Zinc and Chromium removal mechanisms from industrial wastewater by using water hyacinth, eicchonia crassipes

NATIONAL UNIVERSITY OF RWANDA

Zinc and Chromium removal mechanisms from
industrial wastewater by water hyacinth, Eichhornia

crassipes (Mart.) Solms-Laubach

John GAKWAVU RUGIGANA

MSc. Thesis in WREM November 2007

National University of Rwanda

Faculty of Applied Sciences

Department of Civil Engineering

In collaboration with

Zinc and Chromium removal mechanisms from
industrial wastewater by Water hyacinth, Eichhornia

crassipes (Mart.) Solms-Laubach
Master of Science Thesis
in

WATER RESOURCES AND ENVIRONMENTAL MANAGEMENT
(W.R.E.M)

by
John R. GAKWAVU
Supervisors
Mr. B. C. SEKOMO (PhD Research Fellow/ UNESCO-IHE)
I. NHAPI, PhD. (UNESCO-IHE & National University of Rwanda)

A Thesis submitted in partial fulfilment of requirements of the Master of Science degree in Water
Resources and Environmental Management (WREM) at the National University of Rwanda

NUR, November 2007

Statement of originality

I declare that this research report is my own work; unaided work. It is being submitted for the degree of Master of Science in the National University of Rwanda. It has not been submitted before for any degree of examination in any other University.

Gakwavu Rugigana John

Date: November 10th, 2007

Signature:

When wastewaters are not well purified they can seriously damage surface and
ground water. They can also endanger human and animal health.

The findings, interpretations and conclusions expressed in this study do neither necessarily reflect the views of the National University of Rwanda, Faculty of Applied Sciences nor of the individual members of the MSc committee, nor of their respective employers.

Table of Contents

Statement of originality iii

Table of Contents v

List of tables viii

List of figures ix

List of symbols and abbreviations x

Dedication xi

Acknowledgements xii

Abstract xiii

1 INTRODUCTION 1

1.1 Background 1

1.2 Problem description 2

1.3 Objectives 3

1.4 Research questions 3

1.5 Hypotheses 4

1.6 Scope of the research 4

1.7 Report outline 4

2 LITERATURE REVIEW 5

2.1 Overview on use of macrophytes in metal removal 5

2.2 Water hyacinth (Eichhornia crassipens (Mart.) Solms. 6

2.2.1 Systematic position 7

2.2.2 Ecological factors 8

2.2.3 Potentials and constraints in using of water hyacinth 8

2.3 Heavy metals 9

2.4 Wastewater 11

2.5 Foliar absorption 12

2.6 Translocation of metals within plants 12

2.7 Uptake 13

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

3 MATERIALS AND METHODS 15

3.1 Water Hyacinth sampling site description 15

3.2 Methods 16

3.2.1 Description 16

3.2.2 Synthetic wastewater solution preparation 16

3.2.3 Experimental Procedures 16

3.3 Sample Analyses 19

3.3.1 Relative Growth 19

3.3.2 Bioconcentration Factor 19

3.3.3 Metals Accumulation 20

4 RESULTS AND DISCUSSIONS 22

4.1 Variations on plant relative growth 22

4.1.1 Relative growth of water hyacinth plants 22

4.1.2 Discussions on relative growth of water hyacinths 23

4.1.3. Correlation between final fresh weight and relative growth 23

4.2 pH effects and metal concentrations remained in controls (blanks) 24

4.2.1 pH effects in blank samples 24

4.2.2 Zinc concentrations remaining in blank samples 24

4.2.3 Chromium concentrations remained in blank samples 25

4.2.4 Discussions of pH effects on metal concentrations in blank samples 26

4.3 pH variations and Zn(II) and Cr(VI) concentrations in water samples with water

hyacinths 26

4.3.1 Variations of pH on metal removal by the plants 26

4.3.2 Zinc concentrations remaining in water samples after 4 weeks of

experiment. 28

4.3.3 Chromium conc. remaining in water after 4 weeks of experiment 29

4.3.4 Discussions on pH variations and metal removal by the plants 29

4.4 Bioconcentration Factor (BCF) for zinc and chromium 30

4.4.1 Bioconcentration Factor for zinc 30

4.4.2 Bioconcentration Factor for chromium 30

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

4.4.3 Discussions on bioconcentration factor 31

4.5 Bioaccumulation 32

4.5.1 Adsorption of Zinc by water hyacinth plants 32

4.5.2 Total adsorption of zinc 33

4.5.3 Adsorption of chromium by water hyacinth plants 34

4.5.4 Discussions on adsorption mechanism 35

4.6 Uptake mechanism 35

4.6 1 Uptake mechanism for zinc 35

4.6.2 Uptake mechanism for chromium 36

4.6.3 Discussions on uptake mechanism 36

4.7 Translocation Ability (TA) 37

4.7.1 Variation of translocation ability for zinc 37

4.7.2. Variation of translocation ability for chromium 39

4.7.3 Discussions on translocation ability 41

5. CONCLUSIONS AND RECOMMENDATIONS 42

5.1 Conclusions 42

5.2 Recommendations 43

References 44

Appendices 50

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

List of tables

Table 2.1: Maximum growth response of water hyacinth exposed to Cd and Zn 12

Table 2.2: Chromium uptake by water hyacinths during a period of 17 days from Keith

et al., 2006 13
Table 2.3: copper uptake by water hyacinth during a period of 17 days.from Keith et

al., 2006 14
Table 2.4: Arsenic uptake by water hyacinth during a period of 17 days from Keith et

al., 2006 14

Table 4.1: variations on bioconcentration factor of zinc 31

Table 4.2: variations on bioconcentration factor of chromium 31

Table 4.3: Variability in zinc uptake compared to initial concentration & exposure time.

37

Table 4.4: variability in uptake of chromium 37

Table 4.5: Translocation ability of chromium by the plant 40

Table 4.6: variations in translocation ability of zinc 41

List of figures

Figure 2.1: Common aquatic plants (source: Aquatics, 2005) 6

Figure 2.2: Morphology of water hyacinth plant (source: Aquatics, 2005) 8

Figure 3.1: Topographic map showing the location of Nyabugogo swamp and its

influents 15

Figure 3.2: Plan view of experimental set up. 18

Figure 3.3: steps in lab experiment. 18

Figure 4.1: Relative growth of water hyacinth plants vs exposure time for different Zn

and Cr concentrations 23

Figure 4.2: Correlation between Relative Growth of plants and Final Fresh Weight 24

Figure 4.3: variations of pH in blank samples 24

Figure 4.4: Zinc conc. remaining in blank water samples over time 25

Figure 4.5: Chromium conc. remaining in blank water samples over time 26

Figure 4.6: pH variations in plant water samples over time 27

Figure 4.7: Zinc conc. remaining in water samples with water hyacinth plants over time

28

Figure 4.8: Chromium conc. remaining in water samples with water hyacinth plants 29

Figure 4.9: Bioconcentration factor of Zinc 30

Figure 4.10: Bioconcentration factor of Chromium 31

Figure 4.11: Desorption of Zinc after 1 week 33

Figure 4.12: Desorption of Zinc after 2 weeks 33

Figure 4.13: Desorption of Zinc after 4 weeks 33

Figure 4.14: Total desorption of Zinc 34

Figure 4.15: Desorption of Chromium 34

Figure 4.16: Variations of uptake for zinc by the plants 35

Figure 4.17: Uptake of chromium in plant tissues for different initial concentrations 36

Figure 4.18: Translocation ability for Zinc by water hyacinth plants 38

Figure 4.19: Translocation ability for 1 week 38

Figure 4.20: Translocation ability for 2 weeks 39

Figure 4.21: Translocation ability for 4 weeks 39

Figure 4.22: Comparison of roots and shoots in translocation ability 40

Figure 4.23: Correlation of roots vs. shoots 40

List of symbols and abbreviations

AAS: Atomic Absorption Spectrometer

ANOVA: Analysis of variance

APHA: American Public Health Association

AWWA: American Water Works Association

BCF: Bioconcentration factor

BOD5: Biological oxygen demand during 5 days

CGIS: Geographic Information System and Remote Sensing Center.

Cr: Chromium

EDTA-Na2: Ethylen diethyl tetracetate disodium

et al.: Et alii

FFW: Final fresh weight

IFW: Initial fresh weight

ppm: Part per million

RG: Relative growth

SPSS: Statistical Package for the Social Sciences

TA: Translocation ability

US.EPA: United States Environment Protection Agency

WEF: Water Environment Federation

Zn: Zinc

Dedication

To my beloved family.

Acknowledgements

The achievement of this research was possible with the contribution of several persons with their continued remarks, comments, encouragement, their financial and moral supports, etc. to whom I would like to thank you.

First of all, I am very grateful to the Almighty God for his mercy during my studies and research. I'm also grateful to Rwandan Government for the financial support to complete this Master's Programme.

In particular, my heartfelt thanks to my supervisor PhD. candidate SEKOMO BIRAME Christian and my co-supervisor Dr. Innocent Nhapi, for their acceptance to supervise this research, their particular remarks, scientific discussions, critical comments, their availability and their encouragement. My thanks go also to all UNESCO-IHE and NUR staffs who had direct and indirect contributed to my studies at this juncture. I am also indebted to the laboratory personnel of National University of Rwanda, especially Jean Nepomuscene and Dominic of the Department of Chemistry from the Faculty of Sciences, NUR.

I can not close my acknowledgements without to thank all WREM colleagues cohort for their individual contribution, their sharing ideas through certain modules, their scientific discussions during some hard moments, for their cooperation and team spirit.

Furthermore, I greatly acknowledge my family, for being always with me in every single steps of this thesis with their encouragement.

To everyone concerned by this research, please found your place in my grateful thanks.

Abstract

Zinc and chromium are some of environmental pollutants and are toxic even at very low concentrations. Domestic and industrial discharges are probably the two most important sources for chromium and zinc in the water environment. Rwanda is still facing problems of heavy metal discharges into natural ecosystems by factories and household without any prior treatment. The toxic heavy metals are entering the food chain through drinking water, agriculture and fisheries activities and therefore endangering human life.

The general objective of this study is to investigate on the major mechanisms responsible for Cr (VI) and Zn (II) removal form the water phase by macrophyte plants. Water hyacinth have been used in the remediation process in the present work because this plant has elaborate much roots system providing more binding sites for Cr (VI) and Zn (II). Three mechanisms (Adsorption, uptake and translocation) for fixation of Cr (VI) and Zn (II) by macrophytes plants had been reported. The investigation had been conducted on two heavy metals commonly found in polluted industrial wastewater in Rwanda (Cr (VI) and Zn (II)).

Different parameters were studied in this research such as pH effects, plant relative growth, trace metal remaining in water samples, translocation ability, bioconcentration factor, adsorption, bioaccumulation and uptake mechanisms. The pH slightly increase from starting time 0 hr (pH= 6.7) to 48 hr (pH= 7.64 to 7.86); but after 48 hr of experiment, the pH decrease due to the saturation of bound sites so some H⁺ are released in water samples which cause the decreasing of pH. The relative growth significantly decreased (P = 0.05) from 1, 3 and 6 mg/L in 1 week but for 2 and 4 weeks, the relative growth slightly decreased linearity with the increasing (P = 0.05) of metal concentrations due to relatively increasing toxicity in contrast to Cr (VI) and Zn (II) concentration. This study shows that 56.7% of Zn (II) was accumulated in petioles, 27.0 % in leaves and 16.3% in roots whereas for Cr (VI) 73.7% was taken up in roots, 14.1% in petioles and 12.2% in leaves. It was seen that 17.6%, 6.1% and 1.1% were respectively adsorbed for 1 mg/L, 3 mg/L and 6 mg/L of Zn (II) concentrations by water hyacinth plants; but for Cr (VI), 9.0%, 36.4% and 54.6% were adsorbed respectively for 1 mg/L, 3 mg/L and of 6 mg/L. The order of translocation ability for Cr (VI) was leaves<petioles<roots in water hyacinth whereas for Zn (II) was leaves<roots<petioles.

Key words: Chromium, removal mechanisms, wastewater, water hyacinth, Zinc

1 INTRODUCTION

1.1 Background

Heavy metals are environmental pollutants and some of them are toxic even at very low concentrations. Pollution of the biosphere with toxic metals has accelerated dramatically since the beginning of the industrial revolution (Nriogo, 1979). The primary sources of this pollution are the burning of fossil fuels, the mining and smelting of metalliferous, municipal wastes, fertilizers, pesticides and sewage.

Heavy metals are of great concern primarily due to their known toxicity to aquatic life and human health at trace levels (EPA, 2001; EPA, 2002). It was reported that domestic and industrial discharges are probably the two most important anthropogenic sources for metals in the water environment (Stephenson, 1987). However, the lack of a reliable method to predict metals distribution in treatment units is a key weakness in determining metals fate and transport in wastewater treatment processes, and therefore, the development of effective pre-treatment guidelines (Patterson and Kodukula, 1984).

