Arundo Donax L. (Giant Reed) Phytoremediation Function

ARUNDO DONAX L. (GIANT REED) PHYTOREMEDIATION

FUNCTION OF CHROMIUM (Cr) CONTAMINATION

SADEG SALEH NAGI ABDURAHMAN

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Voiland College of Engineering and Architecture

DECEMBER 2016

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To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of

SADEG SALEH NAGI ABDURAHMAN find it satisfactory and recommend that it be accepted.

______Claudio O. Stöckle, Ph.D., Chair

______James B. Harsh, Ph.D.

______Pius M. Ndegwa, Ph.D.

______Indranil Chowdhury, Ph.D.

ACKNOWLEDGEMENT

First of all, I would like to praise and thank ALLAH, the most Gracious and the most

Merciful, who offered me the ability to conduct and complete this research study. This dissertation would not have been possible without the guidance and the support of many sincere people, who helped and motivated me a lot. Firstly, I am heartily thankful to my principle supervisor, Dr.

Claudio Stöckle, whose encouragement, assistance and support from the initial to the final draft enabled me to develop an understanding of the subject. His invaluable help, which includes constructive comments and suggestions throughout the experimental and dissertation work contributed to the success of this research. Special appreciation goes also to Dr. James Harsh for his support and sharing his expertise regarding this research topic. I express my sincere appreciation for the other members of my advisory committee, Dr. Pius Ndegwa and Dr. Indranil

Chowdhury for their assistance.

I would also like to thank Dr. Marc Beutel and Dr. Usama Zaher for their respective support and assistance for the time they served in my advisory committee. I appreciated all precious comments I had from all of them. My acknowledgements also go to the faculty and staff of Biological Systems Engineering Department: Ms. Joanna Dreger and Ms. Dorota Wilk. Thanks go to Mr. Jonathan Lomber for his assistance in the laboratory. Appreciations are also conveyed to my colleagues and friends.

I am indebted to my family, my wonderful wife Siham and my lovely children Saja and

Ahmed for their support, encouragement, and bearing many of my responsibilities during this research. My deepest gratitude also goes to my beloved parents for their endless love, prayers, and encouragement. To my brothers and sisters, to my country State of Libya, to those who indirectly contributed in this research; your kindness means a lot to me. Thank you very much.

iii

Dedication

This dissertation is dedicated to my loving parents and Family members

ARUNDO DONAX L. (GIANT REED) PHYTOREMEDIATION

FUNCTION OF CHROMIUM (Cr) CONTAMINATION

Abstract

by Sadeg Saleh Nagi Abdurahman, Ph.D. Washington State University December 2016

Chair: Claudio O. Stöckle

Anthropogenic activities such as agriculture, industrial processes, and extraction of natural resources produce large quantities of wastewater. Pollutant types and concentrations vary with these activities raising environmental concerns. Heavy metals adversely affect the environment and human health because of their persistence in the environment and carcinogenicity to human beings. The presence of high level of chromium (Cr) from industrial processes is a major health concern and reducing Cr to minimal levels is necessary to protect the environment.

Phytoremediation is considered a more cost-effective approach for remediating soil of heavy metals removal because it has fewer side effects than physical and chemical approaches. This dissertation examines potential phytoremediation capacity of two plant varieties and the environmental variables (pH) affecting uptake of chromium by A. donax from a planting mix-water system in small-scale under greenhouse conditions. Specific objectives were:

1) To assess the capacity of two varieties of Arundo donax for phytoremediation of

chromium contaminated soil-water ecosystem, and

2) To investigate the effect of solution pH on the uptake of chromium by Arundo donax L.

from synthetic wastewater added to an organic nursery mix.

To achieve the first objective, two plant varieties were investigated for their capacity for phytoremediation of Cr contaminated soil using A. donax planted in nursery soil and irrigated with synthetic wastewater containing different chromium concentrations. The results indicated that increases in chromium concentration in growth medium significantly increased chromium concentration in the organs of the plant (roots, stems, and leaves). Chromium concentrations in the aerial parts of the plant were lower than in the roots.

In the second study, the effect of solution pH on the uptake of chromium by Arundo donax

L. from synthetic wastewater added to an organic nursery mix at different pH values was investigated. The results obtained from this experiment showed that the Cr concentration in plant tissues decreased with increased soil pH.

TABLE OF CONTENTS Page

ACKNOWLEDGEMENTS...... iii

ABSTRACT ...... v

LIST OF TABLES...... x

LIST OF FIGURES...... xi

ABBREVIATIONS ……………………...... xii

CHAPTER ONE ...... 1

INTRODUCTION ...... 1

Background ...... 1

Research Objectives ...... 6

Dissertation Structure...... 7

References ...... 8

CHAPTER TWO ...... 11

A REVIEW ON CHROMIUM CONTAMINATION IN ECOSYSTEM AND ITS

PHYTOREMEDIATION VIA ARUNDO DONAX L...... 11

Abstract ...... 11

Introduction ...... 12

Chemistry of Chromium ...... 14

Chromium Concentration in The Environment ...... 18

Remediation Strategies of Chromium ...... 21

Limitation of Phytoremediation ...... 23

vii

Phytoremediation of Chromium ...... 23

Uptake of Chromium by Plants...... 24

Chromium Accumulation and Translocation in Plants ...... 26

Arundo Donax L.: Description and Ecology ...... 27

Chronology of Arundo Donax L...... 28

Arundo Donax L. Characteristics ...... 29

Reproduction and Biomass Yield of Arundo Donax L...... 30

Phytoremediation potential of heavy metals using Arundo Donax L...... 31

Conclusion ...... 37

References ...... 38

CHAPTER THREE ...... 48

ASSESSMENT OF THE CAPACITY OF TWO VARIETIES OF ARUNDO DONAX

FOR PHYTOREMEDIATION OF CHROMIUM CONTAMINATED SOIL-WATER

ECOSYSTEM...... 48

Abstract ...... 48

Introduction ...... 49

Materials and Methods ...... 52

Results and Discussion ...... 59

Conclusion ...... 70

References ...... 71

viii

CHAPTER FOUR ...... 76

EFFECT OF pH ON THE REMEDIATION OF CHROMIUM CONTAMINATED SOIL

BY ARUNDO DONAX L...... 76

Abstract ...... 76

Introduction ...... 77

Materials and Methods ...... 80

Results and Discussion ...... 87

Conclusion ...... 96

References ...... 97

CHAPTER FIVE ...... 103

FINAL CONCLUSIONS AND FUTURE WORK ...... 100

Conclusions ...... 100

Recommendations for Future Work...... 102

LIST OF TABLES

CHAPTER TWO

Table 2.1 Chemical species of Cr in the environment ...... 17

Table 2.2 Selected references on different sources and levels of chromium contamination in soil and Water ...... 20

Table 2.3 Summary of work done by various studies using diverse plant species for the removal of chromium ...... 25

Table 2.4 Selected studies applied phytoremediation potential of some heavy metals using A. donax L...... 35

CHAPTER THREE

Table 3.1 Physicochemical properties of the soil used in the experiment...... 53

Table 3.2 Operating conditions for the Agilent 7500 cx ICP/MS ...... 58

Table 3.3 Color parameters of A. donax L. leaves at various chromium treatments ...... 69

CHAPTER FOUR

Table 4.1 Physicochemical properties of the soil used in the experiment...... 81

Table 4.2 Amount of K2CO3 needed to reach target soil pH ...... 82

Table 4.3 Operating conditions for the Agilent 7500 cx ICP/MS ...... 86

Table 4.4 Relative bioaccumulation factor (BF) of A. donax L. at 1.0 and 2.0 mg/L applied chromium concentrations...... 92

Table 4.5 Relative translocation factor (TF) of A. donax L. at 1.0 and 2.0 mg/L applied chromium concentrations...... 95

LIST OF FIGURES

CHAPTER ONE

Figure 1.1 Heavy metals present in all matrices at Superfund locations ...... 2

Figure 1.2 Conceptual response strategies of metal concentrations in plant tops in relation to increasing total metal concentrations in the soil ...... 5

CHAPTER TWO

Figure 2.1: Diagram for chromium species in solutions…………………………...……16

CHAPTER THREE

Figure 3.1 Respective Cr concentrations in plant tissues: (A) long leaf variety, (B) short leaf variety...... 61

Figure 3.2 (A) Relative bioaccumulation factor (BF) and (B) translocation factor (TF) of A. donax variety (long and short leaf) at different applied chromium concentrations .....64

Figure 3.3 (A) The height of A. donax L (long leaf) and (B) The height of A. donax L. (short leaf) at different applied chromium concentrations ...... 66

Figure 3.4 (A) The Nodes number of A. donax L (long leaf) and (B) The Nodes number of A. donax L. (short leaf) at different applied chromium concentrations...... 67

CHAPTER FOUR

Figure 4.1 (A) Cr content in plant parts in various pH at 1.0 mg Cr/L supplied. (B) Cr content in plant parts in various pH at 2.0 mg Cr/L supplied...... 89

Figure 4.2 Main effects for response of chromium uptake by A. donax tissues in different soil pH...... 90

Figure 4.3 Least squares mean of chromium uptake with 95.0% Confidence Interval...... 93

Figure 4.4 Interaction Plot for Response mean of chromium uptake by A. donax L. at different soil pH and Cr concentration...... 93

LIST OF ABBREVIATIONS

ANOVA Analysis of Variance

Cr Chromium.

-2 CrO4 Chromate

-2 Cr2O7 Dichromate

EC Electrolytic Conductivity

EPA Environmental Protection Agency

ET Environmental Technology

HMS Heavy Metals

ICP-MS Inductively Coupled Plasma Mass Spectrometry

IWWT Industrial Wastewater Treatment

IWA International Water Association

IETC International Environmental Technology Centre mg kg-1 Milligram Per Kilogram mg L-1 Milligram Per Litre

K2CO3 Potassium Carbonate

K2Cr2O7 Potassium Dichromate pH Potential of Hydrogen Ion

RCBD Randomized Complete Block Design

UNED United Nations Environmental Program

xii

CHAPTER ONE

INTRODUCTION

Background

Contamination of the environment by heavy metals, such as arsenic, cadmium, chromium, lead and mercury can lead to accumulation in plants, soils, and water bodies (Alloway, 2013;

Kabata-Pendias, 2010). Contamination might be sourced from land-use change impacts because of domestic, industrial and agricultural activity or naturally from heavy metals leaching from bearing minerals and rocks. Contamination by heavy metals in surface and groundwater is a challenging environmental problem because most heavy metals are bioaccumulative in aquatic food webs (Kabata-Pendias, 2010). Heavy metals are cationic or anionic that might be a required micronutrient at low concentrations or toxic to aquatic life at high levels. For instance, copper, iron, and zinc are essential elements for animals and plants at low concentration; on the other hand, they are toxic at elevated levels (Kadlec and Wallace, 2009). They do not only impact ecosystems but also increase the potential health risk for wildlife and human health (Wang et al., 2010).

The background concentrations of heavy metals in natural aquatic ecosystems are at trace levels, but they have been increased in many areas by generation of wastes, mining activities, and landfill applications. One metal that is of a considerable environmental significance is chromium

(Cr). U. S. Environmental Protection Agency (EPA) has been reported that about 1,000 locations in the United States that stand significant environmental health risks. Approximately 40 percent of these places have been stated to have heavy metal problems (U.S. EPA, 1996b). Heavy metals distributions at Superfund locations are illustrated in (Figure 1.1).

Figure 1.1: Heavy metals present in all matrices at Superfund locations (U.S. EPA, 1996b).

Chromium is a typically occurring element in the ecosystem, but it has become one of the most widespread metal pollutants in the ecosystem (Choppala et al., 2013). Chromium enters the ecosystem in several different forms commonly as chromium metal (0), trivalent chromium (III), and hexavalent chromium (VI) Alloway, 2013. Cr (III) can be found naturally in the ecosystem and is an essential animal nutrient. In contrast, Cr (VI) is highly toxic and carcinogenic (Choppala et al., 2013). Due to the high solubility of Cr (VI) in oxyanionic form, chromate, and dichromate are considered extremely hazardous. Many human activities such as chromium chemical production, electroplating, leather tanning, paint and pigment production, paper production,

stainless steel production, textile production and wood preservation result in Cr contamination in the environment (Kadlec and Wallace, 2009). Conventional tannery processes are used worldwide and lead to the discharge of solutions with high levels of chromium concentrations. Currently, chrome tanning is favored by the leather industry because of the rapidity of processing, cost- effectiveness, leather color, and higher stability of the resulting leather (Hafez et al., 2002).

Conversely, chromium salts are not completely absorbed by the leather. Therefore, elevated levels of chromium are found in the soil-water ecosystem. Estimations range from 3 – 350 mg/L

(Vlyssides and Israilides, 1997) to 2000 – 3000 mg/L (Bajza and Vrcek, 2001). If these contaminants are not treated before release they might lead to severe environmental pollution.

Traditional methods for treating these contaminants are complicated and expensive.

Consequently, many developing countries only utilize primary treatment. Primary treatment may use biological, chemical and physical processes. These treatments however often still leave levels of chromium in the wastewater over the legal limit of discharge for surface water. Reduction of these concentrations to minimal levels are necessary for the enhanced sustainability of this industry, in particularly in Libya. For that reason, in the case of chromium, further treatment is always necessary. Electrolysis treatment systems (Vlyssides and Israilides, 1997), ion exchange methods (Kocaoba and Akcin, 2002) and reverse osmosis systems (Hafez et al., 2002) have all been examined as techniques of further purification. However, these methods are often not considered cost-effective for small sized tanneries. Alternatively, phytoremediation is a well- established and low-cost remediation biotechnology for heavy metals removal from contaminated soil and water.

Phytoremediation of soil and water contaminated with tannery flow using plants with tolerant mycorrhizal fungi has been examined by Khan (2001) and is thought to have high potential. Cr in situ contaminant removal occurs via three mechanisms: the first mechanism is adsorption to soil and organic materials; the second is precipitation as insoluble Cr (III) solids, and the third mechanism is vegetation uptake (Nelson et al., 2006). Therefore, bioavailability of Cr can be decreased by adsorption or precipitation as insoluble Cr (III). Plant selection for treating wastewater is a primary consideration where phytoremediation is to be used. The use of plants to eliminate and remove heavy metals from an ecosystem is a growing field of research in environmental studies because of the advantages of it's low-cost and the ability to harvest the plants for the extraction of absorbed heavy metals that may not be readily biodegraded (Skinner et al.,

2007). The behavior of plants in the presence of heavy metals was investigated. Drost et al. (2007) conducted a study on the toxicity of heavy metals to plants. Terry and Banuelos, (1999) suggested three basic strategies through which plant species tolerances to large concentrations of heavy metals in their environment: metal accumulation, metal indicator plants, and metal exclusion; the general strategies of these plant tolerances are illustrated in (Figure 1.2).

