.2 ± 1.1ab ± 48.2a 82.2 ± 2.2a 7.6 ± 1.83c 89.8 ± 3.3a 50.3 ± 12.6a 4.6 ± 1.1ab 54.8 ± 13.3a 0.5 g CeO 2 nano 0.1%Bulk CeO 2 0.1%Bulk Fe 2O ± 12.8a 5.6 ± 0.32bc 98.3 ± 13.0a ± 35.4a 7.3 ± 3.0ab ± 38.4a ± 23.1a 6.9 ± 0.81bc ± 22.4a 91.7 ± 40.9a 6.0 ± 2.7ab 97.6 ± 43.2a 78.4 ± 14.7a 7.2 ± 0.41bc 85.6 ± 14.5a 85.3 ± 33.3a 7.7 ± 2.4b 93.0 ± 35.4a BulkFeCl ± 8.7a 2.1 ± 0.40a 89.1 ± 9.0a 98.6 ± 24.8a 2.4 ± 0.7a ± 25.5a Means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P < Total As in whole plant The lowest total As concentration in the whole plant was found in the plants grown on soils treated with 0.1% bulk Fe2O3 (91.2 ± 18.6 mg/kg) followed by 0.5 g Fe2O3 nanoparticles (92.2 ± 19.7) mg/kg and 0.5 g CeO2 nanoparticles (97.9 ± 16.7 mg/kg). The highest 37

49 accumulation was observed in the plants which were grown on soils that received no treatment (110.4 ± 11.1 mg/kg), but there was no significant difference between treatments (p = 0.803). Arsenic content for plants grown on soils that did not receive any treatment was lowest (45.0 ± 12.8 μg/pot). The highest As content was found in plants grown on soil treated with 0.05 g CeO2 nanoparticles (126.7 ± 6.2 μg/pot). Plants grown on 0.05 g CeO2 nanoparticles treated soils showed significant differences ( p <0.05) in As content with untreated and 0.5 g Fe2O3 treated soils. No significant differences are observed between the other treatments. Table 4.9 Total arsenic concentration and content in the roots, shoots and whole plant grown on As (V) contaminated soil (Mean ± S.D., n=3) As in root (mg/kg) As in shoot (mg/kg) Total As in plant (mg/kg) As in root (μg/pot) As in shoot (μg/pot) Total As (μg/pot) Control ± 11.8a 6.0 ± 2.0ab ± 11.1a 42.6 ± 12.2b 2.5 ± 1.0b 45.0 ± 12.8b 0.05 g Fe 2O 3 nano 0.5 g Fe 2O 3 nano 0.05 g CeO 2 nano 93.5 ± 10.2a 6.5 ± 0.7b ± 9.7a 73.9 ± 18.6ab 5.3 ± 1.9ab 79.2 ± 20.3ab 86.8 ± 19.5a 5.4± 0.4ab 92.2 ± 19.7a 48.7 ± 8.1b 3.2 ± 1.1b 51.9 ± 9.2b ± 4.1a 6.7 ± 0.4b ± 4.1a ± 6.1a 7.9 ± 0.5a ± 6.2a 0.5 g CeO 2 nano 0.1%Bulk CeO 2 0.1%Bulk Fe 2O ± 17.0a 4.7 ± 0.7ab 97.9 ± 16.7a ± 36.5ab 5.1 ± 1.9ab ± 38.0ab ± 28.9a 6.5 ± 1.3b ± 28.1a ± 54.9ab 6.4 ± 2.1ab ± 56.1ab 84.5 ± 18.0a 6.7 ± 0.7b 91.2 ± 18.6a 74.8 ± 20.7ab 5.9 ± 1.3ab 80.8 ± 21.8ab Bulk FeCl ± 23.9a 3.2 ± 0.9a ± 24.7a ± 49.7ab 3.5 ± 1.8b ± 51.5ab Means within the same column followed by the same letter are not significantly different from each other based on Tukey s test at P <