The rapid industrialization in some developing countries with an enormous and increasing demand for heavy metals, such as zinc (Zn) and chromium (Cr), causes high emissions of these pollutants into water bodies. Unlike organic pollutants, metals in wastewater are not degraded through biological processes, threatening not only the aquatic ecosystems but also human health through contamination of drinking water. The reuse option of the treated wastewater is an important strategy for conserving water resources, particularly in areas suffering from shortage of water.

Several studies have shown that constructed wetlands are very effective in removing heavy metals from polluted wastewaters (Qian et al., 1999). Different wetland plant species differ, however, in their abilities to take up and accumulate various trace elements in their tissues (Rai et al., 1995). Recently, wetland plant species with high capacities of trace element (Cu, Ni, Zn, etc.) removal from water were identified (Zayed et al., 1998a; Zhu et al., 1999) duckweed (Lemna minor L.) and water hyacinth [Eichhornia crassipes (Mart.) Solms-Laubach

Heavy metals may come from natural sources, leached from rocks and soils according to their geochemical mobility or come from anthropogenic sources, as the result of human land occupation and industrial pollution. Depending on their solubility, these metals may eventually become associated to suspended particulates matter and/or accumulate in the bottom sediments (Espinoza-Quinones et al., 2005).

In Rwanda, the problem regarding waste treatment in general is still crucial at one side. At the other side the way to deal with such problem is not easy at all because there is no appropriate technology for waste treatment. The selection of that technology is not also an easy issue to deal with because it must take into account many other important aspects like the financial and social ones. And finally is that selected technology appropriate in order to meet the effluent standards and is it also cost effective for a developing country like Rwanda?

1.2 Problem description

Rwanda is facing problem of pollution in general. In many cases the pollution ends in the water bodies. That pollution contains diverse toxic pollutants (organic and inorganic compounds) coming from household and factories. Heavy metals from factories but also from other sources such as agriculture are the toxic compounds of our interest because they are not only polluting the water sources used for drinking, agricultural and fisheries purposes but they are also entering the food chain and therefore endangering human life.

Wastewater contaminated by heavy metals need an effective and affordable technological solution. In general wastewater from industrial activities must not be allowed to be discharged into our water reservoir. In order to stop the pollution at the production site, the on site treatment of wastewater is the best option recommended for such waste. Several studies indicate that aquatic plants have large potential for removal of organic and inorganic pollutants from wastewater.

Diverse industrial wastes have aggravated the problem of water pollution. This problem
becomes complex because of the differences in pollution according to the industrial
activities and also due to the non-biodegradability of inorganic pollutants like heavy

metals (Srivastav, et al., 1994). Heavy metals produce undesirable effects, even if they are present in extremely small quantities on human and animal life.

1.3 Objectives

The general objective of this study is to investigate on the major mechanisms responsible for Cr (VI) and Zn (II) removal form the water phase by macrophyte plants. Water hyacinth have been used in the remediation process in the present work because this plant has elaborate roots system providing much binding sites for heavy metals.

The specific objectives are:

· Assessment of the Cr (VI) and Zn (II) concentrations adsorbed on the outer surfaces of the roots;

· Assessment of the Cr (VI) and Zn (II) concentrations taken up by water

hyacinths;

· Assessment of the mobility and translocation of Cr (VI) and Zn (II) within water hyacinths.

1.4 Research questions

In order to achieve the above mentioned objectives some research questions need to be addressed:

· Which part of water hyacinth has much contributed in Cr (VI) and Zn (II) removal?

· Which heavy metal between Cr (VI) and Zn (II) have been preferably fixed by water hyacinth plant?

· What was the effect of metals concentration on water hyacinth growth and how was the bioaccumulation factor?

1.5 Hypotheses

· Cr (VI) and Zn (II) are adsorbed, taken up and translocated by water hyacinth. When saturation of binding sites is reached, the plant will no longer be efficient in Cr (VI) and Zn (II) removal.

· Zn (II) is preferably adsorbed and taken up by water hyacinth when compared to Cr (VI) because of the smallest size and his positive charge.

· High metal concentration is toxic to the growth of water hyacinth plant therefore the bioaccumulation factor will increase with a low metal concentration and decrease with the increasing of the metal concentration.

1.6 Scope of the research

The research was aimed to investigate on the mechanisms involved in Cr (VI) and Zn (II) removal by water hyacinth plants in a closed system. That's why this research was limited at a laboratory pilot scale by using an industrial synthetic wastewater prepared in the lab by adding different concentrations of Cr (VI) and Zn (II), water hyacinth plants were used as adsorbent to assess the mechanisms of removal.

1.7 Report outline

This study has been delimited in six chapters according to the general introduction (background, problem description, objectives, research questions, hypotheses and the limitation of the research), literature review (general in formations on heavy metals, water hyacinth, wastewater treatment), materials and methods used, results found, discussions on results found, conclusions and recommendations followed by references and some appendices.

2 LITERATURE REVIEW

2.1 Overview on use of macrophytes in metal removal

Aquatic plants have been used now for decades to remove heavy metal in polluted water (Rai et al., 1995; Denny et al., 1995; Mungur et al., 1997; Zhihong et al., 1997; Obarska, 2001; Cheng et al., 2002; Keskinkan, 2005). These aquatic plants commonly called macrophytes plants belong to different plant species. In general these aquatic plants showed the capacity to remove heavy metal from polluted water by accumulation in their roots or by simple uptake by the plants (Lubberding et al., 1999; Lubberding et al., 2000; Awuah et al., 2000; Lubberding et al., 2001). Many investigations on metal removal have been conducted with the principal aim of cleaning the environment from these dangerous metals. No particular attention to the mechanism involved in the removal process had been investigated to explain what is really occurring in the solution and what should be do to enhance the removal efficiency when macrophytes plants cannot accumulate anymore heavy metal in its roots or leaves.

Considerable interest has developed in the removal of heavy metal in water using macrophytes plants (Prasad and Freitas, 2003). This method of extracting heavy metal from polluted water bodies is called phytoextraction. Plants are used to accumulate and uptake heavy metal from soil, sludge or water. It has been reported that the removal accumulation process occurs via adsorption, uptake and translocation processes (Abdel-Rahman, 1999; Kelderman, 2000; Leman, 2000; Babu, 2001; Meggo, 2001 and Alick, 2002).

Figure 2.1: Common aquatic plants (source: Aquatics, 2005)

In all reported investigations, it has been demonstrated that adsorption was the main mechanism involved in the removal of heavy metal. In some cases uptake and translocation have been observed. (Hasan et al., 2006)

Different species have the ability to remove coliforms, bacteria, metals from wastewater such as Nasturtium officinale to accumulate Copper, Zinc and Nickel (Kara, 2005), the ability of water hyacinth (Eichhornia crassipes) to remove Aluminum by constructed wetland grown under different nutritional conditions is excepted (Jayaweera et al., 2007), by different mechanisms. Vesk et al. (2006) confirm the metal localization within and around roots of water hyacinth growing in a wetland receiving urban run-off.

Several publications (Sarabjeet and Dinesh, 2005; Liu et al., 2007, etc.) shown different plants able to treat wastewater in terms of heavy metals removal such as Lemna minor, Myriophyllum aquaticum, Ceratophyllum demersum, Azolla filiculoides, Salvinia natans, Acanthopanax sciadophylloides, Ilex crenata, Clethra barbinervis, Acanthopanax sciadophylloides, Pieris japonica, Ilex crenata, Rhododendron semibarbatum Acer sieboldianum, Acer rufinerve, Acer micranthum, Lindera erythrocarpa, Clethra barbinevris Macadamia neurophylla, M. augustifolia, Betula verrucosa, Sorbus aucuparia, Clethra barbinervis.

2.2 Water hyacinth (Eichhornia crassipens (Mart.) Solms.

Water hyacinth (E. crassipens) is fast growing perennial aquatic macrophyte (Reddy
and Sutton, 1984). It is a member of pickerelweed family (Pontederiaceae) and its name

Eichhornia was derived from well-known 19th century Prussian politician J.A.F. Eichhorn (Aquatics, 2005). The plants can double its population in only twelve days (APIRIS, 2005). Water hyacinth is also known for its ability to grow in severe polluted waters (So et al., 2003). E. crassipens is well studied as an aquatic plant that can improve effluent quality from oxidation ponds and as a main component of one integrated advanced system for treatment of municipal, agricultural and industrial wastewaters (U.S. EPA, 1988; Sim, 2003). Water hyacinth is often described in literature as serious invasive weed on the world (U.S. EPA, 1988; Maine et al., 1999; Wilson et al., 2005).

2.2.1 Systematic position

a. Taxonomy

Cronquist (1988), Thorne (1992) and Takhtajan (1997) suggest following water hyacinth taxonomic placement (Center et al. 2002):

Division: Magnoliophyta

Class: Liliopsida

Subclass: Commeinidae

Superorder: Commelinanae

Order: Pontederiales

Family: Pontederiaceae

Genus: Eichhornia

Specific epithet: crassipes (Martius) Solms-Laubach.

b. Morphology

Water hyacinth (Eichhornia crassipes) is a perennial, floating macrophyte, freshwater aquatic vascular plant with rounded, upright, shiny green leaves and spikes of lavender flowers (Reed et al., 1997). The petioles of the plant are spongy with many air spaces and contribute to the buoyancy of the hyacinth plant. When grown in wastewater, individual plants range from 0.5 to 1.2 m from the top of the flower to the root tips (Reed et al., 1997).

The plants spread laterally until the water surface is covered and then the vertical
growth increases. Hyacinths are very productive photosynthetic plants. Their rapid

growth is a serious nuisance problem in many slow flowing southern waterways. These same attributes become an advantage when used in a wastewater treatment system.

Figure 2.2: Morphology of water hyacinth plant (source: Aquatics, 2005)

2.2.2 Ecological factors

Water hyacinth is heliophyte plant growing best in warm waters rich in macronutrients. Optimal water pH for growth of this aquatic plant is neutral but it can tolerate pH values from 4 to 10. This is very important fact because it points that E. crassipens can be used for treatment of different types of wastewater. Optimal water temperature for growth is 28-30^oC. Temperatures above 33^oC inhibit further growth (Center et al., 2002). Optimal air temperature is 21-30^oC. So if aquatic systems with water hyacinth are constructed in colder climates it would be necessary to build greenhouses for maintaining optimal temperature for plant growth and development. Low air humidity from 15% to 40% can also be limiting factor for undisturbed growth of water hyacinth (Allen, 1997). E. crassipens tolerates drought well because it can survive in moist sediments up to several months (Center et al., 2002).

2.2.3 Potentials and constraints in using of water hyacinth

Water hyacinth is plant with many advantages firstly because it can be used for many
purposes, but it has one major consequence. E. crassipes is one of the most invasive
weeds that can destroy precious aquatic ecosystems in a short time which can lead to

series of other problems. Because of that it is very difficult to answer the question - Is E. crassipes the golden plant or the world's worst aquatic weed?

One can often read that people have a moral imperative to think about potential utilization of abundantly available biomass of water hyacinth in tropical countries for the benefit of people for whom E. crassipes has created many problems or even has destroyed their lives. There are many examples around the world of how communities or individuals have used water hyacinth to great advantage. In regions where it can be found in abundance water hyacinth can be used like food for people because its leaves are rich in proteins and vitamin A. It can be also utilized as green fertilizer or as mulch, compost and ash in regenerating degraded soils. (Lindsey and Hirt, 1999).

In African countries like Uganda water hyacinth has also influenced on much frequent occurrence of diseases (dysentery, malaria, and schistosomiasis) related to content of different pathogens in water. It has been discovered that water hyacinth's quest for nutrients can be turned in a more useful direction. The plant has been shown to accumulate trace elements such as Ag, Pb, Cd and Zn.

The focus on water hyacinth as a key step in wastewater recycling is due to the fact that it forms the central unit of a recycling engine driven by photosynthesis and therefore the process is sustainable, energy efficient and cost efficient under a wide variety of rural and urban conditions.

2.3 Heavy metals

The designation «Heavy metals» is applied to a group of metals and metalloids with a specific density greater than 5 g / cm³. They are frequently associated with pollution and toxicity in the environment. In general «trace metals» is the term used because they occur in low concentrations in the earth's crust. Element like As, Cd, Cr^VI, Hg and Pb are known to be very toxic. However some metals like Co, Cu, Mn, Se and Zn are essential for living organisms at low concentrations and are vital components of enzymes. Heavy metals occur naturally in the environment, usually at relatively low concentrations as a result of weathering and other pedogenic processes acting on the

rock fragments on which soils develop (Rulkens et al., 1995). These metals are then transported to the aquatic ecosystem through leaching and run-off phenomena.