Figure 1.2: Conceptual response strategies of metal concentrations in plant tops about increasing total metal concentrations in the soil (Terry and Banuelos, 1999).

This study assessed the uptake of chromium concentration from nursery mix peat moss by two varieties of A. donax and evaluated the effect of pH on the Cr accumulation and translocation in A. donax and chromium removal efficiencies. These projects were chosen because knowledge of chromium bioavailability and mobility is critical for predicting chromium accumulation in the environment as water is always present. Effects of soil pH on chromium removal during phytoremediation was investigated to determine the optimum pH for Cr uptake. The reason for that is to help design efficient and well-organized phytoremediation systems. This research is

important because these effects may cause even the most persistent chromium species through the biological, chemical, and environmental factors of the processes, and implementing phytoremediation techniques that remove chromium from an ecosystem can lower the risk of chromium contamination of the environment.

Research Rationale and Hypotheses

It was hypothesized, firstly, that the Arundo donax variety with more biomass can extract more chromium (Cr) than Arundo donax variety with low biomass. The rationale for this hypothesis is that the varieties of A. donax with more biomass has more roots, taller stems, and the number of leaves compared to the varieties with less biomass. Hence more plant volume is available for Cr uptake, translocation and accumulation, and secondly that the efficiency of chromium uptake by Arundo donax improves with moderate pH. The rationale for this hypothesis is that available plant Cr exists in the soil solution in the pH range of 5.0 – 7.0.

Research Objectives

Overall, the significance of this research lies in the ability to remove chromium contamination from soil and wastewater by cost-effective means. The study assessed the efficiency of phytoremediation using A. donax growing in a nursery mix for treating chromium in synthetic wastewater and evaluated the effects of soil pH on the uptake of chromium. Therefore, the main objectives of this research were:

Objective 1: To assess the capacity of two varieties of Arundo donax for phytoremediation of chromium contaminated soil-water ecosystem.

Objective 2: To determine the effect of solution pH on the uptake of chromium by Arundo donax

L. from synthetic wastewater added to an organic nursery mix.

Dissertation Structure

This Dissertation is organized into five chapters, including an introduction, three chapters addressing the dissertation’s specific objectives, and conclusions to present to the reader the use of phytoremediation application to eliminate and control chromium contamination from anthropogenic activities.

 Chapter One provides a concise overview of the importance of phytoremediation of

chromium contamination. It presents the motivation for this research study, the main

objectives, and the dissertation structure as well.

 Chapter Two presents an intensive review of chromium contamination and the use of plant

species (phytoremediation) for remediating contaminated water by chromium.

 Chapter Three covers Objective 1 of this study: to assess the effects of A. donax L. variety

on the capacity to phytoremediation chromium (Cr).

 Chapter Four covers objective 2: to evaluate the effects of solution pH on the uptake of

chromium (Cr) from an organic nursery mix by Arundo donax L.

 Chapter Five discusses and outlines the major conclusions and implications of the results

drawn from the entire research study and offers recommendations for future studies.

REFERENCES

Alloway, B. J. (2013). Introduction. In Heavy metals in soils (pp. 3-9). Springer Netherlands.

Bajza, Z., & Vrcek, I. V. 2001. Water Quality Analysis of Mixtures Obtained from Tannery Waste Effluents. Ecotoxicology and Environmental Safety, 50, 1, 15-18.

Banks, M.K., Schwab, A.P., Henderson, C., 2006. Leaching and reduction of chromium in the soil as affected by soil organic content and plants. Chemosphere 62, 255-264.

Calheiros, C.S.C., Quiterio, P.V.B., Silva, G., Crispim, L.F.C., Brix, H., Moura, S.C., Castro, P.M.L., 2012. Use of constructed wetland systems with Arundo and Sarcocornia for polishing high salinity tannery wastewater. Journal of Environmental Management 95, 66-71.

Calheiros, C.S.C., Rangel, A.O.S.S., Castro, P.M.L., 2009. Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis. Bioresource Technology 100, 3205-3213.

Choppala, G., Bolan, N., Park, J.H., 2013. Chromium contamination and its risk management in complex environmental settings. Academic Press.

Damasio, J., Tauler, R., Barata, C., Teixido, E., Rieradevall, M., Prat, N., Riva, M.C., Soares, A.M.V.M., 2008. Combined use of Daphnia Magna in situ bioassays, biomarkers and biological indices to diagnose and identify environmental pressures on invertebrate communities in two Mediterranean urbanized and industrialized rivers (NE Spain). Aquatic Toxicol. Aquatic Toxicology 87, 310-320.

Doran, J.W., Soil Science Society of America. Division, S., 1992 Defining soil quality for a sustainable environment: proceedings of a symposium sponsored by Divisions S-3, S-6, and S-2 of the Soil Science Society of America, Division A-5 of the American Society of Agronomy, and the North Central Region Committee on Soil Organic Matter (NCR-59) in Minneapolis, MN, 4-5. SSSA: American Society of Agronomy, Madison, Wis.

Drost, W., Matzke, M., Backhaus, T., 2007. Heavy metal toxicity to Lemna minor: studies on the time dependence of growth inhibition and the recovery after exposure. Chemosphere 67, 36-43.

Hafez, A. I., El-Manharawy, M. S., & Khedr, M. A. 2002. RO membrane removal of unreacted chromium from spent tanning effluent. A pilot-scale study, Part 2. Desalination, 144, 1-3.

Kabata-Pendias, A. (2010). Trace elements in soils and plants. CRC press.

Kadlec, R.H., Wallace, S.D., 2009. Treatment wetlands. CRC Press, Boca Raton, FL.

Karlen, D.L., Mausbach, M.J., Doran, J.W., Cline, R.G., Harris, R.F., Schuman, G.E., 1997. Soil quality: A concept, definition, and framework for evaluation. Soil Science Society of America Journal 61.

Khan, Abdul G., & the University of Western Sydney. 2001. Relationships between chromium biomagnification ratio, accumulation factor, and mycorrhizae in plants growing on tannery effluent-polluted soil. UK Pergamon-Elsevier Science.

Kocaoba, S., & Akcin, G. 2002. Removal and recovery of chromium and chromium speciation with MINTEQA2. Talanta, 57, 1, 23-30.

Legates, D., Mahmood, R., Levia, D., DeLiberty, T., Quiring, S., Houser, C., Nelson, F., 2011. Soil moisture: A central and unifying theme in physical geography. Progress in Physical Geography 35, 65-86.

Miretzky, P., Saralegui, A., Cirelli, A.F., 2004. Aquatic macrophytes potential for the simultaneous removal of heavy metals (Buenos Aires, Argentina). Chemosphere 57, 997- 1005.

Nelson, E.A., Specht, W.L., Knox, A.S., 2006. Metal Removal from Water Discharges by a Constructed Treatment Wetland. ENGINEERING IN LIFE SCIENCES 6, 26-30.

Nordmark, D.s.e., Kumpiene, J., Andreas, L., Lagerkvist, A., 2011. Mobility and fractionation of arsenic, chromium, and copper in thermally treated soil. Waste Management and Research 29, 3-12.

Papazoglou, E.G., Karantounias, G.A., Vemmos, S.N., Bouranis, D.L., 2005. Photosynthesis and growth responses of giant reed (Arundo donax L.) to the heavy metals Cd and Ni. Environment International 31.

Rahman, M.H., Okubo, A., Sugiyama, S., Mayland, H.F., 2008. Physical, chemical and microbiological properties of an Andisol as related to land use and tillage practice. Soil & Tillage Research 101.

Shukla, M.K., Lal, R., Ebinger, M., 2004. Soil Quality Indicators for Reclaimed Mine soils in Southeastern Ohio. SOIL SCIENCE 169, 133-142.

Skinner, K., Wright, N., Porter-Goff, E., 2007. Mercury uptake and accumulation by four species of aquatic plants. Environmental Pollution 145, 234-237.

Terry, Norman, and Gary S. Banuelos, (Eds) 1999. Phytoremediation of contaminated soil

and water. CRC Press.

U.S. EPA (1996b), Report: Recent Developments for In Situ Treatment of Metals contaminated Soils, U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response, draft.

Vlyssides, A. G., & Israilides, C. J. 1997. Detoxification of tannery waste liquors with an electrolysis system. Environmental Pollution (Barking, Essex: 1987), 97, 1- 2.

Wang, Y., Chen, P., Cui, R., Si, W., Zhang, Y., Ji, W., 2010. Heavy metal concentrations in water, sediment, and tissues of two fish species (Triplohysa pappenheimi, Gobio Hwang he NSIS) from the Lanzhou section of the Yellow River, China. Environ. Monit. Assess. Environmental Monitoring and Assessment 165, 97-102.

CHAPTER TWO

A REVIEW OF CHROMIUM CONTAMINATION IN SOIL-WATER SYSTEMS AND

ITS PHYTOREMEDIATION BY ARUNDO DONAX L.

Abstract

A review is presented for the potential use of macrophyte Arundo donax L. for the

phytoremediation of chromium-contaminated sediments, soil, and water. The review considers

the following aspects: chromium chemistry, chromium concentrations in water, remediation

strategies, including phytoremediation and its limitations, uptake, translocation and accumulation

of chromium by plants, and the specific phytoremediation potential of Arundo donax L. The

review summarizes and compares data from greenhouse, laboratory, and field studies regarding

the ability of A. donax to remediate Cr contaminated water. The macrophyte Arundo donax L.

has been shown to be remarkably effective at cleaning up ecosystems. This review examines and

discusses previous studies that involved Cr remediation by A. donax varieties and the effect of

pH on Cr accumulation and translocation in A. donax. This review focuses primarily on

demonstrated actual results of A. donax used for attempted remediation of chromium-

contaminated water system under a variety of differing conditions and utilizing a variety of

different plant species. A. donax might be utilized for remediating of Cr contaminated-

wastewaters.

Introduction

Contamination of the ecosystem by non-regulated emerging contaminants such as heavy

metals, pesticide residue, and organic compounds is a human and environmental health concern

(Kabata-Pendias, 2010; Choppala et al., 2013). Soil and water resources contaminated by heavy

metals are a growing problem in various locations around the world. Even though at trace levels

several elements are natural components of soils, anthropogenic activities such as mining,

industry, and localized agriculture have contributed to undesirable toxic levels in the ecosystem

(Meers et al., 2005; Lewandowski and Schmidt, 2006). Several locations around the world have

been contaminated by combined heavy metals. (Cui et al., 2004). Chromium accumulation in

soil and water in particular, is a growing worldwide issue. Chromium contamination might

originate from both natural geochemical processes via weathering of ultramaﬁc rocks, and also

by anthropogenic activities such as mining and smelting, combustion of fossil fuels, utilization

of fertilizers and pesticides, and disposal of wastes (Ghosh and Singh, 2005b).

Plants have the ability to absorb various elements from soil and water, some of which have

no known biological function while others are known to be toxic at low concentrations (Rai et

al., 2011). In particular, the accumulation of considerable amounts of trace elements in aquatic

macrophytes has been reported by numerous authors and aquatic macrophytes have been

proposed as pollution-monitoring organisms (Windham et al., 2003; Demırezen and Aksoy,

2004; Duman et al., 2007). The macrophyte Arundo donax L. is one of the most widely utilized

plants as a trace element bioaccumulator (specifically in phytoremediation approaches) because

of its ability to take up pollutants such as heavy metals that cannot be easily biodegraded

(Papazoglou, 2007; Papazoglou et al., 2007; Mirza et al., 2011). In contrast, due to its excessive

acclimation to diverse environmental conditions, A. donax L. is one of the most harmful and aggressive weeds and is extensively distributed in riparian territories around the world (Mal and

Narine, 2004; Mack, 2008; Quinn and Holt, 2008; Spencer et al., 2008; Quinn and Holt, 2009;

Kui et al., 2013).

There is extensive literature on the uptake of trace elements using plant species; however there has been substantially less attention given to the absorption of chromium by A. donax.

Currently , there has been increased focus on this topic due to ecological concerns of chromium toxicity, carcinogenicity, and mutagenicity (Chandra and Kulshreshtha, 2004; Shanker et al.,

2005; Zhang et al., 2010). Similarly, there is growing desire for the potential employment of A. donax for the remediation of contaminated soil and water via phytoremediation biotechnologies, phytoextraction, and phytostabilization (Chaudhry et al., 1998; Papazoglou et al., 2005;

Papazoglou et al., 2007; Mirza et al., 2010; Kausar et al., 2012).

This review summarizes both previous and current examples from the published literature, which demonstrate remediation efficiency of chromium contamination in soil-water systems using macrophyte plants. Recently, phytoremediation has been applied to a host of pollutants such as agrochemicals, organic compounds, and pesticide residues. In addition, researchers have recently developed modeling techniques to predict and clarify macrophyte plant phytoremediation efficiencies. With the exception of citing a few early modeling contributions, this review focuses mainly on demonstrated experimental results.

Chemistry of chromium

Chromium is an element of Group VIB of the Periodic Table and a d-block transition metal.

Its atomic number is 24, ionic radius 124.9 pm, the atomic weight of 51.996 g mol-1, and an

electronic configuration [Ar] 3d5 4s1. Out of its 28 known isotopes, four stable isotopes are found

in nature: Cr50, Cr52, Cr53, and Cr54. All other chromium isotopes are radioactive; the most

common stable of radioactive chromium isotopes is Cr51. It has a half-life of 27.8 days.

Chromium metal is gray and brittle and can be highly polished (McGrath and Smith, 1995;

Choppala et al., 2013). Chromium has different valence states ranging from Cr (0) to Cr (VI),

but trivalent Cr (III) and hexavalent Cr (VI) states are the most stable and the most important

forms in the environment (Figure 2.1) (Nieboer and Jusys, 1988; Katz and Salem, 1994; Rai et

al., 1989; Palmer and Wittbrodt, 1991; Zayed and Terry, 2003). The intermediate states of Cr

(IV) and Cr (V) are metastable and are rarely encountered in soil (Choppala et al., 2013). The

classified chemical species of chromium in the environment and their occurrences in nature are

listed in Table 2.1.