50 5. Discussion 5.1. Release of Fe in pore water from soils irrigated with As (III) and As (V) contaminated water Iron mainly exists as ferric oxides and hydroxides under aerobic conditions in the soil. Under submerged conditions anaerobic/anoxic reactions are initiated and Fe (III) oxides undergo reductive dissolution. As a consequence, the concentration of Fe (II) increases in solution. Hence, this leads to a higher solubility and availability of Fe in the soil solution. In this study also under submerged soil conditions in the early stage of the experiment, there was a release of Fe in the pore water for the untreated soils (54.5 ± 24.5 mg/l for As (III) and 76.9 ± 1.2 mg/l for As (V) irrigated soils). When the Fe release of Fe2O3 nanoparticle treated soils was compared with untreated soils it did not differ significantly (Table 4.4 and 4.5). This could be due aggregation of nanoparticles in the upper layers of the soil as they were supplied with irrigation water which may lead to the formation of flocks, making them unable to migrate to the soil water. Flock formation in the water layer on top of the soil was indeed observed, as depicted in Figure 5.1. Fig Fe2O3 nanoparticles forming flocks in the water layer above soil in the pots. 39

51 For the bulk soil amendment 0.1% bulk Fe2O3 also no significant differences were observed in Fe release from the untreated soils and nanoparticle treated soils. Nevertheless, a significant release of Fe in the pore water was observed for soils treated with bulk FeCl3 (157.3 ± 15.4 mg/l for As (III) and ± 28.0 mg/l for As (V) irrigated soils), although the amount of Fe added in this treatment equaled the amount added through 0.1% Fe2O3. The difference between 0.1% bulk Fe2O3 and bulk FeCl3 could be due to the lower solubility of Fe2O3 in the soil water phase. Moreover, addition of FeCl3 to the soil caused its dissociation and formation of Fe hydroxides, which releases protons via hydrolysis, thus resulting in a decrease of soil ph (Makino et al 2010). At low ph heavy metal solubilization increases which may result in an increase of Fe availability. The differences in the Fe concentrations in the pore water between nanoparticle treatments and bulk soil amendments could be attributed to the inability of nanoparticles to reach the pore water at the place of sampling, as they were aggregated in the top soil layer. This could be due to the high ionic strength of the soil solution which decreases the repulsive forces between particles and between particles and the soil surfaces in accordance to the DLVO theory, thus leading to aggregation and sorption of particles to the soil (Tourinho et al., 2012). An increase in the Fe release was observed for all treated and untreated soils in the second week due to formation of reducing/anoxic conditions. The highest and most significant release was measured for bulk FeCl3 treated soils (459.7 ± 42.5 mg/l for As (III) and ± 42.1 mg/l for As (V) irrigated soils). However, from the second week onwards a sudden decrease in Fe concentration in the soil solution was observed for the treated and untreated soils even though the conditions were still considered to be reducing. This result is in accordance with what was reported by Ultra et al. (2009) who used three levels of laboratory-synthesized Am-FeOH (0, 0.1 and 0.5% w/w) to treat paddy soils submerged in 5 mg/l As (V). They found that the Fe concentration in soils after 63 days of cultivation did not differ between the treatments, which were attributed to the continuous precipitation of Fe on the roots of the plants in the form of Fe-plaque or its deposition at the oxic soil surface (Fig 5.2). Another possible explanation may be the precipitation of Fe (II) in the soil solution to form iron sulfide compounds such as FeS (troilite) and FeS2 (pyrite) when the Eh further decreases due to reducing conditions. Furthermore, also under submerged paddy soil conditions, the diffusion of gases is restricted 40

52 and O2 depletion and CO2 accumulation occurs. This leads to formation of (FeCO3) siderite (Zhang et al. 2012). Among all treatments bulk FeCl3 treated soils showed the highest Fe release in soil pore water. This can be attributed to the fact that it was added as a salt to the bulk soil, being more readily available in the soil pore water at any sampling location. Salt added as bulk to the soil is in direct contact with the pore water samplers and does not need to migrate anymore from the place of application to the place of sampling, as is the case when amendments are added as part of the irrigation water. Figure 5.2 Brownish red deposition of Fe-plaque on the rice roots and iron oxide deposition on the oxic soil surface and sides of the pots 41