Human activities have increased the amount of heavy metals released to the environment. Metals have been exploited at an alarming rate because of their economic value. The negative consequences of this situation have only been realized within the last decades. Because of that, many researches on the interaction of heavy metals with various components of the environment have been conducted and still going on with the main objective of finding suitable ways of solving and avoiding heavy metals pollution on the surrounding environment.

The existence of heavy metals in the environment represents a very significant and long-term environmental hazard. Even at low concentrations these metals can be toxic to organisms, including humans. In particular, chromium is a contaminant that is a known mutagen, teratogen and carcinogen (Chang, 1996; Young et al., 2006).

The removal of heavy metals from aqueous solutions has therefore received considerable attention in recent years. However, the practical application of physicochemical technology such as chemical precipitation, membrane filtration and ion exchange is sometimes restricted due to technical or economical constraints. For example, the ion exchange process is very effective but requires expensive adsorbent materials (Lehmann et al., 1999; Volesky, 2001).

The use of low-cost waste materials as adsorbents of dissolved metal ions provides economic solutions to this global problem and can be considered an eco-friendly complementary (Volesky et al., 1995; Mullen et al., 1989). At present, emphasis is given to the utilization of biological adsorbents for the removal and recovery of heavy metal contaminants. (Young et al., 2006).

Aquatic macrophytes are known to remove metals by surface adsorption and/or absorption and incorporate them into their own system or store them in a bound form (Rai et al., 1995). The uptake of trace metals by the root systems of aquatic plants depend both on the kind of metal and on the species of plant absorbing the metal (Samecka-Cymermann and Kempers, 1996).

2.4 Wastewater

Wastewater is a general term that encompasses a myriad of wastes in the water medium originating from diverse sources. Normally, the two major sources of concern are of domestic and industrial origin but also agriculture.

Several authors shown results from Iron bridge water hyacinth system in Florida, USA demonstrated that phosphorus removal was from 35 to 80 % (U.S. EPA, 1988). The same facility successfully removed about 60% of BOD5 and 43% of suspended materials from wastewater. These systems can also remove heavy metals like chromium, cadmium, copper, zinc and other effectively. In their experiments Maine et al. (1999) have shown that 72% of cadmium was removed from wastewater by water hyacinth. Accumulated nutrients and heavy metals are removed from aquatic systems by plant harvesting and sediment dredging (Reddy and Sutton, 1984; U.S. EPA, 1988).

There are many speculations on the use of water hyacinth upon harvesting. According to some authors (Lindsey and Hirt, 1999) it can be use like food for people or fodder. But it is not recommended to consume water hyacinth if it was used for removal of heavy metals, rare earth elements or other toxic substances that can cause problems if they enter food chain (Chua, 1998). Upon harvesting water hyacinth can be used for composting, anaerobic digestion for production of methane, and fermentation of sugars into alcohol (U.S. EPA, 1988). These operations can help in recovering expenses of wastewater treatment.

Aquatic macrophytes are known to remove metals by surface adsorption and/or absorption and incorporate them into their own system or store them in a bound form. The uptake of trace metals by the root systems of aquatic plants depend both on the kind of metal and on the species of plant absorbing the metal

Table 2.1 shows that the effective response of water hyacinth after exposed to cadmium and zinc in different concentrations is different depending on metal. As it shown, zinc was more adsorbed and taken up by the plant than the cadmium.

Table 2.1: Maximum growth response of water hyacinth exposed to Cd and Zn

Parameter	Cd	Zn
Relative growth	0.85^a	0.89^c
Metal accumulation (mg/kg) shoot	113.2^a	1926.7^c
root	2044^a	9652.1^c
Residual concentration (mg/L)	0.185^a	6.29^c
BCF	622.3^b	788.9^d

a: 4 mg/L Cd, b: 2 mg/L Cd, c: 40 mg/L Zn, d: 5 mg/L Zn.
(Source: Xiaomei et al., 2004)
BCF : bioconcentration factor

2.5 Foliar absorption

In addition to root absorption, plants can also derive low amounts of some elements through foliar absorption. Foliar absorption of solute depends on the plant species, its nutritional status, the thickness of its cuticle, the age of the leaf, the presence of stomata guard cells, the humidity at the leaf surface and the nature of the solutes (Marschner, 1986). Metal antagonism, such as Cu and Zn, can occur in foliar absorption as well as in the root (Channel, 1986). Aerosol deposited lead does not penetrate the cuticle of higher plants, but tend to adhere to the surface of leaves. They can however be absorbed through the cuticle of some bryophytes (Alloway, 1990).

2.6 Translocation of metals within plants

Once the ions have been absorbed through the roots or leaves and have been transported to the xylem and phloem vessels. There is a possibility of movement throughout the whole plant (Alloway, 1990; Streit and Stumm, 1993). The mobility of different metal ions varies and the rate and extent of movement within plants depends on the metal, the plant organ and the age of the plant. Zn, Cu and Pb fall in the category of metals which are readily, immediately and least translocated respectively. In the xylem heavy metals will usually only be transported if special chelates are formed. For example, zinc may be transported by chelation to organic acids, copper transported in complex with amino acids, nickel can be transported as nickel peptide complex and lead may be transported as a Pb-EDTA complex (Greger, 1999; Saxena et al., 1999).

2.7 Uptake

The uptake process is a mechanism by which metal ions are transported across the cell membrane and can be used in a building of new biomass or stored in vacuoles. Streit and Stumm (1993) reported that little is known about the mechanism involved in the absorption and translocation of heavy metal to the host plant root cells. The presence of carboxyl groups at the roots system induces a significant cation exchange capacity and this may be the mechanism of moving heavy metal in the roots system where active absorption takes place.

Table 2.2 shows that water hyacinth plants are able to remove the maximum of chromium concentration from wastewater at low concentration (72.3 %). When metal concentration increases in wastewater, the removal capacity of water hyacinth plants decrease linearity.

Table 2.2: Chromium uptake by water hyacinths during a period of 17 days from Keith et al., 2006

Sample ID Chromium % removal vs. control

Ctr. w/ plant 0.067

Ctr. w/ o plant 0.014 0

7 ppm w/ plant 1.98

7 ppm w/ o plant 7.16 72.3

14 ppm w/ plant 10.4

21

14 ppm w/ o plant 13.1

28 ppm w/ plant 20.7

28 ppm w/ o plant 25.6 19.1

Comparing to the above results in Table 2.2, the Table 2.3 depicts the phenomenon for copper removal which follow the same trend as for chromium, but the removal capacity by the plants is less than for chromium (53 %).

The most important parameter to consider is the pH (Kelly, 1988). Generally when the pH decreases, the toxicity of metal ions increases because the proportion of the adsorbed ion on the root system decreases (Harding and Whitton, 1977).

Table 2.3: copper uptake by water hyacinth during a period of 17 days.from Keith et al., 2006

Sample ID Copper % removal vs. control

Ctr. w/ plant 0.567

Ctr. w/ o plant 0.266 0

2.5 ppm w/ plant 0.949

53

2.5 ppm w/ o plant 2.02

5 ppm w/ plant 3.45

0

5 ppm w/ o plant 2.9

10 ppm w/ plant 5.38

0

10 ppm w/ o plant 2.39

Table 2.4 presents the results of Arsenic removal by water hyacinth plants and it is shown that water hyacinth is not able to remove Arsenic from wastewater even if at low concentration.

Table 2.4: Arsenic uptake by water hyacinth during a period of 17 days from Keith et al., 2006

Sample ID Arsenic % removal vs. control

Ctr. w/ plant 0.056

0

Ctr. w/ o plant 0.03

5 ppm w/ plant 5.4

4.8

5 ppm w/ o plant 5.67

10 ppm w/ plant 10.2

4.7

10 ppm w/ o plant 10.7

20 ppm w/ plant 20.4

20 ppm w/ o plant 19.6 0

The results show how a floater plant like water hyacinth affects arsenic, chromium, and copper. Water hyacinths do not seem to remove large amounts of arsenic or copper from contaminated water.

The water hyacinth appeared to be a good choice for removing chromium from polluted water. At low concentrations, the plant removed about 70% of the chromium in the water (Table 2.2). As the concentrations increase, the plant appeared to be unable to take up as much as possible percent chromium (21%). In conclusion, the effectiveness removal order of these metals was arsenic<copper<chromium.

3 MATERIALS AND METHODS

3.1 Water Hyacinth sampling site description

The water Hyacinth plants were collected in Nyabugogo swamp which is located in capital city (Kigali) of the country. The part which is shown as Nyabugogo swamp on the map is one which is not exploited by the population for agriculture and is the one considered as natural wetland, receiving wastewaters of Kigali City. Its surface area, according to CGIS, is 60.09 ha (CGIS, 2007).

Figure 3.1: Topographic map showing the location of Nyabugogo swamp and its influents. (Source: CGIS, 2007)

3.2 Methods

3.2.1 Description

The methodology developed in this research taken the approach consisting in the laboratory pilot scale experiment. Major mechanisms of metal removal were explained on basis of experimental results and available information in the literature review. Adsorption and uptake, translocation and foliar absorption tests were performed to assess the metal removal using water hyacinth. Three replicates were done during the lab experiment.

3.2.2 Synthetic wastewater solution preparation.

1 mol of ZnCl2 contains 1 mol of Zn (II) and also 1 mol of _K2Cr2O7 contains 2 mol of Cr(VI). Then knowing that the load of 1 mol ZnCl2 is 136.2 g ,1 mol _K2Cr2O7 is 294 g, 1 mol Zn is 65.2 g and 2 mol of Cr is 2* 52 g = 104 g , we calculated the load of each salt to be weighted and dissolved into 1 liter of aqueous solution. Thus for zinc, the calculation has been done as follows: 1g * 136.28 g / 65.2 g = 2.1 g of ZnCl2 and for chromium we did it as follows: 1 g * 294g / 104 g = 2.8 g of _K2Cr2O7 .As we decided to prepare 100 ml of solution, 0.21g of ZnCl2 and 0.28 g of _K2Cr2O7 were dissolved into 100 ml of solution. For 1 mg/l of Zn (II) and Cr(VI) preparation, we abstracted 1 ml from these 100 ml and dilute up 1000 ml of solution. We did the same for 3 mg/l and 6 mg/l by abstracting respectively 3 ml and 6 ml from the 100 ml of solution and dilute up 1000 ml. The pH of the solution was then adjusted between 6 #177; 0.7 by addition of dilute HNO3 or NaOH as required.

3.2.3 Experimental Procedures

The Water Hyacinth plants (Eichhornia crassipes) were collected from Nyabugogo wetland in Kigali city, were rinsed with tap water and distilled water to remove any epiphytes and insect larvae grown on plants. The plants were placed in big plastic containers with water under natural sunlight for several weeks to let them adapt to the new environment, then the plants were selected and weighted by sensitive balance. The experimental set-up was consisting in the use of small plastic container buckets of 16

cm of diameter and 14.5 cm in height. All experiments were run in a batch system using a nutrient solution constituted by 500 ml of tap water from the valley located at Butare near Pharmacopée centre, 500 ml of wastewater from the Nyabugogo wetland plus quantity of Ca(NO3)2 .4H2O, NaNO3, NH4Cl, K2HPO4 respectively 20, 20, 20 and 40 mg. The fresh weight of the plants in each bucket was measured by using sensitive balance before starting each growing time: 1, 2 and 4 weeks.

A stock solution (1,000 mg/L) of Zn (II) (ZnCl2) and Cr (VI) _(K2Cr2O7) was prepared in distilled water, which was later diluted as required. The plants were maintained in water supplemented by Heavy metals by pouring a certain volume of the metals stock solution in order to get the final concentration of 1, 3 and 6 mg/L of Cr and Zn respectively in different plastic buckets containing water hyacinth plant in three replicates.

Plastic buckets with zinc and chromium concentrations without water hyacinth plants served as control. Distilled water was added in order to compensate for water loss through plant transpiration, sampling and evaporation. Water samples were taken and pH measurements by pH meter were taken every 60 minutes for the first day during 6 hours and for the following days one sample after time period was taken during 1, 2, and 4 weeks of exposure to metal solution. All samples were filtered using 0.45 um cellulose acetate filters (wathman papers) and acidified with 5 drops of nitric acid (HNO3 65%) in the laboratory for storage of water samples in volumetric flasks (250 ml) before Atomic Absorption Spectrometer analyses.

The Figure 3.2 shows the plan view of laboratory experimental set up developed during our research in National University of Rwanda, Faculty of Sciences.

Figure 3.2: Plan view of experimental set up.