As can be seen in Fig 2.1, Cr(III) and Cr(VI) occur in solution under different pH and Eh conditions. Cr(III) solubility in solution is pH-dependent. At pH < 3, Cr+3 is a significant species of dissolved Cr(III) and declines at pH > 4.0, when can form compounds of chromium hydroxides

- such as Cr(OH)3(s) and Cr(OH)4 (aq). However, species of Cr(VI) such as hydrogen chromate

- -2 -2 (HCrO4 ), chromate (CrO4 ) and dichromate (Cr2O7 ) are much more soluble than species of

Cr(III). The distribution of these species is pH and redox potential dependent. At pH < 6.0 and

- high oxidation-reduction potential (Eh), HCrO4 is the predominant dissolved species. At pH > 6.0,

-2 - -2 -2 CrO4 occurs in pH-dependent equilibrium with another species HCrO4 and Cr2O7 . CrO4 is

more mobile and easily sorbed by clays and hydrous oxides. The relation between the species of

Cr(III) and Cr(VI) is strongly dependent on pH and oxidation-reduction conditions, but in most cases Cr(III) is the predominant species.

Chromium species existing in any ecological conditions are determined by different chemical and physical processes such as adsorption and complexation, hydrolysis, precipitation, and redox reactions (Rai et al., 1987; Deshpande et al., 2005). Because the solubility of Cr(III) is very low, it has a high propensity to adsorb on surfaces (Rai et al., 1987). At pH > 7.5, Cr(III) can form complexes with organic ligands such as citrate, diethylenetriamine pentaacetate (DTPA) and ethylene diamine tetra acetic acid (EDTA) (James and Bartlett, 1983). In environment systems, limited oxidants can oxidize species of Cr(III) to species of Cr(VI) due to the positive redox potential of the Cr(VI)/Cr(III) couple. Trivalent Cr(III) oxidation by manganese oxides and dissolved oxygen has been reported (Johnson and Xyla, 1991). Chromium is expected to be remediated in the solution as precipitation of chromium hydroxide Cr(OH)3(s) at pH > 6, or in the existence of Fe(III), removed as (Cr, Fe) (OH)3(s) (Deshpande et al., 2005).

- -2 -2 The oxyanionic species of Cr(VI), HCrO4 , Cr2O7 and CrO4 can be adsorbed to positively charged soil colloids like aluminum (Al) and iron (Fe) oxides at pH below their point of zero charge and at positive charge positions on plant root surfaces (Ainsworth et al., 1989).

Adsorption depends on the quantity and type of soil components, pH of the solution, and the

-2 presence of competing ligands like phosphate (PO4 ). Cr(VI) species adsorption is pH-dependent, increasing as pH and decrease. Cr(VI) forms oxyanionic compounds that have an adsorption on positive proton – specific mineral surfaces, even though their adsorption is incomplete in soil-

-2 - water systems by the existence of competing anions such as sulphate (SO4 ) and phosphate (PO4

2) (Zachara et al., 1987).

Toxicity of chromium is a condition whereby an organism cannot tolerate the high concentration Cr by direct utilization or storage (Shanker et al., 2005). Cr(III) is known to have a low toxicity with respect to plant growth (Megharaj et al., 2003). In mammalian systems, Cr (VI) is known to be a highly toxic, hazardous species showing both carcinogenicity, and mutagenicity in humans (Skovbjerg et al., 2006). Cr(VI) compounds are extremely toxic to plant species and are damaging to their growth and development (Davies et al., 2002) who reported that Cr compounds are toxic to plants at 100 µM kg-1 dry weight.

Figure 2.1: Diagram for chromium species in solutions (Rai et al., 1987).

Table 2.1: Chemical species of Cr in the environment ( Zayed and Terry 2003).

Chemical species Oxidation state Examples Remarks Elemental Cr Cr(0) ------Does not occur naturally Divalent Cr Cr(II) CrBr2, CrCl2, Relatively unstable and is readily CrF2, CrSe, Cr2Si oxidized to the trivalent state. Trivalent Cr Cr(III) CrB, CrB2, CrBr3, Forms stable compounds and CrCl3.6H2O, occurs in nature in ores, such as CrCl3, CrF3, CrN, ferrochromite (FeCr2O4). KCr(SO4)2.12H2O

Tetravalent Cr Cr(IV) Cr dioxide CrO2, Does not occur naturally and tetrafluoride CrF4 represents an important intermediate that influences the rate of reduction of the Cr(V) form. Chromium (IV) compounds are less common. The Cr(IV) ion and its compounds are not very stable and because of short half-lives, defy detection as reaction intermediates between Cr(VI) and Cr(III). Pentavalent Cr Cr(V) Tetraper- Does not occur naturally and oxochromate potassium represents an -3 CrO4 , intermediate that influence the perchromate rate of reduction of the Cr(VI) form. Chromium (V) species are -3 derived from the anion CrO4 and are long-lived enough to be observed directly. However, there are relatively few stable compounds containing Cr(V). Hexavalent Cr Cr(VI) (NH4)2 CrO4, The second most stable state of BaCrO4, CaCrO4, Cr. However, Cr(VI) rarely K2CrO4, K2Cr2O7 occurs naturally but is produced from anthropogenic sources. It occurs naturally in the rare mineral crocoite (PbCrO4).

Chromium concentrations in the environment

Chromium and its compounds occur in all phases of the ecosystem including air, soil and water. In natural soil, Cr generally ranges from 10 to 50 mg kg-1 depending on the parental material.

In ultramafic soils, it can reach up to 125 g kg-1 (Adriano, 2001). Natural levels in uncontaminated water range from 0.1 to 117 µg l-1, while in seawater, Cr levels generally range from 0.2 to 50 µg l-1. In soil-water system, chromium exists mostly as Cr (III) and Cr (VI) species, which are widely different in physico-chemical properties and biological reactivity. When the pH increases,

+3 + hydrolysis of Cr(H2O)6 (aq), which is a moderately strong acid, deprotonates into Cr(OH)2 (aq) and Cr(OH)3 (s) respectively within pH 4 – 10. In contrast, Cr(VI) is stable at positive redox

-2 potential under alkaline conditions and is a strong oxidizing agent that exists as chromate CrO4

- is the main species at pH > 6, while hydrogen chromate HCrO4 is predominant species at pH < 6.

(Tandon et al., 1984).

Chromium compounds are involved in various industrial practices. They are extensively utilized in leather processing and finishing (Choppala et al., 2013) as well as in the production of refractory steel, drilling muds, electroplating cleaning agents, catalytic manufacture, and in the production of chromic acid and specialty chemicals. Hexavalent chromium compounds are used in industry for metal plating, cooling tower water treatment, hide tanning and, until recently, wood preservation. These anthropogenic activities have led to the widespread contamination of Cr in the environment and have increased its bioavailability and mobility. A detailed review on the critical assessment of Cr in the environment has previously been published (Kotaś and Stasicka, 2000).

The leather industry is the major cause for the high influx of Cr to the environment, accounting for 40% of the total industrial use (Barnhart, 1997), these wastewater need to remediated before discharging to the environment and phytoremediation application might be the proper technique for wastewater treatment. In India, about 2000–32,000 tons of elemental Cr annually escape into the environment from tanning industries. Even if the recommended limit for

Cr concentration in water are set differently for Cr(III) (8 µg l-1) and Cr(VI) (1µg l-1), it ranges from 2 to 5 g/L in the effluents of these industries (Chandra et al., 1997; Chandra and Kulshreshtha,

2004). In the United States, 14.6 µg l-1 in ground water and 25.9 g kg-1 in soil have been found in the vicinity of chrome production sites (Zayed and Terry, 2003). In most soils, chromium occurs in low concentrations (2 - 60 mg kg-1), but values of up to 4 g kg-1 have been reported in some uncontaminated soils, with only a fraction of this chromium being available to plants (Choppala et al., 2013). It is not known whether chromium is an essential nutrient for plants, but all plants contain the element (up to 0.19 mg/kg on a wet weight basis). Measurements of the amount of total chromium and chromium speciation in ecosystems reported in the literature are listed in Table 2.2.

(Choppala et al., 2013).

Table 2.2: Selected references on different sources and levels of chromium contamination in soil and Water (Choppala et al., 2013)

Country Species Media Source of Cr Contamination Concentration level (ppm) Reference New Jersey, USA Cr(total) Water Mining 30 Burke et al., 1991 New Jersey, USA Cr(total) Soil Mining 53.000 Burke et al., 1991 Glasgow, UK Cr(total) Water Mining 169 Whaley et al., 1999 Iraja river, Brazil Cr(total) Sediment Electroplating 210 – 70.000 Pfeiffer et al., 1982 Brazil Cr(total) Sediment Tannery 2878 Jordao et al., 1997 Skeleton Creek, Cr(total) Sediment ------5.1 India (Tamilandu) Cr(total) Soil Chromate Factory 250 – 510 Imam Khasim et al., 1989 India (Tamilandu) Cr(total) Water Chromate Factory 160 – 1240 Imam Khasim et al., 1989 Mexico Cr(total) Soil Geogenic 274 Robles-Camacho and Armieta, 2000 USA (Oregon) Cr(VI) Water Chromate Plating 14,600 Palmer and Wittbrodt., 1991 USA (Oregon) Cr(VI) Soil Chromate Plating 25,900 Palmer and Wittbrodt., 1991 USA (New York) Cr(VI) Water ------40 Palmer and Wittbrodt., 1991

20 USA (Oregon) Cr(total) Soil Electroplating 60.000 Sturges et al., 1991

USA (Oregon) Cr(VI) Water Electroplating 19,000 Sturges et al., 1991 Australia Cr(total) Soil Industries 91 – 270 Tiller., 1992 Australia Cr(total) Soil Tannery 100 – 62,000 Naidu et al., 2000 Poland Cr(VI) Water Tannery 0.68 Stepniewska and Bucior., 2001 Poland Cr(total) Water Tannery 1.18 Stepniewska and Bucior., 2001 Poland Cr(total) Soil Tannery 5.79 Stepniewska and Bucior., 2001 Slovenia Cr(VI) Water Galvanization 0.175 Brilly et al., 2008 China Cr(VI) Water Alloy and petroleum Plants 0.02 – 5.0 Zhang and Li., 1997 Russia Cr(VI) Water Cr Processing Plant 0.922 Leslie et al., 1999 Italy Cr(total) Soil Industries 67 – 5490 Viti and Giovannetti., 2001

Remediation Strategies of Chromium

Traditional methods for treating chromium are very complicated and very expensive.

Electrolysis treatment system (Vlyssides and Israilides, 1997), ion exchange methods (Kocaoba and Akcin, 2002) and reverse osmosis systems (Hafez et al., 2002) have all been examined as techniques of further purification. However, all these methods are very costly and are often not considered cost-effective for small sized tanneries and other anthropogenic activities, which use chromium. Therefore, there is a need to search for more cost-effective alternatives for remediating chromium contaminated soil and water systems such as phytoremediation.

For treating Cr-contaminated soil, various remediation technologies for treating chromium(VI) have been considered to remove Cr from soil. For instance, permeable reactive barrier with electro-kinetics (Zhang et al., 2012), reduction of Cr in contaminated soil using ferrous sulphate (FeSO4) and nanoscale zero-valent iron (Di Palma et al., 2015), and ex-situ remediation of Cr(VI) contaminated soil (Kathiravan et al., 2011). However, most of these treatment methods need knowledge of site-specific settings, elasticity in remediation design and optimization strategies. Therefore, there is a need to continuing researching for more cost- effective alternatives for remediating chromium contaminated soil and water systems such as phytoremediation. Consequently, phytoremediation is a well-established and cost effective

remediation biotechnology for heavy metals removal. Phytoremediation of soil and water contaminated with tannery effluent using trees hosting tolerant mycorrhizal fungi has been investigated by (Khan, 2001), and is considered to have high potential. This review discusses the potential of phytoremediation system on treating chromium removal by plant species, chemistry of chromium and chromium speciation in soil-water system.

Phytoremediation is considered one of the most cost-effective biotechnologies for remediation of contaminated sites and landfills (Flathman and Lanza, 1998). The capital costs for phytoremediation generally ranges from one third to one fifth to that of conventional technologies.

Additionally, this technology has lower operation and maintenance costs (Singh et al., 2003). In particular, the lower capital, operation, and maintenance costs make phytoremediation more suitable and attractive for non-point source contaminations. Other benefits of phytoremediation include reduction of wind and water erosions along with the production of undesirable wastes by- products (Singh et al., 2003). On the other hand, net infiltration of surface water is also reduced by some plant species which curbs leaching of contaminants into groundwater (Mirck et al., 2005).

In addition to these benefits, soil conditions are highly improved by increasing soil organic carbon, enhancing microbial and fungal communities, and humifying metals and recalcitrant organic through metal complexation with soil organics (McCUTCHEON and Schnoor, 2004).

Phytoremediation has been recognized as ecologically compatible (Raskin and Ensley,

2000). This process can be applied separately as a single technique or it can be combined with other conventional technologies. For instance, contaminated soil can be collected through excavation and then treated in engineered phytoremediation treatment units (Salt et al., 1995;

McIntyre, 2003). Similarly, groundwater can also be pumped from a location through conventional

technologies such as air stripping or bioreactor (Ram et al., 1993). In Beaverton, Oregon, a landfill is irrigated for hybrid poplars with effluent from treatment works owned by public works, which has been planned as an alternative cover for the city landfill (Erickson et al., 1994).

Limitations of Phytoremediation

Despite the advantages of phytoremediation, it has disadvantages for some environmental applications. Phytoremediation is a long-term remedial technology in most cases which takes several years, and can be implemented only when the contaminants are found within 20 feet depth

from the ground surface (Chappell, 1997; Prasad, 2003). For the case of ground extraction, the

contaminants must be located within a few feet of water table surface. Some plants are known to have adapted to some of these inhospitable conditions. However, this remediation method will not be successful if the contaminants are phytotoxic. Furthermore, all phytoremediation plants may not be appropriate in certain applications (Prasad, 2003).

Phytoremediation of Chromium

Phytoremediation of heavy metals can be impractical for special cases (Garbisu et al.,

2002; Paz-Alberto and Sigua, 2013). For instance, the issue of the biogeochemical cycle must be considered as up taking of toxic heavy metals from soil and water will enter into the food chain

(Kabata-Pendias, 2010). In addition, toxic heavy metals can be mobilized downward and move from rhizosphere and enter into groundwater and pose threat to water contamination (Salt et al.,

1998; Garbisu et al., 2002). Hence as technology improves, some of these disadvantages will be minimized while new limitations will be identified (Kabata-Pendias, 2010). High concentrations of Cr can be found naturally in some soils originating from cretaceous shale bedrock.