53 5.2. Release of Ce in pore water from soils irrigated with As (III) and As (V) contaminated water Even though the indigenous Ce concentration present in the soil is quite high (408.2 ± 5.95 mg/kg), it was observed that the Ce concentration in the soil solution of untreated and 0.1% bulk CeO2 treated soils remained below the detection limit (0.5 µg/l) (Table 4.6 and 4.7). According to Laveuf and Cornu (2009) geogenic Ce is sparingly soluble in soils, mainly because it is precipitated as phosphate minerals. This explains low Ce concentration in pore water of untreated soils despite having high Ce concentration in the soil. Low Ce concentration in soils treated with 0.1% bulk CeO2 may also be due to low solubility of bulk CeO2 in the soil solution. Cornelis et al. (2011) also studied the solubility and retention of CeO2 nanoparticles, Ce (III) and Ce (IV) ions and bulk CeO2 in 16 different Australian soils and found that the bulk CeO2 has higher Kd values (partitioning coefficient) which means it was very sparingly soluble in soil. Only when the soil was treated with CeO2 nanoparticles, Ce could be detected in the soil solution Thus, addition of nanoparticles in comparison to bulk CeO2 released more Ce in the soil solution. The fate of CeO2 nanoparticles and ionic Ce in sediment suspensions was also reported by Van Koetsem et al., (2014) who demonstrated that nanoparticles were more mobile and were less strongly bound to sediments compared to ionic Ce. However, the Ce concentrations measured in the soil solution of soil treated with CeO2 nanoparticles were still very low in our study. This implies that all cerium amendments are effectively removed from the water phase (e.g., by precipitation or adsorption to the soil phase) or they may have aggregated and be retained by the filter material contained within the samplers (having a pore size of around 0.1 µm). Moreover, similar to the Fe2O3 nanoparticle treatments CeO2 nanoparticle also formed aggregates in the upper layers of the soil as they were supplied with irrigation water. This may lead to the formation of flocks, making them unable to migrate to the soil water. Also Tourinho et al. (2012) stated that the dissolution of CeO2 nanoparticles is very low in different types of soil. The slow dissolution of CeO2 nanoparticles can be explained by the fact that the reduction rate of Ce (IV) to Ce (III), which is more soluble than Ce (IV), is slow. Presence of phosphates in soil can also affect the dissolution and release of Ce into the soil solution. Phosphates were previously reported to alter the surface properties of CeO2 nanoparticles or precipitate them as sparingly soluble phosphates, thus affecting their dissolution in soil (Cornelis et al. 2011). Thus it can 42

54 be said that the behaviour of nanoparticles in the soils was mainly influenced by the soil properties. 5.3 Effects of treatments on As concentrations in soil solution for soils irrigated with As (III) and As (V) contaminated water. Arsenic concentrations were measured in the soil solution in weekly intervals for all soils irrigated with either As (III) or As (V) and given different mitigation treatments (Table 4.2 and 4.3). It has been demonstrated in several studies that As has a high affinity towards oxides and hydroxides of Fe, Al and Mn. Therefore, the amount of As in the soil solution is mainly influenced by the amount of Fe in the soil (Warren and Alloway 2003). Studies using Fe (III) oxides, such as amorphous ferric oxide, revealed their ability to remove and adsorb both As (V) and As (III) from aqueous solutions. In the beginning of the experiment, during the first week, it seemed that reducing conditions were not yet established in the soils and it was still oxic. We observed that As concentrations in the soil solution of soils which were treated with bulk FeCl3 were lower during the first week (approximately 70 μg/l). This can be attributed to the precipitation of added FeCl3 as ferric oxide under the oxidizing conditions which are still prevailing during the first week. This may lead to As precipitation as well. However, as the conditions became anoxic from the second week onwards, the As availability in the soil solution seemed to increase again in the FeCl3 treated soil. On the one hand, reductive dissolution of iron (hydr)oxides in the paddy field may have started to occur, with the release of adsorbed As to the soil solution being a consequence. On the other hand, As (V) may have been converted to As (III) under these conditions. Since arsenite is less strongly adsorbed to the soil, it has a greater tendency to partition into the solution phase, thus increasing As release in the soil solution. As the reducing conditions became more dominant from the third week onwards, it was noted that the concentration of As started to decrease and/or stabilize in the soil solution. However, the soils were still irrigated with As contaminated water (containing 100 µg As), which is expected to result in increasing As concentrations if adsorption to the solid soil phase would be absent. In the submerged paddy soils redox properties differ in different parts of the soil, 43