After each test duration (1, 2 and 4 weeks), final fresh weight for each water hyacinth plant was taken; plants were harvested for other analyses. They were separated into petioles, roots and leaves and were analysed for relative growth, metals accumulation, translocation ability, bioconcentration factor (BCF) and adsorption on the outer surface of roots. For adsorption, roots were immersed in EDTA-Na2 for metal desorption. All parts of the plants were dried in drying oven at 105°C for 24 hours. In addition, the metals remained in the solution were measured to assess the removal potential of water hyacinth plants.

The figure 3.3 depicts different steps developed in the laboratory for data collection and analyses.

Figure 3.3: steps in lab experiment.

3.3 Sample Analyses

3.3.1 Relative Growth

Relative growth of control and treated plants was calculated to assess the effects of zinc and chromium concentrations on water hyacinth plant growth in relationship with time. The formula bellow was used to calculate the relative growth:

RG =	FFW (3.1) IFW

Where FFW denotes final fresh weight (g); IFW denotes initial fresh weight (g) and RG denotes the relative growth of water hyacinth plants which is dimensionless.

3.3.2 Bioconcentration Factor

The BCF (bioconcentration factor) provides an index of the ability of the plant to accumulate the metal with respect to the metal concentration in the substrate. The BCF was calculated as follows:

Concentration of metal in plant tissue

BCF =

Initial concentration of metal in external solution (3.2)

(source: Xiaomei et al., 2004)

or

BCF = (P/E)i (3.3)

(Source: Liao and Chang, 2004)

Where I denotes the heavy metals, BCF the bioconcentration factor and P represents the trace element concentration in plant tissues (mg.Kg^-1), E represents the trace element concentration in the water (mgL^-1) or in the sediment (mgkg^-1 dry wt). A larger ratio implies better phytoaccumulation capability.

3.3.3 Metals Accumulation

Metals accumulation in plant and water samples was measured. Digestion of samples in this study was performed according to the Standard Methods by APHA.7 (APHA/AWWA/WEF, 2005) Plant biomass samples was decomposed to dry matter by heating at 105°C for 24 hours in a hot air oven and the fine particles were digested with nitric acid (HNO3) and hydrogen peroxide (H2O2), filtered through a wathman paper filter into a volumetric flask before Atomic Absorption Spectrophotometer analyses. The two following mechanisms were performed in analyses to differentiate the metal adsorbed and up taken by water hyacinth during experiment period.

a. Adsorption

The adsorption consists on metal attached to the outer surface of the roots. To quantify the metal adsorbed by water hyacinth after the plant exposure to different concentrations of chromium and zinc in different periods of times (1week, 2weeks and 4 weeks). After test duration of observation, the adsorption was determined by putting roots of water hyacinth plant in nine beakers containing 20 ml of EDTA-Na2 respectively for 5, 10, 15, 20, 25, 30, 35, 40 and 45 min for removal of zinc and chromium trace elements on the outer surface of the roots. Those EDTA-NA2 solutions were filtered, acidified by 5 drops of Nitric acid (HNO3) and analysed by Atomic Adsorption Spectrophotometer (AAS) for zinc and chromium adsorbed by the plants.

b. Uptake

The uptake process is a mechanism by which metal ions are transported across the cell membrane and can be used in a building of new biomass or stored in vacuoles. To assess this mechanism during our research; after period observation, water hyacinth plants were taken out form the small buckets, roots, petioles and leaves were separated, dried in dry oven at 105°C during 24h. Plant samples were digested and analyzed by AAS to identify the zinc and chromium concentrations in plant biomass (roots, leaves and petioles).

c. Translocation ability (TA)

The translocation ability shows the ability of water hyacinth plants to transport across the metal ions in the shoot tissues. It was calculated by dividing the concentration of a trace element accumulated in the root tissues by that accumulated in shoot tissues (Wu and Sun 1998). TA is given by:

TA = (Ar Ú As)I (3.4)

Where i denote the heavy metal, TA is the translocation ability and is dimensionless. Ar represents the amount of trace element accumulated in the roots (mg.Kg^-1 dw), and As represents the amount of trace element accumulated in the shoots (mg.Kg^-1 dw).

Statistics were used to assess the variations and correlations between parameters studied. The following were used: standard deviation, regression analyses, analyse of variance 2 (ANOVA 2) with replications and other tools in MS-Excel such as average, mean values, etc.

4 RESULTS AND DISCUSSIONS

The laboratory experiments for this research were started in March 2007 and ended at the end of October 2007. Water hyacinth plants were collected several times but unfortunately some of them died due to rigorous conditions, more than 1,000 samples (water and plant mixed) were analyzed and the results for this research are average values.

4.1 Variations on plant relative growth

4.1.1 Relative growth of water hyacinth plants

The relative growth was calculated to estimate the effects of zinc and chromium concentrations on plant growth according to exposure time. The relative growth indicates the tolerance of the plants to different concentrations of metal and exposure time to these metals. It was observed that the final fresh weight increased compared to the initial fresh weight in the first week but it decreased when the metal concentration increased, mostly for 6 mg/L. There is a slight stagnation for the second week and fourth week in plant growth in terms of metal concentration. The growth decreased with increasing time and metal concentrations due to a decreasing of essential macro and micro nutrients for the plants in the experimental water in the small buckets.

The growth of water hyacinth at different concentrations of chromium and zinc is shown on Figure 4.1. It was noted that the relative growth of the plants decreased considerably in the first week only and slightly decreased with an increasing metal concentration and exposure time. There was constant trend in the plant growth after 1 week of experiment. However, at higher concentrations of these metals, plant growth was always inhibited. The growth of water hyacinth plants significantly increased (P = 0.05) with the passage of time according to the Figure 4.2.

Relative growth values	5 4 3 2 1 0
		1 wk, 1 1 wk, 3 1 wk, 6 2 wks, 1 2 wks, 3 2 wks, 6 4 wks, 1 4 wks, 3 4 wks, 6 Exposure time (week) and initial conc.

Figure 4.1: Relative growth of water hyacinth plants vs exposure time for different Zn and Cr concentrations

4.1.2 Discussions on relative growth of water hyacinths

For water hyacinth plants treated with Zn and Cr, the relative growth significantly decreased (P = 0.05) from 1, 3 and 6 mg/L in 1 week but for 2 and 4 weeks, the relative growth slightly decreased linearly with the increasing (P = 0.05) of metal concentrations. In the case of zinc and chromium, however, the relative growth exhibited an exponential decrease caused by relatively increasing toxicity in contrast to chromium and zinc concentrations.

The ANOVA 2 shows that for 1 week exposure time, there is a high effect (difference is significant) of initial concentrations (1, 3 and 6 mg/L) to the growth of the plants (P = 0.05), but for 2 and 4 weeks according to initial concentrations, the difference is not significant (P = 0.05).

4.1.3. Correlation between final fresh weight and relative growth

Figure 4.2 shows that there is no correlation between the final fresh weight and the relative growth of water hyacinth plants. The non existence of correlation is reported by the stagnation in plant growth due to the decreasing nutrients and increasing metal concentrations. It was considered that a decrease in the growth was induced by metal toxicity.

Final Fresh weight vs Relative growth

y = 0.009x + 1.4994
R2 = 0.2977

30 50 70 90

Final Fresh Weight (g)

4

3

2

1

0

Relative growth

Figure 4.2: Correlation between Relative Growth of plants and Final Fresh Weight

4.2 pH effects and metal concentrations remained in controls (blanks) 4.2.1 pH effects in blank samples

Figure 4.3 shows the variations of pH in blanks which are due to elements contained in blank samples such as bacteria, phytoplanktons, zooplanktons, also the variations of temperature can affects pH in blank samples. The correlation between metal removal and the role of experimental containers exists. The increasing or decreasing of pH in blank samples without water hyacinth plants indicates that some elements of metal were fixed on the internal surface of experimental buckets.

Oh

1 hr

3 hr

6 hr 10 hr 15 hr 21 hr 33 hr 57 hr 105 hr 177 hr 273 hr 393 hr 537 hr 705 hr

Exposure time (h)

pH

pH, 1mg/L pH, 3mg/L pH, 6mg/L

10

8

6

4

2

0

Figure 4.3: variations of pH in blank samples

4.2.2 Zinc concentrations remaining in blank samples

Figure 4.4 shows the trend of zinc concentration in blank water samples at different
initial concentrations (1, 3 and 6 mg/L) and at different periods of time. It was shown

that experimental small buckets may fix some trace elements of zinc on internal surface of buckets or some trace elements were accumulated in sediment because of the variation in metal concentration during the exposure time. For 1 mg/L, the removal of zinc follows a linear trend of decreasing concentration with the increasing of exposure time.

0.2

0.18		Zn2+, 1mg/L Zn2+, 3mg/L Zn2+, 6mg/L
0.16

Exposure time (h)

1 hr

Oh

6 hr

3 hr

33 hr

21 hr

15 hr

10 hr

57 hr

105 hr

177 hr

537 hr

393 hr

273 hr

705 hr

0.14

0.12

0.1

0.08

0.06

Conc. (mg/L)

0.04

0.02

0

Figure 4.4: Zinc conc. remaining in blank water samples over time

4.2.3 Chromium concentrations remained in blank samples

Figure 4.5 shows that chromium was quickly fixed on the internal surface of experimental buckets and also due to phytoplankton, zooplanktons in water samples. Some trace elements were analyzed in water from 1 to 10 hr only and after 177 hr, the internal surface releases chromium concentration in water and other trace elements were accumulated in sediment.

Exposure time (h)

Conc. (mg/L)

0.16

Cr6+,1mg/L Cr6+, 3mg/L Cr6+,6mg/L

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

Oh

1 hr

3 hr

6 hr

10 hr

15 hr

21 hr

57 hr

33 hr

105 hr

177 hr

273 hr

705 hr

537 hr

393 hr

Figure 4.5: Chromium conc. remaining in blank water samples over time

4.2.4 Discussions of pH effects on metal concentrations in blank samples

It was reported that pH variations affect metal concentrations in blank samples. According to Barron et al. (1982), if metals are present in wastewaters that contain hexavalant chromium, this chromium must be reduced prior to metal removal. In general, hydroxides usually prove to be the controlling species for adsorbing metal from industrial or domestic wastewater.

The ANOVA analyses show that there is no effect of exposure time to metal concentrations in blank samples for 1 and 3 mg/L (0.7 < 2.1; 1.6 < 2.1 respectively) but for 6 mg/L, the exposure time shows a significant effect on metal concentrations (2.7 > 2.1). According to pH variations, type of metal (zinc and chromium) and concentrations (1, 3 and 6 mg/L), there is a high significant difference (P = 0.05) observed during the experiment.

4.3 pH variations and Zn(II) and Cr(VI) concentrations in water

samples with water hyacinths

4.3.1 Variations of pH on metal removal by the plants

The pH is an important parameter affecting the rate and the extent of biosorption of metal ions onto bioadsorbents such as water hyacinth plants. The variation of pH may affect the surface charge of roots of water hyacinth plants and also the solubility of metal ions. Some metal ions are known to be adsorbed or absorbed in the form of

hydroxides at high pH values such as pH>6. For this reason the effects of initial pH on biosorption of Zn (II) and chromium (VI) ions onto water hyacinth plants was investigated for the initial pH values equals to 6.7.

The variations of pH, zinc and chromium concentrations in water samples with water hyacinth plants after 4 weeks of lab experiments in which was observed an increasing in pH up to pH > 7.5 at 105 hr for 1 mg/L, 3 mg/L and 6 mg/L and then the situation changes after 105 hr of exposure to metal.

It was shown that pH variations affects metal removal during the experiment. Figure 4.6 showed that the pH slightly increased from the starting time (0 hr) (pH= 6.7) to 105 hr (pH= 7.64 to 7.86). However, after 105 hr of experiment, the pH decreased due to the saturation of adsorption sites, so some H⁺ are released in water samples that caused the decreasing of pH.

Zinc and chromium ions removal from solution was almost completed within 105 hours for pH values above 6 and bellow 8 because of adsorption. The pH of 6 is the critical point for zinc ions because of zinc hydroxide adsorption or absorption. Therefore, it can be said that the optimum pH for adsorption and absorption of zinc (II) and chromium (VI) ions by water hyacinth plants in lab experimental set up is about pH =7.5.