Unfortunately, existing chemical, microbiological, and electrochemical treatments are not economically feasible and very often produce large amounts of unwanted hazardous by-products which need to be disposed of in a toxic landfill dumps. Thus, phytoremediation, a comparatively cheaper and environmentally friendly, an emerging technology for cleaning up trace toxic metals in soil and water has received increasing attention (Salt et al., 1995; Chen et al., 2000; Nedelkoska and Doran, 2000; McGrath et al., 2001). Chromium accumulating plants can be effective in removing Cr from soil and water.

Uptake of Chromium by Plants

The uptake of heavy metals takes place mostly through canals and transporters in the root

membrane of plasma. Nevertheless, most vascular plants absorb toxic heavy metals through their

roots in varying amounts (Pulford et al., 2001; Loeppert et al., 2003). Several studies have

investigated the ability of plants species to uptake chromium (Table 2.3). Some studies have

indicated that plants uptake Cr(VI) more easily than Cr(III) (Vazquez et al., 1987; Lytle et al.,

1998; Gardea-Torresdey et al., 2005; Ghosh and Singh, 2005a). In a research of Cr uptake

through oat. McGrath, 1982 utilized a streaming culture strategy as a part of which equivalent

concentrations of dissolvable Cr(VI) and Cr(III) were kept up, and found that the plants absorbed

Cr(VI) and Cr(III) as well. The pathway of Cr absorbed by plant tissues is not yet clarified (Zayed

and Terry, 2003). The uptake of Cr(VI) by plant tissues is assumed to be an active mechanism

achieved by carriers for the absorption of essential elements such as phosphate (Kim et al., 2006;

Cervantes et al., 2001).

Table 2.3: Summary of work done by various studies using diverse plant species for the removal

of chromium

Plant Species Media Cr (ppm) Summary Reference Arundo donax L. Hydroponic 0.05 – 0.9 mg Cr/L (A. donax could Kausar et al. absorb Cr from (2012) hydroponic solution) Aptenia cordofolia L. Peat soil 0.05 – 1mmol Cr/L Budak et al. Alyssum maritime L. Peat Soil 0.05 – 1mmol Cr/L (The increases in the (2011) Brassica juncea L. Peat Soil 0.05 – 1mmol Cr/L concentration of Cr in Brassica oleracea L. Peat soil 0.05 – 1mmol Cr/L irrigation water increased the accumulation of Cr in

plant)

B. decumbens Gravel 10 and 20 mg Cr/L (All the system Mant et al. P. australis Gravel 10 and 20 mg Cr/L achieved removal (2006) P. purpureum Gravel 10 and 20 mg Cr/L efficiencies of 97- 99.6%)

Pistia Stratiotes Soil 1, 2, 4, 6 mg Cr/L (It was shown that Maine et al. Salvinia Herzogli Soil 1, 2, 4, 6 mg Cr/L both plants could (2004) effectively Reduce Cr content)

P. australis Soil 268 mg Cr/kg (Both plants present Windham et S. alterniflora Soil 268 mg Cr/kg different patterns of al. (2003) Cr accumulation in tissue types)

Plant species contrast essentially in Cr uptake proficiency and dispersion inside the plant.

(Grubinger et al., 1994) investigated Cr uptake by different dicotyledonous and monocotyledonous

plants and found that dicots (particularly green verdant vegetables) (Zayed et al., 1998) consumed

more Cr and translocated more Cr to leaves than did monocots (corn and grain). The difference

between the two plants is because dicotyledonous has more leaves and roots than monocotyledonous and leaves are different in shape and size.

Chromium Translocation and Accumulation in Plants

Plants have to be able to translocate chromium from the roots to shoots, in order for the plant to carry on the absorption of a toxic trace metal from the medium (Loeppert et al., 2003). There are two physiologic benefits to the plant for translocation. First, translocation decreases metal concentration and consequently reduces potential toxicity to the root. Second, translocation to the

shoot is one of the mechanisms of getting rid of Cr from plants when leaves either drops off or are

harvested (Loeppert et al., 2003).

Previous investigations into phytoaccumulation focused on high biomass producing species that have an ability to accumulate high chromium content, described as hyperaccumulators

(Rai et al., 2004; Zhang et al., 2007). Ultimate research utilizing non-hyper accumulator vegetation has demonstrated that chromium is essentially accumulated in the roots, and a much smaller amount of the total chromium in the plant is in the leaves (Loeppert et al., 2003; Oliveira,

2012). The distribution of chromium between the root and shoot in hyperaccumulator plants show that the leaves sometimes contain a much greater chromium concentration than that of non-hyper accumulator vegetation. The amount of chromium originating in the shoot tended to

-2 be elevated with increasing concentration of chromate CrO4 in solution, but stayed almost similar for Cr(III) (Loeppert et al., 2003; Naidu, 2003).

Arundo Donax L.: Description and Ecology

The macrophyte A. donax L. is a perennial rhizomatous plant domestic to the freshwater areas of Eastern Asia, but recently considered as a sub-cosmopolitan species due to its global and worldwide presence. It is a hydrophyte that has three varieties native to warmer tropical and temperate areas of the world, growing along drains, lakes, streams, and other wetlands locations

(Bell, 1993). The genus A. donax can grow 2.5 to 3 inches per day, reach 20 ft. in height under optimal conditions, and is among the quickest developing terrestrial plants with ability to produce more than 50 t ha-1 aboveground dry biomass (Günes and Saygın, 1996). These characteristics make A.donax an attractive species for bio-fuels, energy, build materials, paper pulp, etc. (Rieger and Kreager, 1989).

Arundo donax L. demonstrates remarkable absorption and concentration of noxious chemicals from contaminated soil and water with no adverse effect to its health and growth (Bell,

1998). Arundo donax is thus one of the plants generally utilized as a heavy metal bio-accumulator due to its absorption ability of metal contaminants that can't be effectively biodegraded. Arundo donax can grow in environments with extreme pH, salts, drought, and still uptake metals without any adverse effects on its growth. Nevertheless, due to its excessive flexibility to diverse ecological conditions, A. donax is considered harmful and obtrusive weed in riparian environments throughout the world.

Chronology of Arundo Donax L.

Literature indicates that the macrophyte Arundo donax L. is native to the Mediterranean area, but some sources suggest that A. donax originated from subtropical parts of the world; for instance, China, India and in the southern USA with some genotypes adapting to cooler climate conditions (Bell, 1993). A. donax normally grows along banks of lakes, rivers, streams, creeks, and wetlands, but it develops similarly well on generally dry and unfertile soils (roadsides) and is utilized to mark field borders (Spencer et al., 2008). Arundo donax L. is known by different names:

Giant cane, Carrizo, Arundo, Spanish cane, Colorado River Reed, and Wild Cane. Giant reed is the common name which was approved by the science community (Bell, 1998).

Reports indicate that A. donax was imported to California in the early 1820’s by Spanish for erosion control (Bell, 1993; Iverson, 1993). Similarly, A. donax was used in Spanish world for several purposes such as a construction material, animal fodder and firewood (Frandsen, 1997).

Moreover, administrative organizations have urged agriculturists to plant A.donax for erosion control in drainage canals (Boose and Holt, 1999). A. donax was initially introduced in Australia in 1788 by the first armada bringing the British to Australia for colonization (Lee, 2009). In

Australia, A. donax has been utilized particularly for windbreaks around agriculture activities, to balance out sand floats, and for draft stores, and grass for ruminants when shoots are youthful and shorter than 1.8m (Williams and Biswas, 2010).

Arundo Donax L. Characteristics

Arundo donax L. is a robust erect variety of perennial grass that grows up to 15 m height under ideal development conditions; developing in several stemmed individuals, tough and hollow

stems 3 to 5 cm thick that have a cane-like appearance like bamboo with alternate leaves 30 to 60 cm long and 2 to 6 cm broad, tapered tips and hairy tuft, at base. A. donax has a boundless system of rhizomes beneath the soil surface, 5 to 30 cm in depth. Stems generated throughout the first emerging season are unbranched and photosynthetic (Bell, 1998). The stringy roots initiating from rhizomes can be able to grow into the soil to 5 m in depth in certain wet soils with most roots being

> 0.1 m in depth. The rhizomes can achieve 3 to 8 cm in width and 10 to 25 cm in length, and develop extreme stringy and long tap roots (Frandsen, 1997; Spencer et al., 2008). The stem of A. donax L. is an empty, divided culm that ranges from 1 to 4 cm in diameter and can branch. The

culms' dividers range from 2 to 7 mm in thickness, and the internodes can achieve 30 cm long.

This stem structure can help the erect position of such a tall plant as its mechanical dependability is not subject to turgor weight (Spatz et al., 1997). Numerous stems grow from the rhizome buds throughout all the vegetative season, forming thick bunches.

In the basin of Mediterranean, where the warm, mild atmosphere is portrayed through gentle, wet winters and hot, dry summers, new shoots of A. donax rise out of buds on rhizomes.

This growth occurs in late winter or early spring, attaining most extreme growth rates in mid- summer. A. donax grows up from horizontal rootstocks beneath the soil and forms dense stands on disturbed sites, sand dunes, in wetlands and riparian habitats. A. donax has the capacity to grow in a broad scope of soil, including evidently unwelcoming and peripheral areas; it can grow in sandy, gravelly soils, and substantial clay. After the first year of growth, A. donax can grow in areas of high dampness and saltiness, including wetlands (Perdue, 1958; Spencer et al., 2006). A. donax can be growth throughout the year, but ideal growth emerges through February to October; it grows

well when the water table is adjacent the soil surface but is hindered if soil is not moist in the first year of growth (Frandsen, 1997).

The drought has little impact on the initiated stands which are in the second or third year of growth. A. donax grows in low temperatures in the torpid winter season but is impeded by ice events after the start of spring growth. These characteristics make A. donax a viable potential competitor against another plant species. Once planted, A. donax has a tendency to cover expansive zones with thick clusters, bargaining the vicinity of local vegetation not ready to contend (Bell,

1998; Coffman et al., 2010).Although the grass A. donax is a C3-grass, it demonstrates high photosynthetic rates and unsaturated photosynthetic potential in contrast with C4 plants (Rossa et al., 1998; Papazoglou et al., 2005). Notwithstanding being a C3 plant species, the biomass yield of A. donax has observed to be greater than for C4 plant species (Angelini et al., 2009; Kering et al., 2012).

Reproduction and Biomass Yield of Arundo Donax L.

The macrophyte A. donax L. reproduces vegetation via rhizomes and also by stems, which will root at the nodes along the stalk (Lewandowski et al., 2003). Rhizomes in perennial grasses are underground stems that produce adventitious roots and shoots (Marton and Czako, 2007).

Stems produce adventitious roots and shoot near the tips of branches, or from sprouts at the stem base or stump. It flowers in late summer and produces a plume-like flower. Although A. donax produces seeds, they are usually infertile making its multiplication through seeds difficult (Marton and Czako, 2007). Numerous articles have been reported that A. donax produces infertile seeds in several regions around the world such as Europe (Lewandowski et al., 2003; Mariani et al., 2010),

Asia (Popov and Belyaeva, 1987), the USA (Balogh et al., 2012), and Australia (Williams et al.,

2008) implying that conventional breeding through sexual hybridization is impossible.

Since A. donax is an asexually reproductive species due to seed sterility, it’s genetic variability and the chances for finding new genotypes or varieties are low (Lewandowski et al.,

2003) and requires in vitro cell and tissue culture for large-scale propagation of plantations. Marton and Czako (2007) have developed a universal cell culture procedure, in the U.S., which establish embryonic cell cultures, micropropagation, and genetic engineering protocols for successful use in many species of monocots such as the grass A. donax. The plant varieties develop bunches, however, can additionally spread enthusiastically because of its long woody rhizomes which attack regular plant groups under certain natural conditions. Günes and Saygın, 1996 reported that dry matter of biomass yields of 53 t ha-1 was accounted for from wild stands of A. donax in Turkey.

Phytoremediation Potential of Heavy Metals Using A. donax L.

As a perennial plant, Arundo donax L. is widely utilized as energy material and paper pulping and has received considerable attention for remediation of soils polluted by multi-metals due to its capacity of thriving in a various range of adverse conditions with rapid growth and high yield (Lewandowski et al., 2003). The adaptability to extreme soil conditions combined with rapid and vigorous growth makes A. donax an attractive candidate for environmental studies on phytoremediation treatments. The use of plants to remove contaminants from polluted soil and water can be an advantageous strategy, which can also be utilized to remediate metals that usually cannot be efficiently removed.

Numerous studies (Table 2.4) demonstrated that A. donax L. may have a potential utilization for phytoremediation purposes (Papazoglou et al., 2005; Papazoglou, 2007; Mirza et al., 2010; Mirza et al., 2011; Kausar et al., 2012; Yang et al., 2012). Because its transpiration rate is extremely high, A. donax L. can uptake large amounts of nutrients and water from wastes and has a potential for treating saline wastewaters or pollutants (Idris et al., 2012). A. donax is able to efficiently transfer arsenic (As), absorbed from the growing medium and efficiently accumulate it into the plant tissues, showing good tolerance to the presence of metal (Mirza et al., 2010). A. donax is a plant only slightly affected by the presence of metals such as arsenic (As), cadmium

(Cd), nickel (Ni) and lead (Pb) in rhizosphere, and because of this trait it can be a potential crop

for contaminated soil capable of high biomass production in polluted areas (Papazoglou et al.,

2005; Papazoglou, 2007; Papazoglou et al., 2007; Guo and Miao, 2010).

A. donax is able to grow well in trace metal polluted soils, and since these trace metals were not stress factors, they did not inhibit stomatal opening and did not affect the function of the photosynthetic machine of A. donax (Papazoglou, 2007; Papazoglou et al., 2007). Yang et al (Yang et al., 2012) examined the influence of diverse five adjustments such as acetic acid, citric acid, ethylene diamine tetraacetic acid (EDTA), sepiolite and phosphogypsum on (Cd) and lead (Pb).

They found that the concentration of As, Cd, and Pb in shoots of A. donax was remarkably elevated when 2.5 mmol kg-1 acetic acid and citric acid, 5.0 mmol kg-1 EDTA, and 4.0 g kg-1 sepiolite were connected to the soil as contrasted with the control. Moreover, accumulations of arsenic and cadmium were significantly increased under these conditions, whereas the aboveground lead accumulation was noticeably enhanced via adding 4.0 g kg-1 sepiolite and 8.0 g kg-1 phosphogypsum to soil, respectively.