55 i.e. an oxic surface soil, an anoxic subsurface soil, and the rhizosphere. In the oxic soil surface and rhizosphere zone Fe (II) formed via reductive dissolution starts precipitating to Fe (III) oxides thus binding the soluble As from the soil solution. This leads to formation of reddish-brown deposits on the soil surface, sides of pots and iron plaque formation on the roots (Fig. 5.2). This could be one of the reasons for the observed decrease in As concentrations in the soil solution despite addition of fresh As contaminated water each week. Another reason could be sulphide formation due to continuous flooding: sulphate ions are converted into sulphide ions and react with As in the soil solution, thus precipitating it in the form of arsenic sulphide. This makes As less bioavailable for plant uptake. Even in the untreated soils a lower presence of Arsenic suggested that the poorly crystalline Fe oxides already present in the soil (indigenous Fe oxides) can also absorb some of the added arsenic. These results are in accordance with results reported by Ultra, et al. (2009) who gave amorphous iron (hydr)oxide treatments to their soil along with 5.0 mg/l arsenate and found that the arsenic concentrations were decreasing in the untreated soils. In addition, this result implies that the application of Fe based treatments could alleviate As toxicity more drastically in soils containing smaller amounts of indigenous Fe oxides. At the end of the experimental period, the 0.5 g Fe2O3 and CeO2 nanoparticle treatments seemed to be more effective in reducing the arsenic concentrations in As (III) contaminated soils compared to the lower doses of nanoparticles and the bulk soil amendments. However, for As (V) contaminated soils only 0.5 CeO2 nanoparticle treatments reduced As concentration in the last weeks of the experiment. According to Fendorf et al. (2008), As (III) exhibits a limited affinity for most soil minerals except iron oxides/hydroxides. We have illustrated that As (III) is more effectively removed by Fe2O3 nanoparticles added to the irrigation water compared to bulk Fe2O3 added to the solid soil phase. When soils were irrigated with As (V) contaminated water bulk FeCl3 treated soils resulted in lower arsenic concentrations. The iron based treatment may have created high redox potentials (Eh) in the soil, and induced sorption of arsenic and precipitation of low solubility arsenic compounds (Yoshiba, M., et al. 1996). This slows down the decrease of Eh under flooding conditions, and may suppress the reduction of arsenate to arsenite and the dissolution of arsenic from soil solids. Additionally, FeCl3 may assist in the formation of iron plaque in the root zone which have affinity for binding arsenic (V), thus reducing also its 44

56 availability in the soil solution. It was observed that adding CeO2 nanoparticles to the soils irrigated with either As (III) or As (V) at a dose of 0.5 g resulted in lower concentrations of arsenic in the soil solution, whereas this was not the case for 0.1% bulk CeO2. The Ce nanoparticles probably bind the arsenic by adsorption and help to reduce its concentration in the water phase, as they are effectively retained by the solid soil phase. Average As concentrations in soil solution for all treatments and soils irrigated with either As (III) or As (V) contaminated water are presented in Fig For the soils irrigated with As (III) contaminated water the lowest As concentration was observed in soils treated with bulk FeCl3 (172.7 ± 50.7 μg/l) followed by 0.5 g Fe2O3 and CeO2 nanoparticle treated soils, whereas the highest As concentrations were observed for the other bulk soil amendments. Soils contaminated with As (V) water had lowest As in soils treated with 0.5 g CeO2 nanoparticles (159.4 ± 25.5 μg/l) followed by bulk FeCl3 treated soils (166.3 ± 45.7 μg/l). These results clearly indicate that among all the treatments bulk FeCl3 and 0.5g CeO2 nanoparticle treatments were performing best in reducing As concentrations in soil solution thus influencing its bioavailability for plant uptake. 5.4 Effect of treatments in reducing impact of As on the plant growth parameters To study the effect of metal tolerance in plants, various vegetative response end points such as root length, shoot height, root, shoot, and total biomass (root and shoot) may be used. We also measured them for plants grown on As (III) and As (V) irrigated soils (Fig 5.4 and 5.5 a,b). The root, shoot and total dry weight of the plants clearly showed that soils irrigated with As (III) contaminated water and treated with 0.5 g CeO2 nanoparticles had the highest total dry weight. Whereas, the root dry weight was highest for bulk FeCl3 treated soils. Among all treatments 0.05 g CeO2 nanoparticle treated soils resulted in the lowest dry weights. On soils irrigated with As (V) contaminated water the dry weights were highest for plants grown on soils treated with CeO2 nanoparticles. In contrast to what was observed for As (III), in this case the 0.05 g CeO2 nanoparticle dose performed best. When compared to the biomass yield of the plants grown on soil that did not receive any treatment it was noted that these results were significant (Fig. 4.1 and 4.3). 45