Oh

1 hr

3 hr

6 hr

10 hr

15 hr

21 hr

33 hr

57 hr

105 hr

177 hr

273 hr

393 hr

537 hr

705 hr

Exposure time (h)

pH, 1mg/L pH, 3mg/L pH, 6mg/L

pH

4

9

8

7

6

5

Figure 4.6: pH variations in plant water samples over time

4.3.2 Zinc concentrations remaining in water samples after 4 weeks of experiment.

Figure 4.7 plotted out the remaining zinc concentration in water samples after four weeks of lab experiments. It was observed that about 60% of zinc (II) was removed within 21 hours. Water hyacinths are effective plants for zinc (II) ions removal form wastewater in the range of concentration between 1 to 6 mg/L. Because the outer surfaces of water hyacinth roots are negatively charged with some acetate groups and metal ions are positively charged, roots attract metal ions, but when adsorption sites of roots are saturated, it is expected that metal ions can be released in water samples. The detention time must be determined. The passage in time influence the metal removal, as the plant can once again release the metal ion back into the water column when the adsorption sites become saturated. The decrease of pH in water samples related to growing time is an important factor in metal absorption and adsorption mechanisms by the plants. It is very clear that after 21 hr, little trace elements of metal are still present in water samples with water hyacinth plants.

0.25

Exposure time (h)

57 hr

Oh

1 hr

537 hr

6 hr 10 hr

177 hr
273 hr

705 hr

393 hr

105 hr

33 hr

21 hr

15 hr

3 hr

Zn2+, 1mg/L
Zn2+, 3mg/L
Zn2+, 6mg/L

0.2

0.15

Conc. (mg/L)

0.1

0.05

0

Figure 4.7: Zinc conc. remaining in water samples with water hyacinth plants over time

4.3.3 Chromium conc. remaining in water after 4 weeks of experiment.

Figure 4.8 highlights the capacity of water hyacinth plants in chromium removal from wastewater. From this figure, it was observed that chromium was fixed on the outer surface of plant roots, but this fixation was not effective because of some trace elements of chromium in water samples. After a certain time, the plant begins to release this metal again in the water due to the saturation of adsorption sites on root system.

Oh

1 hr 3 hr 6 hr

10 hr 15 hr 21 hr 33 hr 57 hr 105 hr 177 hr

273 hr 393 hr 537 hr 705 hr

Exposure time (h)

Conc. (mg/L)

0.16

0.14

0.12

0.08

0.06

0.04

0.02

0.1

0

Cr6+,1mg/L Cr6+, 3mg/L Cr6+,6mg/L

Figure 4.8: Chromium conc. remaining in water samples with water hyacinth plants

4.3.4 Discussions on pH variations and metal removal by the plants

Metal can precipitate as hydroxides simply by raising the pH of the wastewater to the range of pH 8 to 11 (Barron et al., 1982). As a result, the extent of adsorption or absorption was rather low at low pH values. However, in the equilibrium solid phase, Zn (II) and Cr (VI) ion concentrations increased with increasing pH because of increasingly negative charges on the surfaces of the roots at high pH values that attracted positively charged Zn (II) and Cr (VI) ions more strongly.

The ANOVA showed that for 1 mg/L, there is no effect of exposure time (P = 0.05) but a
high effect of pH on metal remained in water samples (P = 0.05). For 3 mg/L, the trend is
the same; no effect of exposure time (P = 0.05) and high effect of pH effects on metal

remaining (P = 0.05). For 6 mg/L there was no effect of exposure time (P = 0.05) but high difference between pH effects and metal remaining (P = 0.05).

4.4 Bioconcentration Factor (BCF) for zinc and chromium

The bioconcentration factor (BCF) is a parameter showing the ability of plant materials to accumulate metals in tissues. It was seen that when the concentration of metal increases, water hyacinth plants are not able to accumulate metal ions. The plants have the limit for metal accumulation in their tissues.

4.4.1 Bioconcentration Factor for zinc

Bioconcentration factor (BCF) is a useful parameter to evaluate the potential of the plants in accumulating metals and this value was calculated according to dry weight basis. Figure 4.9 shows that the bioconcentration factor of zinc decreases while the metal concentration increases. This demonstrates that water hyacinth is able to accumulate zinc at low concentrations, which contributes particularly to plant cells building.

Bioconcentration Factor of Zinc.

initial conc.(mg/L) conc.in plant BCF

tissues (mg/L)

Variation of Zinc conc.

conc. in mg/L

4

8

6

2

0

1 mg/L
3 mg/L
6 mg/L

Figure 4.9: Bioconcentration factor of Zinc

I.C: Initial conc. in mg/L conc./PT: conc. in plant tissues in mg/Kg

BCF: bioconcentration factor

4.4.2 Bioconcentration Factor for chromium

Figure 4.10 plotted the bioconcentration factor of chromium and shows the trend as for zinc. The increase in concentration reduces the ability of the plant to accumulate more trace elements of metals. The Bioconcentration factor of chromium appears to be constant, independent to the initial concentration.

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

BCF of Cr

10

6

4

2

8

0

I.C Conc./P.T BCF

IC vs Conc. in plant tissues

BCF of Chromium

1 mg/L 3 mg/L 6 mg/L

Figure 4.10: Bioconcentration factor of Chromium

I.C: Initial conc. in mg/L conc./PT: conc. in plant tissues in mg/kg

BCF: bioconcentration factor

4.4.3 Discussions on bioconcentration factor

In comparing the two metals studied (Zn (II) and Cr (VI)); the BCF of zinc seems to be higher than the chromium's BCF for 1 and 3 mg/L, but very low for 6 mg/L for zinc. It was seen that the plant accumulated more low concentrations than the high ones.

Tables 4.1 and 4.2 show the variations on bioconcentration factors of zinc and chromium and it is reported that there is no significant difference both for zinc and chromium when comparing initial concentrations to the concentrations in plant tissues and bioconcentration factors (P = 0.05) for zinc and chromium.

Table 4.1: variations on bioconcentration factor of zinc BCF of Zinc ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
Initial concentrations Conc./PT & BCF Error Total	1.3 0.8 0.4 2.5	2 1 2 5	0.7 0.8 0.2	3.3 3.7	0.2 0.2	19.0 18.5

Table 4.2: variations on bioconcentration factor of chromium

BCF of Chromium ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
initial concentrations Conc./PT & BCF Error Total	3.5 4.5 3.6 11.6	2 1 2 5	1.7 4.5 1.8	1.0 2.5	0.5 0.3	19.0 18.5

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

4.5 Bioaccumulation

Metal accumulation in plant and water samples was measured. Digestion of samples in this study was performed according to the Standard Methods (APHA/AWWA/WEF, 2005). Plant biomass samples were decomposed to dry matter by heating at 105°C for 24 hours in a hot air oven.

According to Lin and Zhang (1990), metal accumulations by macrophytes can be affected by metal concentrations in water and sediments. The accumulation of metal in plant material is expressed as mg of metal per kg of dry matter. The data for accumulation of chromium and Zinc are expressed in different forms such as uptake, translocation ability and adsorption, both for the top (shoots) and the roots of plants exposed to metal-containing water. The zinc concentration in both the roots and the shoots tended to increase with increasing concentration of zinc and also with the passage of time.

4.5.1 Adsorption of Zinc by water hyacinth plants

The adsorption mechanism was performed by using EDTA-Na2 to remove metals fixed on outer surface of the roots performed the adsorption mechanism. This mechanism help to understand the ability of water hyacinth plants to fix metals on the roots.

The adsorption behavior of zinc was assessed by immersing roots in different volumetric beakers with 100 ml EDTA-Na2 at different periods of time for desorption. The Figures 4.11, 4.12 and 4.13 shows that the metal concentration decreases when the passage in time of desorption increases (Figure 4.13). Except some differences observed in Figures 4.11, and 4.12, the situation looks to be the same in general. The high concentration adsorbed in 1 week was around 0.036 mg/L (1 mg/L initial concentration), 0.16 mg/L for 2 weeks (3 mg/L) and 0.2 mg/L for 4 weeks (1 mg/L).

conc. in mg/L

0.04

0.03

0.02

0.01

0

5 min

Desorption of Zinc for 1week

10 min

Period of time for removal

15 min

1 mg/L 3mg/L 6 mg/L

20 min

25 min

30 min

35 min

40 min

45 min

Figure 4.11: Desorption of Zinc after 1 week

Desorption of Zinc for 2 weeks

5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min

Period of time for removal

conc. in mg/L

0.2

0.15

0.1

0.05

0

1 mg/L 3 mg/L 6mg/L

Figure 4.12: Desorption of Zinc after 2 weeks

Desorption of Zinc for 4 weeks

1 mg/L 3 mg/L 6 mg/L

0.25

0.2

0.15

0.1

0.05

0

conc. in mg/L

45 min

5 min

10 min

15 min

20 min

35 min

30 min

25 min

40 min

Period of time for removal

Figure 4.13: Desorption of Zinc after 4 weeks

4.5.2 Total adsorption of zinc

Figure 4.14 show that the adsorption for the 1 mg/L zinc initial concentration increased with the exposure time, but higher concentrations (3 and 6 mg/L) reduce the ability of water hyacinth plants to adsorb metal. The majority of molecules are adsorbed onto the roots.

	1 0.8 0.6 0.4 0.2 0	Total desorption of Zinc		1 mg/L 3 mg/L 6 mg/L
conc. (mg/L)
		1 week 2 weeks 4 weeks

Exposure time (week)

Figure 4.14: Total desorption of Zinc

4.5.3 Adsorption of chromium by water hyacinth plants

The adsorption of chromium presented on Figure 4.15 shows a decrease in concentration desorbed on external surface of roots according to time. The capacity of water hyacinth plants to adsorb trace elements of chromium depend on several factors which can affect this mechanism This means that more trace elements of chromium was removed in 5 to 15 minutes and the high concentration observed for 6 mg/L was around 2 mg/L, for 3 mg/L was around 1.6 mg/L and for 1 mg/L was around 0.3 mg/L.

It shows the same situation as for zinc that 5 to 15 minutes are sufficient to remove the maximum quantity of chromium on roots and the concentration of trace elements decreases with the passage of time of desorption.

Period of time for removal (min.)

conc.in (mg/L)

1 mg/L 3 mg/L 6 mg/L

3

2

2

1

1

0

5 min

15 min

10 min

20 min

35 min

30 min

25 min

40 min

45 min

Figure 4.15: Desorption of Chromium

4.5.4 Discussions on adsorption mechanism

The adsorption ability of water hyacinth plants seems to be different when zinc and chromium are compared. It was seen that for zinc 17.6% of 1 mg/L was adsorbed by the water hyacinth plants, 6.1% of 3 mg/L was adsorbed and the plants adsorbed 1.1% of 6 mg/L. Whereas for chromium, 9.0% of 1 mg/L, 36.4% of 3 mg/L and 54.6% of 6 mg/L were adsorbed on the roots of water hyacinth plants.

4.6 Uptake mechanism

4.6 1 Uptake mechanism for zinc

The uptake mechanism was observed to identify which part of water hyacinth plant contributes much in metal ions accumulation. The variation of uptake versus metal dosage for zinc are shown in Figure 4.16 and exhibited linearity at the low level of exposure time (1 week for petioles and leaves); however, the linearity trend could not be established with confidence for leaves and roots for 1 and 4 weeks. The regression coefficients for zinc (II) were found to be 0.6379 for 1 and 3 mg/L, 0.3195 for 3 and 6 mg/L and 0.3660 for 1 and 6 mg/L during all the experimental period. Thus, the uptake process apparently followed an increasing trend with a linear increase of metal concentrations in petioles for 1 week and 4 weeks but in 2 weeks, the pattern of uptake changes. It was observed that petioles are important parts for metal ions accumulation in water hyacinth plants

exposure time (wk) vs initial conc. (mg/L)

roots petioles leaves

Uptake mechanisms

0,6

0,5

0,4

0,3

0,2

0,1

0

Conc. (mg/Kg)

1 mg/L

1 mg/L

1 mg/L

3mg/L

3mg/L

6mg/L

3mg/L

6mg/L

6mg/L

4 weeks

2 weeks

1 week

Figure 4.16: Variations of uptake for zinc by the plants

Figure 4.16 depicted the uptake of zinc (II), which shows to be in normal distribution according to metal concentration, but it exhibits the changes when exposure time increases. Thus, the present observations showed that the extent of metals (Zn) uptake by plant was dependent on the concentration of the metal in the solution as well as the length of exposure to the plants.