Research has documented that increasing heavy metal concentrations in soil is toxic to the biochemical and physiological plant systems, and on growth (Shanker et al., 2005; Mirza et al.,

2010; Bonanno, 2012). High Cd concentrations have been reported in surface soil adjacent to metal preparing industrial projects ranging from 3.2-1781 mg Cd/kg in Belgium and 26-1500 mg Cd/kg in the USA (Kabata-Pendias, 2010). Concentrations of Ni up to 10,000 mg/kg have been accounted for in soils from some ultra-fundamental molten rocks (He et al., 2005). Papazoglou (2007) tested the tolerance of A. donax plants under a watering system laden with metals. A 2-year pot investigation was held in the field and a mechanized drip watering system framework was built to

furnish every day the plants with fluids of Cd and Ni in concentrations of 5, 50 and 100 mg/L,

besides the control. The concentration of cadmium (Cd) and nickel (Ni) in soil was 973.8 ppm for cadmium and 2543.3 ppm for nickel. The morphological features, which included: quantity of the branches, plant height and fresh and dry weight of branches, leaves, stem, root system, above ground part and total plant did not vary amongst treated and control plants, signifying the high tolerance ability of A. donax to the examined concentrations of cadmium and nickel (Papazoglou et al., 2007). Kausar et al, 2012 evaluated the ability of A. donax growth in chromium, which showed excessive potential for growing in nutrient solution supplemented with increasing concentrations of chromium (Cr) up to 900 µg l-1 for three weeks under greenhouse conditions. In general, results demonstrated that the distribution of chromium in different plant organs was as follows: roots > stems > leaves. These results most likely demonstrate conceivable utilization of the grass A. donax to remediate polluted streams.

Mirza et al. (2010) has evaluated the potential of Arundo donax for phytoremediation of arsenic from hydroponic solutions and concluded that the highest estimated values of

bioaccumulation and translocation factors were BF = 15.00 and TF = 4.93 (Mirza et al., 2011).

Arundo donax (Giant reed) planted in constructed wetlands for polishing high salinity of tannery wastewater showed robust growth and ability to uptake nutrients (Calheiros et al., 2012).

Furthermore, the potential of Arundo donax to remediate chromium contamination was assessed in hydroponic solutions and concluded that both BF and TF factors were above the reference value

(1.0), with the highest BF = 2 and TF = 4, suggesting that Arundo donax might be used to treat wastewater contaminated by chromium (Kausar et al., 2012). There are several environmental factors influencing Cr (VI) reduction in the variety of phytoremediation plants which are biomass

density, initial Cr (VI) concentration, carbon source, organic matter content, pH, temperature,

dissolved oxygen, competing electron acceptors and, soil composition.

Table 2.4: Selected studies applied phytoremediation potential of some heavy metals using A. donax L.

Contaminants Experiments and Conditions Substrate Period of experimental Concentrations (ppm) Reference Cd and Ni Pots (Field) Soil 2 Years (5, 50, 100 mg/L) Papazoglou, (2007)a , Papazoglou et al. (2005; 2007)a As, Cd and Pb Plastic tackle (Greenhouse) Soil 70 days (13.7 – 414mg As/kg) (1.10 – 126mg Gou et al. (2010)b Cd/kg) (52.4 – 2552 mg Pb/kg) As and Hg Pots (Greenhouse) Soil 2 months (150, 250 and 350mg As/kg) (2500, Mirza et al. (2010b)c 3000 and 3500 mg Hg/kg)

As Pots (Greenhouse) Hydroponic 3 Weeks (50, 100, 300, 600 and 1000µg As/L) Mirza et al. (2010b)c As Pots (Field) Soil 3 Weeks (50, 100, 300, 600 and 1000µg As/L) Mirza et al. (2010b)c Cd Pots (Laboratory) Hydroponic 2 months (1 and 2 mg Cd/L) Sagehashi et al. (2011)d As, Cd and Pb Pots (Greenhouse) Soil 3 months (80 mg As/kg, 10mg Cd/kg and Miao et al. (2010a)e 500mg Pb/kg) Cr Pots (Greenhouse) Hydroponic 3 Weeks (50, 100, 200, 400, 600 and 900 µg Kausar et al. (2012)f Cr/L) Al, As, Cd, Cr, Cu, Sampling plants (Field) Sediment and 2 Years (< 0.5 µg Cr/L in water and Bonanno et al. (2012,

35 Hg, Mn, Ni, Pb Water 19.2 mg Cr/kg in sediment) 2013)g

and Zn Cu Tubes (sterile conditions) Hydroponic 6 Weeks (0, 1, 2, 3, 5, 10, and 26.8 mg Cu/L) Elhawat et al (2013)h

Notes: a: These studies proved that high soil cadmium and nickel concentration were not stressed factors, as they did not inhibit stomatal opening and did not affect the the function of the photosynthetic machine of A. donax. b: This study demonstrated that the macrophyte A. donax has the capacity to absorb toxic heavy metals. c: These studies showed that A. donax has the ability to clean up soil contaminated with arsenic and mercury. d: This study evidenced that A. donax has great efficiency for absorbing cadmium in hydroponic solution. e: This study proved that A. donax is able to uptake arsenic, cadmium and lead in contaminated soil by using organic acids that could be considered ideal soil amendments for phytoremediation approach of A. donax. f: This study investigated the efficiency of A. donax to remediate chromium concentration in hydroponic solution. A. donax is a promising plant to absorb Cr. g: This study demonstrated that A. donax can be used as a potential biomonitor of heavy metals in ecosystems. h: Cu removal rate ranged between 96.6 to 98.8 % for BL ecotype and 97 to 100 % for 20SZ ecotype.

Conclusion

The absorption of chromium and other heavy metals by A. donax grown in hydroponic solutions indicates its potential to remediate chromium-contaminated wastewater. The use of A. donax as a phytoremediation plant is cost-effective compared with other commonly used technologies such as electrolysis treatment system, ion exchange methods, and reverse osmosis.

However, extensive research needs to be conducted to reduce some of the limitation of phytoremediation and to increase performance and efficiency. Specifically, further study needs to be done to assess the efficiency of A. donax L. for remediating chromium contaminated soils.

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CHAPTER THREE

ASSESSMENT OF TWO VARIETIES OF ARUNDO DONAX FOR REMEDIATION OF

A CHROMIUM CONTAMINATED SOIL-WATER ECOSYSTEM

Abstract

Chromium is harmful to the environment due to its toxicity and transference through air, food, and water resources to plants, animals, and people. In this study, chromium uptake by two varieties of Arundo donax L. was determined. Arundo donax plants were grown under greenhouse conditions in pots containing a peat moss-based nursery mix and irrigated with synthetic wastewater spiked with dichromate. The treatments included a control (no chromium) and three

-1 doses of potassium dichromate (K2Cr2O7) 0.1, 1.0 and 2.0 mg Cr l applied to plants for one month.

The phytoextraction capacity of A. donax plants by translocation and bioaccumulation were evaluated. Growth parameters and color degree values were measured. Generally, chromium absorption by roots, stems and leaves increased with increasing chromium concentrations. The highest Cr concentration in roots of A. donax (long leaf) was 0.69 ± 0.10 mg kg-1 and 0.22 ± 0.09 mg kg-1 in the stems for the 2.0 mg l-1 treatment. In the case of leaves, the maximum Cr concentration was 0.18 ± 0.06 mg kg-1 at 2.0 mg l-1 Cr concentration. In the case of A. donax (short leaf), Cr concentration in roots was 0.38 ± 0.10 mg kg-1 at 2.0 mg l-1 Cr concentration. While, Cr concentration in stems was 0.09 ± 0.07 mg kg-1 and 0.07 ± 0.06 mg kg-1 in the leaves at 2.0 mg l-1 supplied Cr. There were significant differences (p < 0.05) within plant tissues and among the treatments for both long and short leaf varieties of A. donax. Overall, Cr concentrations translocated to the shoots of tested varieties of A. donax were relatively low compared to plants

that classified as Cr-hyperaccumulators in literature that have TF > 1. Thus, the test varieties of A. donax could not be classified as Cr-hyperaccumulators under these conditions of the experiment.

Introduction

Plants have the ability to uptake various chemical elements from the surrounding ecosystem. Some of these elements have no recognized essential nutritional role, and some are known to be toxic at trace levels. plants are capable of employing numerous approaches for tolerance when exposed to heavy metals (Alloway, 2010; Kadlec and Wallace, 2009). Chromium contamination of sediment, soil, and water resources is a growing global environmental issue.

Even though at low concentrations chromium is a natural component of soil. Anthropogenic activities such as agriculture, industrially processes, and mining have contributed to undesirable toxic levels in the ecosystem (Lewandowski et al., 2006). Chromium contamination might originate from both natural geochemical processes (weathering of rocks) and anthropogenic activities such as agricultural applications (utilization of fertilizers and pesticides), industry processes (combustion of fossil fuels, disposal of wastes, mining and smelting, stain steel production, and tannery factories) (Kabata-Pendias, 2010).

Chrome tanning is used by the leather industry due to the pace of processing, cost- effectiveness, color and better stability of the resulting leather (Hafez et al., 2002). In addition, chromium salts are not completely absorbed by the leather. Thus, high levels of chromium are found in the discharge. Approximate levels in such discharges range from 3 – 350 mg l-1 (Vlyssides and Israilides, 1997) to 2000 – 3000 mg l-1 (Bajza and Vrcek, 2001). If these contaminants are not remediated before discharge they might lead to severe environmental pollution. Conventional

methods such as electrolysis treatment system, ion exchange, and reverse osmosis for remediating these contaminants are technologically complex and expensive. On the other hand, phytoremediation is a well-established and low-cost remediation biotechnology for chromium removal from contaminated soil-water ecosystem.

Research has shown that various harmful chromium species are removed by trees and grasses (Ghosh and Singh, 2005). Phytoremediation is the utilization of macrophyte plants to effectively remediate contamination in the ecosystem, including various strategies for the degradation, immobilization, or removal of contaminants (Lone et al., 2008; Mirza et al., 2010).

Phytoremediation is a promising method that can be cost-effective, environmentally friendly, and sustainable (Malik, 2007; Kausar et al., 2012). Plant species have the ability to uptake heavy metals, accumulate them into their roots and then translocate them to stems and leaves.

Specifically, findings of significant accumulation of trace elements by aquatic macrophytes suggests that aquatic macrophytes can be useful as contamination absorbing organisms (Mishra et al., 2014).

Arundo donax L. is one of the most utilized macrophytes because of its capacity for taking up heavy metals (Papazoglou et al., 2007; Mirza et al., 2011). Arundo donax L. is a tall perennial rhizomatous grass (Poaceae family), natural to locations of freshwater in Eastern Asia and

Mediterranean; Arundo donax L. has extensive application in paper manufacturing and energy production (Papazoglou, 2005; Borin et al., 2013). Worldwide, Arundo donax L. seeds are unproductive and thus usually propagates via rhizomes (Angelini et al., 2005; Balogh et al., 2012).

The macrophyte Arundo donax L. shows the unique biological ability to capture and accumulate toxic metals from contaminated sediment, soil, and water resources with no noticeable effect on

its health and growth (Papazoglou, 2005). The potential of A. donax as a phytoremediation plant has already been evaluated in several studies. It was grown in hydroponic solutions containing chromium concentrations up to 0.9 mg l−1 (Kausar et al. 2012), arsenic concentrations up to 1.0 mg l−1 (Mirza et al. 2011) and cadmium concentrations up to 2.0 mg l−1 (Sagehashi et al. 2011).

Only one previous study was found in which A. donax grew on soil contaminated by arsenic, cadmium and lead (Miao et al. 2012).

A basic reason for using A. donax is that there are different varieties that are producing different amounts of biomass. Thus, assessment of these varieties of A. donax for remediation of chromium contamination is important because although much research has been conducted on A. donax, little literature is available about the efficiency of different varieties. Elhawat et al. (2014) evaluated the capacity of two varieties of A. donax L., American (BL) and Hungarian (20SZ), grown in hydroponic solutions having various copper (Cu) concentrations up to 26.8 mg l−1, and results showed that the 20SZ variety could take up more Cu (79 mg kg-1 d.w) than the BL variety

(48 mg kg-1 d.w).

This study will evaluate the capacity of two varieties of Arundo donax to bioaccumulate

Cr. The hypothesis was that the Arundo donax with more biomass would be able to extract more chromium (Cr) than A. donax with low biomass. The rationale for this hypothesis is that the varieties of A. donax with more biomass has more roots, taller stems, and a number of leaves as compared to the varieties with less biomass. Therefore, more biomass is available to former varieties to increase the ability of plant to absorb and translocate Cr to the aerial portion of the plant and then accumulation. This issue is significant because a lot of research has been carried on

A. donax, but little literature is available about the efficiency of different varieties. It is also

important because selection and cultivation of more potent A. donax varieties under field conditions can enhance the soil-water system treatment efficiency. Consequently, the specific objective of the present research was to assess the Cr uptake capacity of two varieties of A. donax grown in a peat moss-based nursery mix and irrigated with a Cr-spiked aqueous solution.

Materials and methods

Plant material

The plant materials utilized for this research were two varieties of Arundo donax L. with the aim to assess their capacity to absorb chromium from nursery soil irrigated with synthetic wastewater. The plants were ordered from Tennessee (TN tree nursery, USA) http://www.tnnursery.net/. The two varieties of the macrophyte Arundo donax L. are (i) Arundo donax L., with long leaves and high biomass. (ii) Arundo donax L., with short leaves and low biomass.

Nursery mix of peat moss

The nursery mix of peat moss used in this study was obtained from the Agriculture

Research Center and Plant Growth Facilities, Washington State University. Peat moss was selected due to its high moisture-holding capacity and low pH (4.7) in water. The nursery mix consisted of

50% peat moss and 50 % perlite. Peat moss has a high organic matter that plays an important role in the bioavailability and mobility of Cr in soil (Banks et al., 2006). The physical and chemical characteristics are presented in (Table. 3.1).

Table 3.1. Physicochemical properties of the nursery mix used in this study.