57 Fig 5.3 Average As concentrations (μg/l) in the soil solution for the different treatments during the whole experimental period. Comparison of soils irrigated with As (III) and As (V) contaminated water showed a significant difference (p <0.05) in root dry weight for untreated soils and soils treated with 0.05 g Fe2O3, 0.05 g CeO2 nanoparticles. For shoot dry weights and total plant dry weights a comparison between As (III) and As (V) contaminated soils showed a significant difference in untreated and soils treated with 0.05 g CeO2 nanoparticles. Shoot height was not enhanced significantly for both As (III) and As (V) irrigated soils. However, a better root growth was observed for soils contaminated with As (V) and treated with 0.05 g CeO2 nanoparticles. Abedin et al., (2002a), (2002b) in their experiment used arsenate contaminated water (0-8 mg/l) for irrigation of rice plants and concluded that As concentrations decreased the plant height, root growth and rice yield. Abedin and Meharg (2002) also reported a decrease in root tolerance index and relative shoot height for rice seedlings due to increased arsenite and arsenate concentrations. However, in our study application of the nanoparticle treatments seem to enhance the growth parameters in comparison to untreated ones. Comparison of soils irrigated with either As (III) or As (V) clearly showed that among all treatments CeO2 nanoparticles were performing the best in reducing the negative impact of As on the dry matter production. Especially in case of As (V) contaminated soils, the lower dosage of CeO2 nanoparticles was more effective in enhancing the plant growth parameters. This could be 46

58 attributed to the association of As with the CeO2 nanoparticles in the soil solution, thus reducing their toxic effects on the plants. According to Hu et al., 2002 CeO2 in dissolved form is sometimes used a fertilizer for the soil thus promoting plant growth this could be another reason why plants grown on CeO2 nanoparticle treated soil had better growth. 5.5 Effect of treatments in reducing As accumulation in rice plants Arsenic concentration (mg/kg) and content (μg/pot) in roots Arsenic concentration and content in roots of As (III) irrigated soils did not show any differences between treatments. Soils irrigated with As (V) contaminated water also did not result in any significant differences in As concentration between treatments. However, a high As content in roots was observed in soils treated with 0.05 g CeO2 nanoparticles and it differed significantly from the untreated and 0.5 g Fe2O3 nanoparticle treated soils. Comparison of As concentration in roots grown in As (III) and As (V) contaminated soils showed a significant difference (p <0.05) in 0.05 g CeO2 nanoparticle treated soils, with As (III) treated soils having lower As concentrations in the roots (Fig. 5.6a,b). When the As content in roots was compared between As (III) and As (V) contaminated soils a significant difference (p < 0.05) was observed for untreated, 0.05 g Fe2O3 and 0.05 g CeO2 nanoparticle treated soils, with 0.05 g CeO2 nanoparticle treated soils having a lower root As content in As (III) treated soils compared to As (V) treated soil, and vice versa for the other two treatments Arsenic concentration (mg/kg) and content (μg/pot) in shoot For As (III) contaminated soils shoot As concentration was lowest in bulk FeCl3 and 0.5 g Fe2O3 nanoparticle treated soils and highest in 0.05 g CeO2 nanoparticle treated soils. Similarly, in As (V) contaminated soils also bulk FeCl3 treated soils had the lowest shoot As concentration. However, a comparison in shoot As concentration between As (III) and As (V) shows no significant difference (Fig5.6c, d). Arsenic content in shoots of plants grown on As (III) contaminated soils also showed lowest As in bulk FeCl3 and 0.05 g CeO2 nanoparticle treated soils. In the case of As (V) contaminated soil, a low As content was observed in untreated, 0.5 g Fe2O3 nanoparticle and bulk FeCl3 treated soils, whereas 0.05 g CeO2 nanoparticle treated soils had highest As content. Comparison of As (III) with As (V) showed 47