4.6.2 Uptake mechanism for chromium

The figure 4.17 describes the uptake mechanism which demonstrates the important part of water hyacinth plant in metal accumulation. As seen from this figure, roots are important parts for chromium accumulation in the plants. This show a difference with zinc, which was more accumulated in petioles. This Figure 4.17 continues to show the behavior of chromium in plant tissues and it is clear that roots are the important parts for the accumulation of chromium in the water hyacinth plants. When chromium is mixed with zinc in the same water samples, zinc is more mobile than chromium, so zinc will be absorbed very quickly than chromium. Petioles come in second position in metal uptake for 3 mg/L. The uptake is linear according to concentration for roots and leaves but less for petioles.

dry weight
(mg/kg)

4

2

3

0

1

Uptake mechanism for chromium

roots petioles leaves

1 mg/L 3 mg/L 6 mg/L

Initial conc. (mg/L)

Figure 4.17: Uptake of chromium in plant tissues for different initial concentrations

4.6.3 Discussions on uptake mechanism

The discussion on uptake mechanism for zinc was reported that 56.7% of zinc was
accumulated in petioles, 27.0% in leaves and 16.3% in roots. Table 4.3 indicates that
there is no significant difference (p<=0.05) according to initial concentration and

exposure time (p<=0.05) in uptake mechanisms of zinc, but a high difference (p<=0.05) (significant) was observed in plant parts (p<=0.05) in uptake processes.

Table 4.3: Variability in zinc uptake compared to initial concentration & exposure time.

ANOVA
Source of Variation	SS^"	df^c	MSd	F^e	P-value^f	F crit
I.C^a & exposure time Plant plants Error Total	0.05 0.22 0.06 0.33	8 2 16 26	0.01 0.11 0.00	1.68 28.75	0.18 0.00	2.59 3.63

a: initial concentration; b: square sums; c: degree of freedom; d: means squared; e: Fischer test; f: probability value.

However, for chromium it was observed that 73.7% was taken up in roots, 14.1% in petioles and 12.2% in leaves. This shows the preference of the plant to store chromium more in roots than in petioles and leaves. Table 4.4 shows that no significant difference (p<=0.05) existed between plant parts (p<=0.05) and also between initial concentrations in uptake processes for chromium (p<=0.05). The inhibition in the uptake was perhaps because of the competition of both the metals for the same site of the plant during metabolism processes of the plants.

Table 4.4: variability in uptake of chromium ANOVA
Source of Variation	SS^"	df^c	MSd	F^e	P-value^f	F crit
plant parts I.C^a. Error Total	4.57 2.36 2.14 9.07	2 2 4 8	2.28 1.18 0.53	4.27 2.21	0.10 0.23	6.94 6.94

a: initial concentration; b: square sums; c: degree of freedom; d: means squared; e: Fischer test; f: probability value.

4.7 Translocation Ability (TA)

4.7.1 Variation of translocation ability for zinc

The translocation ability is a parameter, which shows the ability of the aquatic macrophytes to take up the trace elements in the top part of plants (leaves, petioles and flowers). Most times, the translocation ability of roots/leaves seems to be high when compared to roots/petioles, the reason is that more trace elements were accumulated in

petioles. When concentration accumulated in roots compared to one accumulated in leaves is high than roots concentration compared to petioles concentration.

Figure 4.18 shows that the high translocation ability for 1 mg/L was observed for roots/leaves during 1 week, for 3 mg/L for 4 weeks (roots/leaves) and for 6 mg/L was observed for 2 weeks for roots/leaves, this can be explain by a little concentration of metal accumulated in leaves during plants' exposure to zinc.

1.0

0.8

0.6

0.4

values

0.2

0.0

1 week

2 w eeks

4 w eeks

1.2

roots vs	roots vs	roots vs	roots vs	roots vs	roots vs
petioles	leaves	petioles	leaves	petioles	leaves

Time (week) vs TA

1 mg/L 3 mg/L 6 mg/L

Figure 4.18: Translocation ability for Zinc by water hyacinth plants

The Figures 4.19, 4.20 and 4.21 show the correlations between the translocation ability, the metal concentrations and the exposure time of plants to zinc. This behavior indicates positive or negative correlation between the above parameters. It was shown that there is no correlation for 1 week between translocation ability and metal concentrations. For 2 weeks, a negative correlation was found (R square = 0.89) and for 4 weeks, a high positive correlation (R square = 0.97) was observed. This can be explaining by the key role of exposure time versus metal translocation ability by the plants.

1.5

y = -0.7505x + 0.9256
R2 = 0.0133

0.5

0.0

0.0 0.2 0.4 0.6

zinc concentration (mg/L)

roots vs leaves Linear (roots vs leaves)

1.0

TA

Figure 4.19: Translocation ability for 1 week

39

R. J. GAKWAVU (2007) MSc Thesis

TA

y = -10.817x + 5.7303
R2 = 0.8929

1.5

1.0

0.5

0.0

0.42 0.44 0.46 0.48 0.50

Zn conc. (mg/L)

roots vs leaves Linear (roots vs leaves)

Figure 4.20: Translocation ability for 2 weeks

y = 2.0929x - 0.0386
R2 = 0.9752

0.0 0.2 0.4 0.6

Zn conc. (mg/L)

roots vs leaves Linear (roots vs leaves)

1.5

TA

0.5

0.0

1.0

Figure 4.21: Translocation ability for 4 weeks

As shown on the above figures, the positive correlation between translocation ability and zinc concentration increase progressively when the exposure time increases, according to the regression coefficients observed.

4.7.2. Variation of translocation ability for chromium

Figure 4.22 presents the translocation ability of chromium; which is too high when compared to zinc translocation ability. It is explained by the fact more concentration of chromium was in roots, because the translocation ability is to analyze the capacity of plant parts storage.

Translocatioon ability of chromium

1 mg/L 3 mg/L 6 mg/L

Initial concentration

8

6

4

2

0

Translocation
ability

roots/petioles roots/leaves

Figure 4.22: Comparison of roots and shoots in translocation ability

It seems that the translocation ability of chromium is too high as shown in Table 4.5 compared to the zinc's translocation ability. The ability of plants to translocate trace elements of chromium is increased for roots/leaves (5.3 times for 1 mg/L, 6.5 times for 3 mg/L and 6 times for 6 mg/L). The number of times for roots/petioles decreases (4 times for 1 mg/L, 4 times for 3 mg/L and 7 times for 6 mg/L) because the order of storage was leaves<petioles<roots.

Table 4.5: Translocation ability of chromium by the plant

	I.C^a of chromium (VI)
Roots/shoots	1 mg/L	3 mg/L	6 mg/L
roots/petioles roots/leaves	4.104^b 5.288^b	3.663^b 6.487^b	6.831^b 5.965^b

a: initial concentration; b: times of storage in roots compared to shoots.

The Figure 4.23 reports that the correlation between roots and petioles is high (R square = 0.6) compared to the correlation between roots and leaves (R square = 0.3), this is because less quantity of Cr (VI) was translocated in leaves.

correlation between roots and shoots

8 y = 1.3635x + 2.139

R2 = 0.6314

6

4

y = 0.3385x + 5.2363
R2 = 0.317

0

0 1 2 3 4

conc. (mg/L)

roots/petioles roots/leaves

Linear (roots/petioles) Linear (roots/leaves)

x times roots/shoots

2

Figure 4.23: Correlation of roots vs. shoots

4.7.3 Discussions on translocation ability

Table 4.6 indicates the ANOVA 2 which shows the variability in translocation ability for zinc. It can be seen that the difference is not significant (p<=0.05) between metal concentration (p<=0.05) and no significant difference (p<=0.05) between roots and shoots translocation.

Table 4.6: variations in translocation ability of zinc ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
metal concentration roots/shoots Error Total	0,42 0,13 1,01 1,56	5 2 1 0 17	0 , 08 0,07 0,10	0,84 0,65	0,55 0,54	3,33 4,10

Stratford et al. (1984) found that the metals accumulations in water hyacinth increased linearly with the solution concentration in the order of leaves<petioles<roots in water hyacinth. For this research, the situation is different because the following order was observed: leaves<roots<petioles. When the concentration is high, the water hyacinth plant can accumulate little concentration in plant cells. The high translocation ability was observed for roots/leaves (1.114) for 1 week and the low translocation ability was observed for roots/petioles (0.109) for 4 weeks. Most of times, the translocation ability of roots/leaves seems to be high when compared to roots/petioles. The reason is that more trace elements were accumulated in petioles. When concentration accumulated in roots compared to one accumulated in leaves is high than roots concentration compared to petioles concentration.

Stratford et al. (1984) found that the metals accumulations in water hyacinth increased linearly with the solution concentration in the order of leaves<petioles<roots in water hyacinth. This agrees with the results of this study in the case of chromium concentration accumulation in water hyacinth plants, where the high concentration was accumulated in roots followed by petioles and then leaves.

5. CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

The removal mechanisms of zinc and chromium by water hyacinth plants showed that the plant concentrated a high amount of metals. The aquatic plant water hyacinth have shown promising potential for the removal of Cr (VI) and Zn (II) from industrial synthetic wastewater of three different concentrations (1, 3 and 6 mg / L).

Zn (II) was much more mobile than Cr (VI) due to their sizes and charges. The accumulation of metals in the roots and shoots of water hyacinth has been shown in laboratory experiments in which water hyacinth was used as a biological adsorbent. It was recoded that Zn (II) was more accumulated (leaves<roots<petioles) in petioles whereas Cr (VI) was more accumulated in roots (leaves<petioles<roots). This phenomenon can explain by the mobility of Zn (II) when compare to Cr (VI) and also Zn (II) at low concentration contributes to the plants cells building.

It was observed the Bioconcentration factor decreases while the Zn (II) and Cr (VI) concentrations increase. This expresses that aquatic macrophytes water hyacinths are able to accumulate Zn (II) at low concentration and which contributes particularly to plant cells building. The bioconcentration factor of Cr (VI) shows the same trend as for Zn (II) that the increasing in concentration reduces the ability of the plant to accumulate more trace elements of Cr (VI) and looks to be stagnant independently to the initial concentration.

The translocation ability of aquatic macrophytes water hyacinth plants look to be high for Zn (II) in petioles whereas is high in roots for Cr (VI) due to size of elements and their respective charges.

5.2 Recommendations

Further research is necessary to establish a practical scale demonstration system. The information from the laboratory pilot scale research should also be used to establish a best practice environment for integrated wastewater treatment by aquatic plants and to study also the effects of decrease in pH after 48 h on metal removal as the solution becomes more acidic and a release of trace metals can be expected;

The experiments conducted in this research were carried out using a laboratory synthetic wastewater. It is important to repeat the experiments with a real industrial wastewater on field and to assess the treatment performance.

Further experiments with other heavy metals and other aquatic macrophytes could be more benefits to optimize the mechanisms involved in heavy metals by aquatic macrophytes.

A combination of different types of macrophytes plants could be useful in order to increase the contact between the polluted water and the bioadsorbent for metal fixation.

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Appendices

Appendix I: Initial fresh weight and final fresh weight vs relative growth

Exposure time	Initial water	Final water	Relative growth
(week) with Zn & Cr	hyacinth fresh	hyacinth fresh
concentrations	weight (g)	weight (g)
1 wk, 1 mg/L	32.33	96.91	3.00
1 wk, 3 mg/L	34.50	85.23	2.47
1 wk, 6 mg/L	26.38	50.07	1.90
2 wks, 1 mg/L	19.23	39.96	2.08
2 wks, 3 mg/L	29.75	55.92	1.88
2 wks, 6 mg/L	42.90	80.18	1.87
4 wks, 1 mg/L	41.15	90.17	2.19
4 wks, 3 mg/L	43.21	93.05	2.15
4 wks, 6 mg/L	45.09	96.16	2.13

Appendix II: pH, zinc and chromium concentrations remaining in blank samples during 4 weeks of experiment

Time (hr)	pH	Blank,1mg/L Zn2+ Cr6+		Blank, 3mg/L pH Zn2+		Cr6+	pH	Blank, 6mg/L Zn2+ Cr6+
start	6.70	1.000	1.000	6.70	3.000	3.000	6.70	6.000	6.000
1 hr	7.07	0.062	0.006	7.04	0.068	0.080	7.11	0.070	0.118
3 hr	7.24	0.062	0.084	7.16	0.158	0.107	7.20	0.058	0.081
6 hr	7.23	0.050	0.108	7.17	0.022	0.090	7.21	0.019	0.077
10 hr	7.29	0.032	0.074	7.23	0.065	0.039	7.29	0.018	0.029
15 hr	7.45	0.008	0.000	7.35	0.000	0.000	7.46	0.030	0.000
21 hr	7.49	0.000	0.000	7.43	0.000	0.000	7.50	0.024	0.000
33 hr	7.82	0.021	0.000	7.68	0.007	0.000	7.82	0.000	0.000
57 hr	7.90	0.004	0.000	7.72	0.040	0.000	7.89	0.000	0.000
105 hr	7.64	0.016	0.000	7.86	0.000	0.000	7.65	0.004	0.000
177 hr	8.03	0.014	0.000	7.93	0.006	0.000	7.72	0.024	0.000
273 hr	6.6	0.005	0.012	7.37	0.015	0.079	7.60	0.032	0.021
393 hr	8.01	0.023	0.000	8.14	0.008	0.000	7.75	0.011	0.000
537 hr	7.12	0.000	0.016	7.73	0.000	0.061	7.25	0.000	0.001
705 hr	8.61	0.000	0.012	8.37	0.000	0.000	7.5	0.000	0.000
total		0.297	0.312		0.389	0.456		0.290	0.327

Appendix III: Variations in pH, zinc and chromium concentrations after 4 weeks of experiment with water hyacinth plants.