Characteristic Value Soil water content, % 12.56 ± 0.43

pH 4.74 ± 0.21

Electrical conductivity (EC) ds m-1 1.42 ± 1.10

Nitrate-nitrogen (NO3-N) ppm 58.54 ± 2.00

Ammonium nitrogen (NH4-N) ppm 34.38 ± 1.91

Copper (Cu) ppm 2.10 ± 0.05

Zinc (Zn) ppm 1.90 ± 0.16

Chemical reagents and equipment

An automatic drip watering system (Clabber Spa, Italy) was set to irrigate pots of Arundo at a rate of 120 mL d-1 using synthetic wastewater spiked with Cr. The following analytical grade reagents were utilized in this study: hydrogen peroxide 30% (H2O2) (J T Baker, USA), inorganic

-1 chromium standard 1000 μg mL (Agilent company), nitric acid (HNO3) (J T Baker, USA), and potassium dichromate (K2Cr2O7) (Johnson Matthey, UK). Mili-Q (18.2 MV cm) water prepared by passing de-ionized water through a nano-pure treatment system used for the preparation of necessary chemical solutions. An analytical balance (accurate to 1.0 mg or better) was used to measure masses, while an Inductively Coupled Plasma Mass Spectrometry (Agilent 7500cx ICP-

MS) was used for chemical analysis of the plant material.

Experimental procedure

The plant growth experiment was carried out under greenhouse conditions at the

Agriculture Research Center and Plant Growth Facilities, Washington State University (WSU)

Pullman, WA, USA. Bare roots of the two varieties of Arundo donax L. were planted in pots filled with nursery mix. Roots measuring 0.10 m in length were selected for uniformity. Each selected root was transplanted to a 20 cm x 24 cm pot, with each pot containing about 2.5 kg air-dried nursery mix.

During growth, plants were irrigated with tap water (350 mL-pot) twice a week and allowed to grow for about four to five weeks. After that, for one month, plants were exposed to chromium through synthetic wastewater. A. donax L. varieties were irrigated every day with synthetic wastewater (120 mL-pot) containing the following concentrations of chromium: 0 (control), 0.1,

1.0 and 2.0 mg Cr L-1. The cultivated pots were set under white fluorescent lamps (40 µmol m2 s-

1photon flux density), and 14/10 h light/dark cycle at temperatures of 25ºC during the day and

20ºC during the night. The average relative humidity about 70%.

Chromium was supplied as potassium dichromate (K2Cr2O7). An automated drip irrigation system with constant flow drippers was used for irrigation to deliver synthetic water to the plants at desired concentrations. Each treatment had four replicates. A completely randomized block experimental design (CRBD) was carried out using three replications for each of the four treatments: 0 (control), 0.1, 1.0, and 2.0 mg L-1 Cr, each having one plant per pot. The treatments included control (no chromium) and three doses of potassium dichromate.

Evaluation of A. donax L. growth, development, and color parameters

The existence of chromium Cr in the exterior ecosystem might influence the roots and the aerial parts growth and development pattern of the plant (Shahandeh et al., 2000; Shanker et al.,

2005). The height of A. donax L. varieties and the nodes number were recorded every week during growing periods. Similar to other trace elements, high concentrations of chromium might affect photosynthesis, reducing the efficiency of plants and eventually leading to death (Panda et al.,

2005; Elhawat et al., 2014). Thus, leaf greening was evaluated as a color degree of the leaves of

A. donax L surface using a colorimeter MINOLTA Chroma Meter (model CR400) that had been standardized, before each measurement, against a white tile (L = 96.78, a = −0.67, b = 2.85). For each measurement run, colorimetric measurements were performed every seven days on fresh leaves’ surfaces. The results were stated as L (brightness/darkness), a (greenness/redness), b

(yellowness/blueness). From the Hunter and a & b values, the hue angle was calculated according

to the equation below. Hue angle (H0) considered the qualitative quality of color, is the characteristic according to which color have been traditionally defined as reddish, greenish, etc. It is used to determine the difference of a particular color regarding the grey color with the same lightness (Barreiro et al. 1997; Martinez and Whitaker, 1995).

푏 퐻 = 푇푎푛−1 ( ) 푎

Where H is the hue angle, a is the (greenness/redness) and b is (yellowness/blueness).

When a is negative which means the leaves are green (- a), the hue angle should be equal 90 < H

≤ 180.

Chromium analysis

At the end of the experiment, both varieties were harvested and soil samples were collected.

Before chromium analysis, plant tissues were separated into roots, stems, and leaves. Plant tissues were carefully washed with tap water, rinsed with distilled water to remove water-soluble chromium and nursery mix particles on plant surfaces, and then air-dried. Afterward, all plant samples were oven dried at 60°C for 24 h. Soil samples were air dried and then oven dried at 60°C for 24 h. The plant tissues and soil samples were ground and homogenized to a fine powder in a mortar to ensure uniform element distribution. Dried soil samples were sieved through a 2mm diameter sieve. All plant tissues and soil samples followed the same digestion procedure and analysis. Wet digestion was carried out using a hot plate digestion apparatus. A sample of 0.300 to 0.450 g was loaded into a glass digestion tube. To this tube, 3 mL of 30% hydrogen peroxide was added and allowed to react with the sample for 24 h. Thirty minutes before digestion, 10 mL of the concentrated (69 - 71%) nitric acid was added to each reaction tube along a glass rod to

reduce bubbles. Samples were digested using a programmed hot plate at 60°C for 2 h (USEPA

1996; Kalra, 1997); after cooling at room temperature, the digested samples were filtered and then diluted to volume (25 mL) using Milli-Q Millipore (double deionized water). Analytical calibration using standard solutions of chromium were prepared over the range of 0 – 1000 µg L-

1 using suitable serial dilutions of chromium stock solution. Chromium concentrations in plant tissues and soil samples were analyzed and calculated based on a dry weight basis using inductively-coupled Plasma mass spectrometry ICP-MS (Agilent 7500cx ICP-MS). The ICP-MS operation conditions are presented in (Table 3.2).

Table 3.2. Operating conditions for the Agilent 7500 cx ICP/MS

Rf-power (W) 1500

Ar Nebulizer gas flow rate (L/min) 0.6 - 0.9

Make-up gas flow rate (L/min) 0.19

Sample uptake (ml/min) 0.5

Sampling depth (mm) 8

Sampler (mm, Pt) 1.0

Skimmer (mm, Pt) 0.4

Ion optics Optimized for sensitivity of 7Li,24Mg, 59Co,

89Y, 140Ce and 205Ti

Isotopes measured 9Be, 51V, 52Cr, 53Cr 55Mn, 57Fe, 59Co, 60Ni,

65Cu, 66Zn, 75As 82Se, 95Mo, 98Mo, 110Cd,

111Cd, 138Ba, 205Ti, 206Pb, 207Pb and 208Pb

Reaction gas flow rate (ml/min) 1.2

Replicates 3

Phytoremediation efficiency

The phytoremediation efficiency of A. donax was assessed using bioaccumulation and translocation factors. Bioaccumulation factor (BF) is the Cr mass concentration ratio in the roots of A. donax to the Cr mass concentration in the soil (Yoon et al., 2006). The translocation factor

(TF) is the Cr mass concentration in the aerial parts of A. donax to the Cr mass concentration in the plant roots (Cui et al., 2007).

Statistical analysis

Data analysis was performed using Minitab 16 © (mean values and standard deviation) from three individual experiments. The data obtained was subjected to a general linear model analysis of variance (ANOVA) for assessing the significance of quantitative changes in the variables as a result of chromium treatments and their respective interaction with varieties of plant.

When a significant difference was observed between treatments, multiple comparisons were made using Tukey’s test. Significant differences were accepted at the level P < 0.05.

Results and discussion

Chromium absorbed by tissues of A. donax L.

Chromium concentrations in the roots, stems, and leaves of each A. donax variety are presented in (Figs. 3.1a-b). Chromium concentrations in various plant tissues generally increased linearly with increasing chromium applied in irrigation water. The increases in the chromium concentration in synthetic wastewater significantly increased (p < 0.05) the Cr absorption in roots, stems and leaves in both varieties of A. donax evaluated in this study. For chromium accumulation

in different A. donax tissues, our finding indicated that A. donax was able to accumulate more chromium in the roots than in the shoots (stems and leaves). Figs. 3.1a-b shows that both varieties exhibited the same tendency where the order of chromium accumulation, in descending order was roots > stems > leaves, these results are in line with those of most plant uptake studies (e.g. Maine et al., 2004; Zayed and Terry, 2003). Higher chromium concentration in the irrigation water increased the plants’ uptake of chromium, also consistent with previous studies (Kausar et al.,

2012; Windham et al., 2003).

A. donax L. (long leaf) produces higher biomass than A. donax L. (short leaf). Hence more biomass is available to long leaf varieties to uptake and accumulate Cr than short leaf varieties.

Our findings indicated that the amount of Cr concentration absorbed by both varieties of A. donax was lower comparing to Cr absorbed by A. donax in hydroponic solutions. The low rate of Cr absorbed by A. donax may have been due to the short term of the experiment. because the time of the experiment was just four weeks, expanding the time of the experiment might enhance the plants’ uptake concentration of chromium (Dragoni et al., 2015).

Organic matter plays a significant role in solubility and bioavailability of chromium in soils

(Banks et al., 2006). Organic matter increases the sours of protons and organic carbon that might be the major factors improving the reduction of the mobile Cr(VI) to the relatively immobile Cr

(III) (Bolan et al., 2003) Solubility of Cr in growth medium is predominantly controlled by the organic matter content, oxidation state of Cr, and pH (Ghosh & Singh, 2005). Because our nursery mix was 50% peat moss, it is possible that some fraction of Cr(VI) was reduced to Cr(III) by organic matter (Banks et al., 2006). Cr(III) forms insoluble (hydr)oxide and this reaction could have limited uptake.

Figure 3.1: Respective Cr concentrations in plant tissues: (A) long leaf variety, (B) short leaf variety.

Chromium bioaccumulation and translocation in both varieties of A. donax L.

The bioaccumulation and translocation factors (BF and TF) for both long leaf and short leaf varieties of A. donax L. are presented in (Figs. 3.2a-b) show that both bioaccumulation and translocation values increased as a function of Cr concentration in both varieties. Overall, BF and

TF values for both varieties of A. donax were in the range of 0.14 – 0.58. These results were in good agreement with those from previous study of Bonanno (2012) that were in the range of 0.08

– 0.58. Statistically significant differences in BF and TF Values (p < 0.05) were observed between the two varieties of A. donax. Although both varieties of A. donax accumulated Cr in their roots and translocated a portion to aerial parts, the values were below that expected of A. donax proficiency, indicating concerns with experimental conditions.

As can be seen, the BF and TF values (Figs. 3.2a-b) fell far below the reference value ≥ (1)

(Terry and Banuelos, 1999), and ratios of BF and TF below this value show that the varieties of A. donax used here were not Cr-hyperaccumulators by Terry and Banuelos’ criterion. Data obtained in previous studies (Elhawat et al., 2014; Kausar et al., 2012; Mirza et al., 2010) using A. donax L. grown in hydroponic solutions indicated that both BF and TF values were above the reference value (1.0). According to Kausar et al. (2012), A. donax grown in hydroponic solutions having various concentration of Cr up to 0.9 mg l-1 produced values of BF and TF were in the range of

2.0–4.0, respectively. Therefore, the varieties in our studies fail to be classified as hyperaccumulators and did not perform as well as A. donax used in the Kausar study.

Numerous factors can control accumulation and bioavailability of chromium including growth medium, environmental conditions, plant species, metal speciation, and redox state

(Kabata-Pendias, 2010). The composition of organic materials, that is mostly composed of hemi celluloses, humic substances and lignin, bearing polar functional groups, such as, carboxylic acids and phenolic hydroxides, which can be involved in chemical bonding. In addition to the characteristics of polar, peat has a large surface area and a highly porous structure. Therefore, it can be utilized in remediating various contaminants such as heavy metals, products of petroleum and surfactants from contaminated soil-water systems (Novoselova and Sirotkina, 2008). Harvest time may also affect the uptake of Cr by A. donax. Thus, this entails that long-term harvest would be required to correct the experiment and improve the efficacy of A. donax in remediating contaminated soil (Dragoni et al., 2015).

A - Bioaccumulation factor of A. donax variety (Long and short leaf)

0.4 A. donax (Long leaf) A. donax (Short leaf)

0.3

0.2

0.1

Bioaccumulation factor value 0.0

0.0 0.5 1.0 1.5 2.0 2.5 Chromium concentrations (mg/L)

B - Translocation factor of A. donax variety (Long and Short leaf)

0.7

A. donax (Long leaf) 0.6 A. donax (Short leaf)

0.5

0.4

0.3

0.2

Translocation factor value 0.1

0.0

0.0 0.5 1.0 1.5 2.0 2.5 Chromium concentrations (mg/L)

Figure 3.2: (A) Relative bioaccumulation factor (BF) and (B) translocation factor (TF) of A. donax variety (long and short leaf) at different applied chromium concentrations.

Growth and development of A. donax L

Plant growth and development might be affected by heavy metals exposure. The number of nodes and plant height throughout the period of the experiment as a function of applied chromium concentrations are presented in Figs. 3.3 and 3.4. The capacity of the plants to remain healthy and continue to grow is a major factor in the assortment of plant species for phytoremediation (Mant et al., 2005). The results revealed that both varieties of A. donax were healthy. Generally, the growth and development of A. donax based on the height and number of nodes recorded every week increased with time throughout the period of the experiment. these results are in good agreement with those in a previous study by Papazoglou et al., (2005). There was significant difference (P < 0.05) between the height and nodes number of two A. donax varieties (Figs. 3.3a-b & Figs. 3.4a-b). Overall, the two A. donax varieties grew without exhibiting symptoms of Cr toxicity. The growth of the A. donax varieties were not significantly affected compared to control plants. Similar height and number of nodes increases were also observed in previous studies (Papazoglou et al., 2005; Mirza et al., 2010; Kausar et al., 2012).

A - A.donax (Long leaf)

1.2 Control 0.1 1.0 1.0 2.0

0.8

0.6

0.4

Height Height of plant (m)

0.2

0.0 0 5 10 15 20 25 30 Time (day)

B - A. donax (Short leaf)

1.2

Control 0.1 1.0 1.0 2.0

0.8

0.6

0.4

Height Height of plant (m)

0.2

0.0 0 5 10 15 20 25 30 Time (day)

Figure 3.3: (A) The height of A. donax L (longleaf) and (B) The height of A. donax L.

(shortleaf) at different applied chromium concentrations.