59 a significant difference in As content between untreated and 0.05 g CeO2 nanoparticle treated soils Arsenic concentration (mg/kg) and content (μg/pot) in whole plant Arsenic concentrations and content in the whole plant did not differ significantly between treatments for As (III) contaminated soils. However, for As (V) contaminated soils a significant difference in As content was observed between untreated and 0.5 g Fe2O3 nanoparticle treated soils. When comparing As (III) and As (V), it was noticed that only 0.05 g CeO2 nanoparticle treated soils differed from each other in terms of reducing the total As concentration (Fig. 5.6e,f), whereas the As content varied significantly for untreated, 0.5 g Fe2O3, and 0.05 g CeO2 nanoparticle treated soils. The results clearly indicate that for soils irrigated with either As (III) or As (V) most of the As was accumulated in the root part. When bulk soil amendments and nanoparticle treatments are compared, the bulk FeCl3 treatment is found to be most effective in reducing the accumulation of As in above ground tissue. This could be due to formation of Fe oxides and Fe-plaque precipitates which are formed in the oxic zone, thus sequestering As and reducing its translocation. However, nanoparticle treatments are still performing better in comparison to their bulk substitutes 0.1% bulk Fe2O3 and 0.1% bulk CeO2 in reducing As uptake. 5.6 Arsenic translocation from root to shoot Factors reflecting trace element translocation from root to shoot help in determining whether the element taken up by the plant root will be transferred to the above ground tissue. In this study, we also calculated the translocation factors for the different treatments (Fig. 5.7). Arsenic translocation from root to shoot was inhibited in soils treated with bulk FeCl3 treated soils most effectively. ANOVA analysis confirmed these results and it was observed that for As (III) contaminated soils, bulk FeCl3 treated soils had significantly less As translocation from root to shoot in comparison to untreated soils and soil treated with 0.05 g CeO2 nanoparticles, 0.1% bulk Fe2O3 and 0.1% CeO2. 48

60 Fig. 5.4 Comparison of root, shoot and total dry weights in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <

61 Fig. 5.5a Comparison of shoot height in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05. Fig. 5.5b Comparison of root length in soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05. However, for As (V) contaminated soils translocation factors significantly differed only between bulk FeCl3 and 0.1% bulk Fe2O3 treated soils. This could be due to formation of Feplaque on the roots of the plants which can bind As and reduce its transfer. Among the nanoparticle treatments 0.5 g Fe2O3 and 0.5 g CeO2 were performing best in reducing As translocation. In comparison to the bulk soil amendments, nanoparticle treatments appear to be more effective. However, a comparison of As (III) and As (V) shows no significant difference between different treatments. 50

62 a b c d Fig. 5.6 Comparison of As concentration (mg/kg) and content (μg/pot) in root (a, b) and shoot (c, d) of soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <

63 e f Fig. 5.6 e,f. Comparison of As concentration (mg/kg) and content (μg/pot) in whole plant of soils irrigated with As (III) and As (V) contaminated water. Bars for the same treatments with different letters show significant difference between As (III) and As (V) at p <0.05 Fig. 5.7 Comparison of root to shoot As translocation factor between different treatments for soils irrigated with As (III) and As (V) contaminated water. Bars with same colour and different letter show significant difference between treatments at p <