Time (h)

1 mg/L

Initial concentrations

3 mg/L

6 mg/L

pH Zn2+ Cr6+

pH Zn2+ Cr6+

pH Zn2+ Cr6+

start

6,70 1.000 1.000

6,70 3.000 3.000

6,70 6.000 6.000

1 hr

6,90 0,022 0,087

7,01 0,176 0,112

6,96 0,084 0,085

3 hr

7,07 0,012 0,115

7,13 0,062 0,080

7,09 0,023 0,098

6 hr

7,13 0,015 0,076

7,17 0,002 0,088

7,11 0,071 0,083

10 hr

7,23 0,002 0,001

7,42 0,000 0,000

7,34 0,014 0,000

15 hr

7,33 0,005 0,000

7,55 0,006 0,000

7,45 0,034 0,000

21 hr

7,46 0,024 0,000

7,61 0,013 0,000

7,52 0,000 0,000

33 hr

7,49 0,000 0,093

7,51 0,000 0,000

7,34 0,000 0,000

57 hr

7,48 0,000 0,000

7,53 0,000 0,000

7,34 0,004 0,000

105 hr

7,57 0,000 0,000

7,75 0,000 0,000

7,51 0,000 0,000

177 hr

7,19 0,003 0,000

7,58 0,001 0,000

6,97 0,004 0,007

273 hr

6,15 0,000 0,006

7,58 0,000 0,000

6,88 0,000 0,000

393 hr

6,61 0,009 0,000

7,6 0,000 0,000

5,67 0,006 0,007

537 hr

5,31 0,000 0,049

7,35 0,000 0,000

4,47 0,004 0,053

705 hr

4,93 0,000 0,000

7,27 0,000 0,000

4,43 0,000 0,000

total

0,092 0,427

0,26 0,28

0,244 0,333

Appendix IV: Adsorption variability of zinc during experimental period.

		Zn^2+, 1 week		Zn^2+, 2 weeks			Zn^2+, 4 weeks
Time for desorption	1 mg/L	3mg/L	6 mg/L	1 mg/L 3 mg/L		6mg/L	1 mg/L 3 mg/L		6 mg/L
5 min	0.036	0.026	0.016	0.046	0.051	0.079	0.110	0.134	0.018
10 min	0.023	0.034	0.006	0.021	0.156	0.049	0.203	0.083	0.038
15 min	0.011	0.026	0.005	0.024	0.051	0.059	0.055	0.077	0.029
20 min	0.017	0.020	0.004	0.045	0.020	0.053	0.046	0.077	0.012
25 min	0.016	0.014	0.007	0.027	0.038	0.064	0.068	0.034	0.049
30 min	0.014	0.019	0.014	0.015	0.030	0.030	0.026	0.049	0.024
35 min	0.020	0.012	0.005	0.017	0.039	0.034	0.032	0.034	0.014
40 min	0.029	0.011	0.006	0.016	0.130	0.017	0.042	0.039	0.014
45 min	0.010	0.020	0.004	0.008	0.110	0.099	0.030	0.038	0.019
Total	0.176	0.182	0.067	0.219	0.625	0.484	0.612	0.565	0.217

Zinc and chromium removal mechanisms from industrial wastewater by water hyacinth (Eichhornia crassipes) (Mart.) Solms-
Laubach

Appendix V: Adsorption variability of chromium during experimental period

Time	1mg/L	3 mg/L	6 mg/L
5 min	0.035	0.147	0.403
10 min	0.035	0.152	0.208
15 min	0.035	0.138	0.156
20 min	0.018	0.105	0.115
25 min	0.034	0.171	0.153
30 min	0.016	0.105	0.147
35 min	0.034	0.123	0.210
40 min	0.033	0.082	0.135
45 min	0.034	0.080	0.130
total	0.274	1.103	1.657

Appendix VI: Variation of uptake mechanism of zinc.

Time	I.C.^a	Roots	Petioles	Leaves	Total
	1mg/L	0.117^b	0.306^b	0.105^b	0.528^b
1 week	3mg/L	0.093^b	0.364^b	0.127^b	0.584^b
	6mg/L	0.080^b	0.487^b	0.209^b	0.776^b
	Total	0.290^b	1.157^b	0.441^b
	1mg/L	0.065^b	0.218^b	0.141^b	0.424^b
2 weeks	3mg/L	0.066^b	0.136^b	0.117^b	0.319^b
	6mg/L	0.145^b	0.330^b	0.146^b	0.621^b
	Total	0.276^b	0.684^b	0.404^b
	1mg/L	0.035^b	0.205^b	0.145^b	0.385^b
4 weeks	3mg/L	0.128^b	0.251^b	0.123^b	0.502^b
	6mg/L	0.042^b	0.386^b	0.165^b	0.593^b
	Total	0.205^b	0.842^b	0.433^b

a: I.C.: Initial Concentration in water samples; b: dry weight in mg/kg

Appendix VII: variation of uptake mechanism of chromium

I.C^a	1 mg/L	3 mg/L	6 mg/L	total
roots	0.550^b	1.531^b	3.436^b	5.517^b
petioles	0.134^b	0.418^b	0.503^b	1.055^b
leaves	0.104^b	0.236^b	0.576^b	0.916^b
total	0.788^b	2.185^b	4.515^b

a: Initial concentration in water samples; b: dry weight in mg/kg

Appendix VIII: Translocation ability of zinc by water hyacinth.

	Roots/ Shoots	1 mg/L	Zn (II) conc. 3 mg/L 6 mg/L
1 week	Roots/petioles	0.382^a	0.255^a	0.383^a
	Roots/leaves	1.114^a	0.732^a	0.164^a
2 weeks	Roots/petioles	0.478^a	0.485^a	0.439^a
	Roots/leaves	0.461^a	0.564^a	0.993^a
4 weeks	Roots/petioles	0.171^a	0.510^a	0.109^a
	Roots/leaves	0.241^a	1.041^a	0.255^a

a: times of zinc storage in roots compared to shoots.

Appendix IX: variations between initial concentrations and plant relative growth

a) Variations for 1 week of experiment

ANOVA
Source of Variation		SS	df		MS		F		P-value		F crit
I.C.(1, 3 & 6 mg/L) IFW, FFW and RG Error Total		547.5 8582.8 677.3 9807.6	2 2 4 8		273.8 4291.4 169.3		7.5 25.3		0.3 0.0		6.9 6.9
b) Variations for 2 weeks of experiment ANOVA															Source of Variation
SS	df			MS		F		P-value		F crit		I.C (1, 3 and 6 mg/L) IFW, FFW and RG Error Total
682.7 4830.1 418.9 5931.7	2 2 4 8			341.4 2415.0 104.7		3.3 23.1		0.1 0.0		6.9 6.9

c) Variations for 4 weeks of experiment ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
I.C.(1, 3 and 6 mg/L) IFW, FFW & RG Error Total	16.2 12453.0 9.5 12478.7	2 2 4 8	8.1 6226.5 2.4	3.4 2628.1	0.1 0.0	6.9 6.9

Appendix X: Variations between exposure time, pH effects and metal remained in water samples.

a) Concentration of 1 mg/L

ANOVA
Source of Variation	SS	df	MS	F	P-value	F crit
Exposure time pH & metal remained Error Total	3.03 465.04 5.94 474.01	14 2 28 44	0.22 232.52 0.21	1.02 1096.78	0.46 0.00	2.06 3.34

b) Concentration of 3 mg/L

ANOVA							Source of Variation SS
df	MS	F	P-value	F crit		Exposure time 0.29 pH & metal remained 542.58
14 2	0.02 271.29	0.68 8780.55	0.77 0.00	2.06 3.34		Error 0.87
28	0.03					Total 543.74
44						c) Concentration of 6 mg/L
ANOVA
Source of Variation SS	df	MS	F	P-value		F crit
Exposure time 4.98 pH & metal remained 448.82	14 2	0.36 224.41	1.01 638.11	0.47 0.00		2.06 3.34
Error 9.85	28	0.35
Total 463.65	44

Appendix XI: Preliminary water tests at the beginning
pH test for used waters:

Buutare valley pH= 7.7 Nyabugogo water pH=7.4

nyabugogo + butare valley pH=7.9 (in small buskets)

nyabugogo + butare valley pH=7.6 (in the big basins)