A - A.donax (Long leaf)

Control 30 0.1 1.0 2.0 25

Nodes number 10

0 0 5 10 15 20 25 30 Time (day)

B - A. donax (Short leaf)

Control 30 0.1 1.0 2.0 25

Nodes number

0 0 5 10 15 20 25 30 Time (day)

Figure 3.4: (A) The Nodes number of A. donax L (Longleaf) and (B) The Nodes number of A. donax L. (shortleaf) at different applied chromium concentrations.

Greenness measurements

The effect of Cr treatments on the color of leaves rate of both A. donax varieties is presented in Table 3.3 In leaves of both varieties of A. donax green color rate exhibited similar responses in respect to the exposure of A. donax to Cr treatments. The results were specified as hue angle (H0).

The hue angle (H) of leaves was in the range from 135.62 – 139.63 and from 134.13 – 139.81

Table 3.3 for both of long and short leaves of A. donax respectively. A. donax was able to accumulate the chromium into its roots and to translocate chromium in the stems and leaves above the root concentration without showing any sign of toxicity. Both varieties of A. donax remained almost unaffected by the presence of Cr in the nursery mix and no statistically significant differences were evident. Throughout the experiment period, both plant varieties of all treatments revealed no damaging or symptoms of toxicity and increased in photosynthesis (Maine et al., 2004;

Papazoglou et al., 2005). Green rate of leaves in both varieties of A. donax showed similar responses in respect to the exposure of plants to Cr treatments (Tiwari et al., 2009; Rodriguez et al., 2012).

Table 3.3. Color parameters of A. donax L. leaves at various chromium treatments

Long leaf Short leaf

Cr (mg l-1) Time (day) (H0) (H0) 1 135.88 135.31 8 135.63 137.65 Control 15 136.48 138.23 22 138.00 138.80 29 138.58 139.81

1 136.01 134.13 8 136.46 137.63 0.1 15 136.31 137.90 22 138.03 138.48 29 139.04 139.58

1 136.02 134.55 8 136.12 136.59 1.0 15 136.37 137.81 22 137.84 138.14 29 139.62 139.15

1 135.42 135.52 8 135.96 136.18 2.0 15 136.31 136.53 22 138.56 138.92 29 139.49 139.11

Conclusion

In this experimental study, two varieties of A. donax L (short and long leaf varieties), were investigated for the phytoremediation to Cr-contaminated soil-water ecosystems. The results indicated that increases in chromium concentration in peat moss significantly increased chromium mass concentration in the tissues of the plant (roots, stems, and leaves). BF and TF values were below reference value (1.0). therefore, the test varieties of A. donax could not be classified as Cr- hyperaccumulators under these conditions of the experiment. The performance of the experiment might be affected by the short duration of experiment and organic matter in nursery mix.

Additional studies are required to improve the experiment conditions such as the long period of the experiment and soil properties to enhance the efficacy of A. donax for remediation of Cr contaminated soil.

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CHAPTER FOUR

EFFECT OF pH ON THE REMEDIATION OF CHROMIUM CONTAMINATED SOIL

BY ARUNDO DONAX L.

Abstract

A study was designed to evaluate the effects of soil pH on the phytoextraction potential of chromium by Arundo donax. A pot experiment was conducted with Arundo donax roots planted in 2.5 kg of nursery mix of peat moss amended with potassium carbonate to pH values of 4.71

(control), 5.5, 7.0, and 8.5. Each pot was irrigated with synthetic wastewater containing 0.0 ppm

(control), 1.0 ppm and 2.0 ppm of Cr. The study was carried out for a period of 4 weeks under greenhouse conditions. Physical-chemical properties of the nursery soil were determined using standard methods. Root, leaf, and stem parts of A. donax were analyzed for Cr uptake after 4 weeks. The results of this study revealed that Cr content in plant tissues was higher at low soil pH

(4.71) than at high soil pH (8.5). The plants accumulated significant Cr in the roots (0.71 mg kg-1) than either in the stem (0.36 mg kg-1) or leaves (0.26 mg kg-1) at soil pH 4.71. The concentration of Cr in the roots was (0.43 mg kg-1), while the concentrations in the stem and leaves were 0.12 mg kg-1 and 0.05 mg kg-1, respectively, at soil pH 8.5 and 2.0 mg l-1. The phytoremediation proficiency of the A. donax was also evaluated in terms of its bioaccumulation factor (BF) and translocation factor (TF). BF values were in range (0.08 – 0.38) and TF values were in range (0.01

– 0.51). This study indicates that selection of appropriate macrophytes plants for potential applications in phytoremediation of contaminated soil and water by heavy metal needs further focus regarding interactions among the uptake performance of plants, soil pH, and heavy metal

translocation. The results obtained further suggest that A. donax could accumulate more Cr in low pH than in high pH.

Introduction

Contamination of soil and water by heavy metals is one of the most significant environmental issues all over the world. Indeed, heavy metals are signiﬁcantly toxic to humans and animals (Alloway et al., 2013). Chromium is considered one of the most toxic elements with respect to human, ecological, and animal health, as well as aquatic ecosystems (Oliveira, 2012).

Chromium species commonly found in soil and water are the trivalent (Cr III) and the hexavalent

(Cr VI) (Avudainayagam et al., 2003). Chromium toxicity depends on its concentration and chemical form, with Cr (VI) being more toxic than Cr (III). The latter is mainly due to the high solubility of Cr (VI), which enhances bioaccumulation. The principal pathways for human exposure are air, soil, and water contaminations as well as consumption of contaminated food that bioaccumulate Cr (Reilly et al., 2008).

Several conventional methods currently applied to remediate chromium contaminated soil and water include: physical-chemical extraction, ion exchange, precipitation, reverse osmosis membrane, land filling, and soil removal, washing, and stabilization (Hawley et al., 2004).

Unfortunately, these methods also lead to secondary pollution and may be very costly when applied to large contaminated sites (Madhavi et al., 2013). Consequently, phytoremediation is a promising and more attractive biotechnology for remediating agricultural and industrial wastewaters contaminated with Cr as well as other toxic heavy metals (Sharma and Pandey 2014).

Plant species accumulate heavy metals in their roots and then translocate them to the steam and leaves. In last 30 years, plant uptake of Cr has received considerable attention. Research with

various plant species has shown the capability for soil and water remediation, with Arundo donax

L. being at the forefront. It is anticipated that the current progress in biotechnology will improve the performance of promising hyperaccumulators by translocating heavy metals hyperaccumulating genes from the roots to the stems and leaves to produce cultivable species

(Lone et al., 2008).

Arundo donax L. shows distinctive functional features in that it easily accumulates and concentrates toxic heavy metals from polluted soil, sediment, and water with no considerable destruction to its growth. It is also one of the commonly employed plant species for phytoremediation of heavy metals bioaccumulation because of its efficiency in up-taking pollutants which are not easily biodegraded. To remediate contaminated soil with Cr, it is essential to consider factors affecting Cr phytoremediation. There are several environmental factors that affect the solubility and bioavailability of Cr as well as the efficacy of phytoremediation. Factors influencing Cr and other metals uptake by plants include the: soil pH, cation exchange capability, clay content, organic matter, and the existence of other ions; soil pH being the most important

(Kabata-Pendias, 2010., Loeppert et al., 2003).

Soil pH affects metal sorption to soil components, therefore both phytotoxicity and uptake of plant can be effected by soil pH (Eleftheriou., 2013). In addition, soil pH controls the bioavailability, solubility, and hydrolysis of metal hydroxides, and influences ion-pair construction and solubility of organic matter, as well as surface charge of iron (Fe), manganese (Mn), and aluminium oxides (Al-oxides), organic matter, and clay edges (Kabata-Pendias, 2010). The mobility and bioavailability of some heavy metals in soil are generally low, particularly in high pH soils. This study was triggered by the latter and because the interaction between plant species

and the concentration of heavy metals in soil at different pH levels is presently unclear. The

– chromate (HCrO4 ) ions are the most bioavailable species of chromium found in soil. They can be removed via numerous plant species and easily infiltrate out into the deeper layers of soil resulting in contamination of groundwater. Some slight amounts of Cr(VI) are bound in soils, depending on the composition metal and soil pH (Stewart et al., 2003). Elangovan et al., 2008 reported that the metal binding positions are deprotonated, then the mobility and bioavailability of anion binding locations decreases when the solution pH increased. In addition, Fomina and Gadd, 2014 reported that at low soil pH values, the protonation of plant binding spots was signiﬁcant, and then improved the mobility and bioavailability of anion binding positions arises; in this circumstance, the uptake of metal cations like aluminium Al+3, cadmium Cd+2, copper Cu+2, nickel Ni+2, and mercury Hg+2 and frequently decreases due to the competition from dissolved cations and solution protons. On

- -2 the contrary, species of metal anionic like hydrogen chromate HCrO4 , chromate CrO4 , and

-2 dichromate Cr2O7 , uptake seems to be improved.

This component of the study will evaluate the effect of pH on the remediation of chromium contaminated nursery mix by Arundo donax. The hypothesis is that the efficiency of chromium uptake by Arundo donax improves with low to moderate pH. The rationale for this hypothesis is that plant bioavailable Cr exists in soil in low pH range of 4.0 – 7.0. However, in very acidic or very basic soils the Cr is cationic or anionic, respectively. This charged Cr speciation in held tightly on the soil particles and is unavailable to plant. Research needs to be carried about this issue because it can bring forth information about primary treatment of wastewater prior letting it out in the environment. Consequently, the main objective of this study was to evaluate the influence of soil pH on the uptake of Cr A. donax L. grown in peat moss irrigated with synthetic wastewater

containing different concentrations of Cr. The effects of pH on bioaccumulation and translocation of chromium in plant tissues were also calculated.

Materials and methods

Nursery mix characterization

The growth substrate used in this study was nursery soil obtained from Agriculture

Research Center and Plant Growth Facilities, Washington state university. Peat moss was selected due to its high moisture-holding capacity and low pH (4.7) in water. The nursery mix consisted of

50% peat moss and 50 % perlite. Peat moss has a high organic matter that plays an important role in the bioavailability and mobility of Cr in soil (Banks et al., 2006). Peat moss is a composition of organic materials, that is mostly composed of hemi celluloses, humic substances and lignin, bearing polar functional groups, such as, carboxylic acids and phenolic hydroxides, which can be involved in chemical bonding. In addition to the characteristics of polar, peat has a large surface area and a highly porous structure. Therefore, it can be utilized in remediating various contaminants such as heavy metals, products of petroleum and surfactants) from contaminated soil-water systems (Novoselova and Sirotkina, 2008). The physical and chemical characteristics of nursery mix are given in (Table 4.1).

Table 4.1: Physicochemical properties of the nursery mix used in the experiment.

Characteristic Value Soil water content, % 11.86 ± 1.52 pH 4.71 ± 0.33

Electrical conductivity (EC) ds m-1 1.19 ± 0.18

Nitrate-nitrogen (NO3-N) ppm 58.54 ± 2.00

Ammonium nitrogen (NH4-N) ppm 34.38 ± 1.91

P(ppm) 42.22 ± 1.57

Cu(ppm) 2.10 ± 0.05

Zn(ppm) 1.90 ± 0.16

Amendment of nursery mix pH

Different quantities of potassium carbonate (K2CO3) were added to adjust soil pH to desired levels based on a preliminary pH value (Table 4.2). Soil pH was monitored periodically by taking 10 g soil for measuring pH. The soil was thoroughly mixed every day to ensure equal distribution of potassium carbonate (K2CO3). Incubation was terminated when pH did not change for 3 consecutive weeks. Next, 500 mL of deionized water was added to each pot (2.5 kg soil) to leach salts from nursery soil. This process was repeated several times. The total amount of water added (2.5 L) was equal to more than 3 times the total soil pore volume in each pot (Small, 1995).

Table 4.2: Amount of K2CO3 needed to reach target nursery mix pH.

Initial peat pH K2CO3 g/Kg soil Target soil pH E.C 4.71 0 Control 1.19 ± 0.31 ds/m

4.71 6 5.5 1.35 ± 0.64 ds/m

4.71 36 7.0 4.86 ± 1.10 ds/m

4.71 120 8.5 7.68 ± 0.95 ds/m

Plant material

The plant material used for this study was the macrophyte of Arundo donax L. with the aim to evaluate the effects of soil pH on its uptake of chromium from nursery soil irrigated with synthetic wastewater. The plant was ordered from Tennessee (TN tree nursery, USA) http://www.tnnursery.net/.

Reagent and equipment

An automated Drip Watering System (Claber spa, Italy was set to irrigate pots of Arundo at a rate of 120 mL d-1 using synthetic wastewater spiked with Cr. An analytical balance (accurate to 1.0 mg or less), a hot plate for sample digestion, and Inductively Coupled Plasma Mass

Spectrometry (Agilent 7500cx ICP-MS) were also used for Cr analysis in plant tissues. Analytical grade reagent of hydrogen peroxide 30% (H2O2) (J T Baker, USA), analytical grade of inorganic chromium standard 1000 μg/ml (Agilent company), Mili-Q (18.2 MV cm) water, were used by passing de-ionized water through a nano-pure treatment system in combination with the analytical reagent-grade chemicals and analytical reagent grade of nitric acid (HNO3) (J T Baker, USA), potassium carbonate (K2CO3), and analytical reagent grade of potassium dichromate (K2Cr2O7)

(Johnson Matthey, UK).

Experimental procedure

The plant growth experiment was carried out under greenhouse conditions at the

Agriculture Research Center Plant Growth Facilities, Washington State University (WSU)

Pullman, WA, 99164. Bare roots of Arundo donax L. were ordered from the TN tree nursery,

Tennessee, USA and then planted in pots filled with nursery soil. Roots measuring 0.10 m in length

were selected for uniformity. Each selected roots was transplanted to a 20 X 24-cm pot. Each pot contained about 2.5 kg of homogenized air-dried nursery soil. During growth, plants were irrigated with tap water twice a week and allowed to grow four to five weeks. After that, for the period of one month, A. donax was exposed to chromium through synthetic wastewater. A. donax L. was irrigated every day with synthetic wastewater (120 ml-pot) containing different concentrations of chromium, whose concentrations in synthetic wastewater were 0 (control), 1.0, and 2.0 mg L-1.

Chromium was supplied as potassium dichromate K2Cr2O7. An automated drip irrigation system with constant flow drippers was used for irrigation to provide plants with aqueous solutions containing chromium at desired concentrations. Each treatment had four replicates. Experiments were carried out in a Randomized Complete Block Design (RCBD), replicated four times using individual pot size of 20 X 24 cm under greenhouse conditions; each having one plant per pot. The treatments included control (no chromium) and two doses of potassium dichromate (VI) K2Cr2O7,

0.0 (control), 1.0, 2.0 mg L-1. A. donax roots were planted in nursery mix for one month under greenhouse conditions. The cultivated pots were set under white fluorescent lamps (40 µmol m2 s-

1 photon flux density), and 14/10 h light/dark cycle and temperatures of 25ºC during the day and

20ºC or less during the night. The average relative humidity was recorded to be approximately

70%.

Chromium analysis

The plants were harvested and peat moss samples were collected after the one month of Cr treatment. Before chromium analysis, plant tissues were preliminarily separated into roots, stems, and leaves. The plants’ tissues were carefully washed with tap water and rinsed with distilled water

to remove any solid particles from the peat moss adhering to the root and then air-dried. The plants’ samples were then oven-dried at 60°C for 24 h. Soil samples were air dried and then oven dried at

60°C for 24 h. The plants’ tissues and soil samples were grounded and homogenized to a fine powder to assure uniform distribution of the elements. Dried soil samples were also sieved through a 2-mm diameter sieve.

All plant tissues and soil samples followed the same digestion procedure and analysis. Wet digestion was carried out using a hot plate. An amount of 0.300 – 0.450 g of each sample (± 0.005 g) was loaded into a glass digestion tube. To this tube, 3 mL of 30% analytical grade of hydrogen peroxide was added and allowed to react with the sample for 24 h. Thirty minutes prior to digestion, 10 mL of concentrated (69 - 71%), analytical grade nitric acid was added to each reaction tube via a glass rod to reduce bubbles. Samples were digested on a programmable hot plate set at

60°C for 2 h (USEPA 1996; Kalra, 1997). The digested samples were cooled at room temperature, filtered, and then diluted to volume (25 mL) using Milli-Q Millipore (double deionized water).

Analytical calibration standard solutions of chromium were prepared over the range of 0 - 1000

µg l-1 via suitable serial dilutions of chromium stock solution obtained from the Agilent company.

Chromium concentrations in plant tissues and soil samples were analyzed and calculated based on a dry weight basis using inductively-coupled plasma mass spectrometry ICP-MS (Agilent 7500cx

ICP-MS). The ICP-MS operation conditions are given in (Table 4.3).

Table 4.3: Operating conditions for the Agilent 7500 cx ICP/MS.

Rf-power (W) 1500 Ar Nebulizer gas flow rate (L/min) 0.6 - 0.9 Make-up gas flow rate (L/min) 0.19 Sample uptake (ml/min) 0.5 Sampling depth (mm) 8 Sampler (mm, Pt) 1.0 Skimmer (mm, Pt) 0.4 Ion optics Optimized for sensitivity of 7Li,24Mg, 59Co, 89Y, 140Ce and 205Ti Isotopes measured 9Be, 51V, 52Cr, 53Cr 55Mn, 57Fe, 59Co, 60Ni, 65Cu, 66Zn, 75As 82Se, 95Mo, 98Mo, 110Cd, 111Cd, 138Ba, 205Ti, 206Pb, 207Pb and 208Pb Reaction gas flow rate (ml/min) 1.2 Replicates 3

Statistical analysis

Data analysis was performed using Minitab 16© (mean values and standard deviation) from three individual experiments. The data obtained was subjected to general linear model analysis of variance (ANOVA) for assessing the significance of quantitative changes in the variables as a result of chromium treatments and their respective interaction with plant and soil pH influence. When a significant difference was observed between treatments, multiple comparisons were made by Tukey’s range test and 95.0% confidence. Significant differences were accepted at the level P ≤ 0.05.

Results and discussion

Effects of soil pH on the uptake of Cr by A. donax L.

The effect of pH on the uptake of Cr by A. donax is presented in Figs. 4.1a-b, which show the chromium concentrations in roots, stems, and leaves of A. donax L. The concentration of Cr found in different tissues of A. donax linearly decreased with increasing of solution pH (Figs. 4.1a- b). In all cases roots showed chromium concentration significantly higher than Cr concentrations found in the stems and leaves of A. donax. In this study, the analysis of chromium accumulation revealed statistically significant differences and positive correlations among treatments and Cr concentration in the plant tissues, but negative correlations between uptake of Cr and pH.

Data in the current study show that plants highest and lowest uptake of Cr by A. donax L. occur at pH 4.71 and 8.5 respectively, in both 1.0 and 2.0 mg Cr/L (Figs. 4.1a-b & 4.2), which was consistent with results obtained in previous study (Ponce et al., 2015) who considered the effect of solution pH (4.0, 6.0 and 7.6) on the uptake of Cr by Salvinia minima grown in synthetic

wastewater contains Cr, they reported that Cr concentration absorbed by Salvinia minima was

88.2% at pH 4.0, 71.6% at 6.0 and 34.1% at 7.6. The solution pH and chromium speciation were both significant factors affecting the uptake process in our study. Research indicates that hexavalent chromium Cr (VI) is easily reduced to trivalent chromium Cr (III) in the presence of solid sorbents and in acid media (Park et al., 2005).

Significant differences in the chromium concentrations in the roots and the stems and leaves might be explained by the low solubility and bioavailability of Cr. Cr solubility and bioavailability in soil is mostly controlled by pH, concentration of Cr cation exchange capacity, organic matter content and oxidation state of the system (Bolan et al., 2003). In this sense, only

Cr(VI) might be discussed in terms of a significant bioavailability to A. donax tissues. Because of its lower solubility and bioavailability, Cr(III) is not as bioavailable to A. donax as Cr(VI). Cr(III) solubility in soil is pH-dependent (Palmer and Wittbrodt, 1991) and declines obviously at pH’s >

4.5. Cr(III) in most soils occurs mainly as less soluble Cr(OH)3 (Loeppert et al., 2003; Jeyasingh et al., 2005). on the other hand, Cr(VI) is recognized more soluble than Cr(III) and usually exists

-2 as CrO4 at pH > 6.0 (Ainsworth et al., 1989). At low pH, the protonation of positive charge

- positions of cell wall becomes significant and HCrO4 is the prevalent anionic species (Fomina and

Gadd, 2014). When the solution pH increases, Cr binding sites are deprotonated and thus the availability of anion binding positions declines (Elangovan et al., 2008).

Figure 4.1: (A) Cr content in plant parts in various pH at 1.0 mg Cr/L supplied. (B) Cr content in plant parts in various pH at 2.0 mg Cr/L supplied.

Figure 4.2: Main effects for response of Cr uptake by A. donax tissues in different soil pH.

The effect of solution pH on chromium bioaccumulation in A. donax L.

The effect of pH on Cr bioaccumulation in A. donax L. is shown in Table 4.4. The bioaccumulation values increased as a function of Cr concentration in nursery mix. However, bioaccumulation values reduced when soil pH increased. In present study, the bioaccumulation values were in the range of 0.079 – 0.321 for Cr treatment at 1.0 mg Cr/L supplied and in the range of 0.166 – 0.376 for Cr treatment at 2.0 mg L-1 supplied. Bioaccumulation factor (BF) of A. donax was below the reference value 1, where the highest estimated value was 0.376 for chromium treatment at 2.0 mg Cr/L and pH value 4.71; while the lowest estimated value was 0.079 for chromium treatment at 1.0 mg Cr/L and pH value 8.5. Chromium bioavailability and solubility in soil is primarily controlled by soil pH, amount of the capacity of metal cations exchange, organic matter content, and oxidation state of chromium (Bolan et al., 2003).

It was observed that bioaccumulation of chromium varies widely among A. donax tissues and nursery mix with different pH values (Chunilall et al., 2005). Estimated values of bioaccumulation factor linearly increased along with increasing Cr concentrations in nursery mix for A. donax L.; however, these values decreased when pH increased (Table 4.4 & Fig. 4.3). The

Cr concentrations obtained in the stems and leaves of tested plant were comparatively low in comparison to the plants classified as Cr hyperaccumulators (Reeves et al., 2000). Thus, A. donax could not be classified as a Cr-hyperaccumulator. Plants revealing an aerial parts bioaccumulation factor less than (1.0) are not applicable for hyperaccumulator. The success of phytoremediation relies on plant biomass, plant chromium concentration and bioavailable concentration of chromium in the soil. Thus, this plant showed the good capability to accumulate chromium in its

roots at pH 4.71, proposing that this plant has a good capacity of phytoextraction, as revealed in elevated translocation factor and small bioaccumulation factor (Rafati et al., 2011).

Table 4.4: Relative bioaccumulation factor (BF) of A. donax L. at 1.0 and 2.0 mg/L applied chromium concentrations.

1.0 mg Cr/L 2.0 mg Cr/L pH Root/Soil (BF) Root/Soil (BF)

4.71 0.321 0.376

5.5 0.261 0.321

7.0 0.174 0.239

8.5 0.079 0.16

Figure 4.3: Least squares mean of chromium uptake with 95.0 % Confidence Intervals.

Figure 4.4: Interaction Plot for Response mean of chromium uptake by A. donax L. at different soil pH and Cr concentration.

The effect of solution pH on Chromium translocation in A. donax L.

The results of the translocation values for aerial parts of A. donax L. in different nursery mix pH are presented in (Table 4.5). The translocation values increased as a function of Cr concentration in the nursery soil. However, translocation values linearly decreased when nursery mix pH increased. Values of translocation factor (TF) in both stems and leaves of A. donax L. were below (1.0). Translocation values were in range of 0.04 – 0.29 for stems and in range of 0.01 –

0.13 for leaves at the 1.0 mg l-1 loading Cr treatment. The highest calculated value for translocation factor A. donax L. varieties was 0.58 for chromium treatment at 2.0 mg l-1, while, the lowest estimated value was 0.26 for chromium treatment at 0.1 mg l-1. The highest calculated value for translocation factor for shortleaf A. donax L. varieties was 0.42 for chromium treatment at 2.0 mg l-1 and the lowest estimated value was 0.24 for chromium treatment at 1.0 mg l-1. As can be seen, all the TF values (Table 4.5) fell far below the reference value ≥ (1) (Terry and Banuelos, 1999), and ratios of BF and TF below this value potentially reduce the efficiency of plants to classify as

Cr-hyperaccumulator. Increases in soil pH can stabilize toxic chromium in soil, subsequently decreasing leaching effects of the toxic chromium in soil and water resources.

Table 4.5: Relative translocation factor (TF) of A. donax L. at 1.0 and 2.0 mg/L applied chromium concentrations.

1.0 mg Cr/L 2.0 mg Cr/L pH Stem/Root (TF) Leaf/Root (TF) Stem/Root (TF) Leaf/Root (TF)

4.71 0.293 0.131 0.512 0.362

5.5 0.206 0.057 0.476 0.22

7.0 0.038 0.014 0.418 0.155

8.5 0.042 0.008 0.287 0.115

Conclusion

The overall goal of this study was to investigate the effect pH on the remediation of chromium contaminated soil by A. donax. The results obtained in this experimental study indicated that increases in the solution pH significantly decreases the concentration of Cr in the roots and aerial parts of A. donax. The values of BF and TF were below the reference value (1.0) and declined as solution pH increased. Further research is necessary to conduct the effects of soil properties on the bioavailability of Cr in soil and its bioaccumulation, translocation and accumulation by A. donax in actual field conditions.

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CHAPTER FIVE

CONCLUSIONS AND FUTURE WORK

Conclusions

The current dissertation has improved the understanding of phytoremediation potential of

Cr-contaminated soil-water system using Arundo donax L. A better understanding of design and operation considerations for the phytoremediation biotechnology to achieve optimum results for cleaning up ecosystem has been established. This dissertation reports experimental results to identify the major changes occurred in the uptake of chromium by A. donax L. Three major studies are presented:

1) A review of chromium contamination in the ecosystem and its phytoremediation by plant species (Arundo donax L.).

2) Assessment of the effects of A. donax variety on the capacity to phytoremediation chromium

(Cr).

3) Evaluation of the effects of solution pH on the uptake of chromium (Cr) by Arundo donax L.

This chapter briefly summarizes major findings and their research implications. The following conclusions can be drawn from this research study:

1. Uptake of chromium by plant species using phytoremediation biotechnology shows

potential to be an effective way to remediate chromium-contaminated environments as it

has some advantages such as environmentally friendly, cost-effect compared with other

commonly used conventional technologies.

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2. A. donax might be utilized in remediation of Cr contaminated-wastewater.

3. It was found that chromium concentrations in plant tissues of the two A. donax varieties

increased with increased Cr levels in the nursery mix.

4. Cr concentration in A. donax root was much higher than that in the stem and leaf,

indicating that most Cr absorbed from the nursery mix was remained in the roots with

only a small amount of them being translocated into above-ground A. donax parts. At a

higher initial chromium concentration, high bioaccumulation and translocation rates were

observed.

5. Growth and development of A. donax plants occurred even with the presence of large

amounts Cr in the nursery mix. The highest Cr concentrations were accumulated in the

roots followed by the stems and leaves.

6. It was evident that Arundo donax is a plant that tolerates increased chromium

concentrations in its rhizosphere, and thus it might be cultivated in contaminated water.

The results of this study expand our knowledge of the Cr tolerance, absorption and

translocation capacity of A. donax.

7. It was found that increased solution pH lowered chromium accumulation in Arundo

donax.

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Recommendations for further work

Within next decade, phytoremediation of chromium contaminated soil and water certainly will be a standard natural treatment. Various plant species, such as A. donax L. is highly promising for the removal of chromium and other heavy metals from contaminated soil and water. The current experimental works conducted were constrained by the period of study for a Ph.D. degree. Many areas of research still require investigation. Therefore, future research should be focused on the following themes that are outlined below:

1. Field investigation of phytoremediation is ongoing wide-reaching, but studies often provide short-term performance data on small scale or non-typical design, and performance is subject to significant uncertainties linked to uncertainties in variations due to differences in design, climate, and loadings. Consequently, further research is needed to estimate the long-term treatment performance of phytoremediation.

2. Further studies are required to assess the uptake capacity of the different growth media to model removal processes of chromium, and the factors that affect their removal regarding the different speciation. Where phytoremediation stagnated, the symbiotic bacteria introduced by plant root systems, need to be studied to establish their role in chromium removal.

3. Further research is required at pilot and field level to assess the potential of phytoremediation of chromium.

4. Further research is necessary to conduct the effect of soil properties on the bioavailability of Cr in soil and its accumulation and translocation by A. donax in actual field conditions.

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