solution of _K2Cr2O7 pH = 7.2 solution of ZnCl2 pH = 6.4

fixed pH : 6.7 at starting in all small plastic buckets

Appendix XII: Raw data from lab experiments

Lab experiments (observation and analyses

No			date of sampling							Period			Sample no			pH sample			Zn (II)		Cr (VI)
			1june 06,2007							after 1h			S1BLK1			7,07			0,062		0,006
2													S1BLK3			7,04			0,068		0,008
3													S1BLK6			7,11			0,07		0,118
4													S1WH1,1			7,08			0,048		0,113
5													S1WH1,3			7,09			0,029		0,099
6													S1WH1,6			6,97			0,045		0,121
7													S1WH2,1			6,59			0,036		0,001
8													S1WH2,3			6,91			0,044		0,107
9													S1WH2,6			6,98			0,026		0,121
10													S1WH4,1			6,9			0,022		0,087
11													S1WH4,3			7,01			0,176		0,112
12													S1WH4,6			6,96			0,084		0,085
13										after 2h			S2BLK1			7,24			0,062		0,084
14													S2BLK3			7,16			0,158		0,107
15													S2BLK6			7,2			0,058		0,081
16													S2WH1,1			7,21			0,007		0,129
17													S2WH1,3			7,17			0,022		0,052
18													S2WH1,6			7,08			0,088		0,112
19													S2WH2,1			6,66			0,008		0,109
20													S2WH2,3			6,99			0,094		0,087
21													S2WH2,6			7,06			0,095		0,105
22													S2WH4,1			7,07			0,012		0,115
23													S2WH4,3			7,13			0,062		0,008
24													S2WH4,6			7,09			0,023		0,098
26										after 3h			S3BLK1			7,23			0,05		0,108
27													S3BLK3			7,17			0,022		0,090
28													S3BLK6			7,21			0,019		0,077
29													S3WH1,1			7,23			0,144		0,075
30													S3WH1,3			7,18			0,011		0,081
31													S3WH1,6			7,1			0,003		0,063
32													S3WH2,1			6,62			0,007		0,093
33													S3WH2,3			7			0,029		0,079
34													S3WH2,6			7,04			0,001		0,107
35													S3WH4,1			7,13			0,015		0,076
36													S3WH4,3			7,17			0,002		0,088
37													S3WH4,6			7,11			0,071		0,083
38								after 4h				S4BLK1			7,29			0,032				0,074
39												S4BLK3			7,23			0,065				0,039
40												S4BLK6			7,29			0,018				0,029
41												S4WH1,1			7,32			0,034				0,064
42												S4WH1,3			7,22			0,045				0,051
43												S4WH1,6			7,12			0,033				0,026
44												S4WH2,1			6,61			0,007				0,023
45												S4WH2,3			7,02			0,007				0,021
46												S4WH2,6			7,06			0,010				0,005
47												S4WH4,1			7,23			0,002				0,001
48												S4WH4,3			7,42			0.000				0.000
49												S4WH4,6			7,34			0,014				0.000
50
								after 5h				S5BLK1			7,45			0,008				0.000
51												S5BLK3			7,35			0.000				0.000
52												S5BLK6			7,46			0,03.0				0.000
53												S5WH1,1			7,44			0,014				0.000
54												S5WH1,3			7,32			0.000				0.000
55												S5WH1,6			7,14			0,098				0.000
56												S5WH2,1			6,7			0,013				0.000
57												S5WH2,3			7,17			0.000				0.000
58												S5WH2,6			7,12			0.000				0.000
59												S5WH4,1			7,33			0,005				0.000
60												S5WH4,3			7,55			0,006				0.000
61												S5WH4,6			7,45			0,034				0.000
62
63								after 6h				S6BLK1			7,49			0.000				0.000
63												S6BLK3			7,43			0.000				0.000
64												S6BLK6			7,5			0,024				0.000
65												S6WH1,1			7,55			0,001				0.000
66												S6WH1,3			7,56			0.000				0.000
67												S6WH1,6			7,48			0,032				0.000
68												S6WH2,1			6,83			0,062				0.000
69												S6WH2,3			7,35			0,014				0.000
70												S6WH2,6			7,32			0,098				0.000
71												S6WH4,1			7,46			0,024				0.000
72												S6WH4,3			7,61			0,013				0.000
73												S6WH4,6			7,52			0.000				0.000
74
74				june 07,2007				after12h				S7BLK1			7,82			0,021				0.000
75												S7BLK3			7,68			0,007				0.000
76												S7BLK6			7,82			0.000				0.000
77												S7WH1,1			7,62			0.000				0.000
78												S7WH1,3			7,47			0.000				0.000
79												S7WH1,6			7,42			0,022				0.000
80												S7WH2,1			6,23			0,012				0.000
81												S7WH2,3			7,13			0.000				0.000
82												S7WH2,6			6,85			0,002				0.000
83												S7WH4,1			7,49			0.000				0,093
84												S7WH4,3			7,51			0.000				0.000
85												S7WH4,6			7,34			0.000				0.000
86				june 08,2007				after24h				S8BLK1			7,9			0,004				0,000
87												S8BLK3			7,72			0,040				0,000
88												S8BLK6			7,89			0,000				0,000
89												S8WH1,1			7,48			0,000				0,000
90												S8WH1,3			7,35			0,000				0,000
91												S8WH1,6			7,37			0,005				0,000
92												S8WH2,1			5,65			0,004				0,000
93												S8WH2,3			6,62			0,000				0,000
94												S8WH2,6			6,28			0,032				0,000
95												S8WH4,1			7,48			0,000				0,000
96												S8WH4,3			7,53			0,000				0,000
97												S8WH4,6			7,34			0,004				0,000
98				june 10,2007				after48h				S9BLK1			7,64			0,016				0,000
99												S9BLK3			7,86			0,000				0,000
100												S9BLK6			7,65			0,004				0,000
101												S9WH1,1			7,8			0,000				0,000
102												S9WH1,3			7,64			0,000				0,000
103												S9WH1,6			7,57			0,000				0,000
104												S9WH2,1			5,52			0,002				0,000
105												S9WH2,3			6,07			0,000				0,000
106												S9WH2,6			6,16			0,016				0,000
107												S9WH4,1			7,57			0,000				0,000
108												S9WH4,3			7,75			0,000				0,000
109												S9WH4,6			7,51			0,000				0,000
110
					june 13,2007						after 72 h			S10BLK1			8,03			0,014				0,000
111														S10BLK3			7,93			0,006				0,000
112														S10BLK6			7,72			0,024				0,000
113														S10WH1.1			7,24			0,011				0,000
114														S10WH1,3			7,21			0,000				0,000
115														S10WH1,6			7,23			0,009				0,000
116														S10WH2,1			5,19			0,017				0,000
117														S10WH2,3			4,94			0,011				0,000
118														S10WH2,6			6,35			0,012				0,000
119														S10WH4,1			7,19			0,003				0,000
120														S10WH4,3			7,58			0,001				0,000
121														S10WH4,6			6,97			0,004				0,007
123
124					june 15,2007						after 96 h			S11BLK1			6,6			0,005				0,012
125														S11BLK3			7,37			0,015				0,079
126														S11BLK6			7,6			0,032				0,021
127														S11WH2,1			4,57			0,024				0,079
128														S11WH2,3			4,5			0,001				0,065
129														S11WH2,6			6,6			0,009				0,045
130														S11WH4,1			6,15			0,000				0,006
131														S11WH4,3			7,58			0,000				0,000
132														S11WH4,6			6,88			0,000				0,000
133
134					june 20,2007						after 120h			S12BLK1			8,01			0,023				0,000
135														S12BLK3			8,14			0,008				0,000
136														S12BLK6			7,75			0,011				0,000
137														S12WH2,1			4,75			0,029				0,000
138														S12WH2,3			4,87			0,008				0,000
139														S12WH2,6			6,48			0,008				0,000
140														S12WH4,1			6,61			0,009				0,000
141														S12WH4,3			7,6			0,000				0,000
142														S12WH4,6			5,67			0,006				0,007
143
					1 mg/L						3 mg/L			6 mg/L
roots	0.324167					0			0
petioles	0.134					0.418			0.503
leaves	0					0			0
0.4582 0.418 0.503
total		% of Cr BCF
0.324167		23.5
1.055		76.5
0		0.0
1.379167		100
144					26-juin-07						after 144h			S13BLK1			7,12			0,000				0,016
145														S13BLK3			7,73			0,000				0,061
146														S13BLK6			7,25			0,000				0,001
147														S13WH4,1			5,31			0,000				0,049
148														S13WH4,3			7,35			0,000				0,000
149														S13WH4,6			4,47			0,004				0,053
150
151					3-juil-07						after 168h			S14BLK1			8,61			0,000				0,012
152														S14BLK3			8,37			0,000				0,000
153														S14BLK6			7,5			0,000				0,000
154														S14WH4,1			4,93			0,000				0,000
155														S14WH4,3			7,27			0,000				0,000
156														S14WH4,6			4,43			0,000				0,000

Appendix XIII: Bioconcentration Factor of zinc

Part	1 mg/L	3 mg/L	6 mg/L	total	% of BCF of Zn
roots	0.032	0.081	0.812	0.925	16.5
petioles	0.793	1.491	0.75	3.034	54.1
leaves	0.171	1.095	0.383	1.649	29.4
	0.996	2.667	1.945	5.608	100.0

Appendix XIV: Bioconcentration Factor of chromium

Appendix XV: Synthetic wastewater and Nutrient solutions

A) Heavy metals concentration in synthetic wastewater

Substances	Concentration (mg / L)
Zn (mg/l)	5
Cu (mg/l)	0,4
Pb (mg/l)	1
Cr (mg/l)	6

B) Heavy metals concentration in synthetic wastewater

Substances	Concentration (mg / L)
Zn (mg/l)	5
Cu (mg/l)	0,4
Pb (mg/l)	1
Cr (mg/l)	6
SO4^2- (mg/l)	200 - 2000
OC (mg/l)	50 - 500

C) Hutner solution (Leman, 2000)

Substances	Concentration (mg / L)
NH4Cl	20
K2HPO4	40
Ca(NO3)2 4H2O	20
MgSO4 4H2O	50
FeSO4 7H2O	2,5
MnCl2 4H2O	1,5
ZnSO4 7H2O	6,5
H3BO3	1,5
Na2MoO4 2H2O	2,5
CuSO4 7H2O	0,4
CoSO4 7H2O	0,1
EDTA (Na)2	50

Appendix XVI: Protocol used in laboratory experiments

The destruction of plants and fish tissue for the determination of Cd, Cr, Cu, Pb, Mn,

Fe and Zn with the atomic absorption technique

2.2.1 Apparatus

a) Destruction-bloc with destruction tubes made of borosilicate glass

b) Nichiryo pipet model 3100 with removable tips

2.2.2 Reagents, all with a low metal content

a) Nitric acid, 65% HNO3

b) Hydrogen peroxide, 30% H2O2

c) Pumice

2.2.3 Glassware

All rinsed with 1 + 1 HNO3

Measuring cylinder, 50 mL

Funnels with a diameter of 6 cm Volumetric flasks of 250 mL

2.2.4 Procedure

1. Transfer not more than 1.250 g dried sample (24 hours at 103°C) to the destruction tube, add 25 mL HNO3, three boiling chips and place a funnel on

top of the destruction tube.

2. a) Heat the tube to 100°C and maintain for 1 hour b) Heat to 125°C and maintain for 15 minutes

c) Heat to 150^°C and maintain for 15 minutes

d) Heat to 175^°C and maintain for 15 minutes

e) Heat to 200^°C and add, if necessary (if no volume is left), 5 mL HNO3

3. Concentrate to about 5 mL

4. Add, after cooling, 1 mL 30% _H2O2 and destruct for 10 minutes, repeat 1 x

5. Add, after cooling, 3 mL 30% _H2O2 and destruct again for 10 minutes

6. Add 25 mL water, mix and heat till boiling

7. Cool and transfer the whole sample to a 250 mL volumetric flask, fill up to the mark, mix and let settle during at least 15 hours

8. Measure the absorbance of the clear supernatant.

Note: 1. Duration of procedure steps 1-7 at least 7 hours 2. Determine two blancs

2.3 The destruction of soil and sludge for the determination of Cd, Cr, Cu, Pb,

Mn, Fe and Zn with the atomic absorption technique

2.3.1 Apparatus

a) Destruction-bloc with destruction tubes made of borosilicate glass

b) Nichiryo pipet model 3100 with removable tips

2.3.2 Reagents

All reagents with a low percentage of heavy metals

a) Hydrochloric acid, 37% HCl

b) Nitric acid, 65% HNO3

c) Hydrogen peroxide, 30% H2O2

d) Pumice

2.3.3 Glassware

All rinsed with 1 + 1 HNO3

Measuring cylinder, 500 mL Measuring cylinder, 50 mL Funnels with a diameter of 6 cm

1 L flask for the acid-mixure, see note

Volumetric flasks of 250 mL

2.3.4 Procedure

1. Transfer not more than 1.250 g of a ground air-dried sample to the destruction tube, add 50 mL H2O and three boiling chips

2. Add 50 mL HCl/HNO3 3:1, mix, and place a funnel on top of the destruction

tube

3. a) Heat the tube to 100°C and maintain for 1 hour b) Heat to 125°C and maintain for 15 minutes

c) Heat to 150^°C and maintain for 15 minutes

d) Heat to 175°C and maintain for 15 minutes

e) Heat to 200^°C and add, if necessary (if no volume is left), 5 mL HNO3

4. Concentrate to about 5 mL

5. Add, after cooling, 1 mL 30% H2O2 and destruct for 10 minutes. Repeat 1 x

6. Add, after cooling, 3 mL 30% _H2O2 and destruct again for 10 minutes

7. Add 50 mL water and 25 mL HCl, mix and heat till boiling

8. Cool and transfer the whole sample to a 250 mL volumetric flask, fill up to the mark, mix, and let settle during at least 15 hours

9. Measure the absorbance of the clear supernatant.

Note: 1. Prepare a fresh acid-mixture and do not close the container!!

2. Duration of procedure steps 1-7 at least 7 hours

3. Determine two blancs

4. Sludge samples will decompose almost completely

2.4 Preliminary Digestion for Metals in water

To reduce interference by organic matter and to convert metal associated with particulates to a form (usually the free metal) that can be determined by atomic absorption spectrometry or inductively-coupled plasma spectroscopy, use one of the digestion techniques presented below. Use the least rigorous digestion method required to provide complete and consistent recovery compatible with the analytical method and the metal being analyzed.

Nitric acid will digest most samples adequately. Nitrate is an acceptable matrix for both flame and electrothermal atomic absorption. Some samples may require addition of perchloric, hydrochloric, or sulfuric acid for complete digestion. These acids may interfere in the analysis of some metals and all provide a poorer matrix for electrothermal analysis. Confirm metal recovery for each digestion and analytical procedure used. As a general rule, HNO3 alone is

adequate for clean samples or easily oxidized materials: HNO3-H2SO4 or
HNO3-HCl digestion is adequate for readily oxidizable organic matter; HNO3-
HClO4 or HNO3-HClO4-HF digestion is necessary for difficult-to-oxidize organic

matter or minerals. Dry ashing is helpful if large amounts of organic matter are present, but yields highly variable precision and bias, depending on sample type and metal being analyzed. Dry-ash only samples that have been shown to yield acceptable precision and bias.

2.4.1 Dry ashing procedure, use only if necessary

Mix sample and tranfser a suitable volume into a platinum or high-silica glass evaporating dish. Evaporate to dryness on a steam bath. Transfer dish to a muffle furnace and heat sample to a white ash. If volatile elements are to be determined, keep temperature at 400 to 450°C. If sodium only is to be determined, ash sample at a temperature up to 600^°C. Dissolve ash in a minimum quantity of conc HNO3 and warm water. Filter diluted sample and

adjust to a known volume, preferably so that the final HNO3 concentration is about 1%. Take portions of this solution for metals determination.

2.4.2 Nitric acid digestion procedure

Apparatus

a) Hot plate

b) Conical flasks, 100 mL, acid-washed and rinsed with water

Reagents

Nitric acid, HNO3, conc. Procedure

Mix sample and transfer a suitable volume (50 to 100 mL) to a 100 mL conical
flask or beaker. Add 5 mL conc. HNO3 and a few boiling chips. Bring to a slow

boil and evaporate on a hot plate to the lowest volume possible (about 10 to 20

mL) before precipitation occurs. Continue heating and adding conc HNO3 as

necessary until digestion is complete as shown by a light-colored, clear solution. Do not let sample dry during digestion.

Wash down flask or beaker walls with water and then filter if necessary. Transfer filtrate to a 100 mL volumetric flask with two 5 mL portions of water, adding these rinsings to the volumetric flask. Cool. dilute to mark, and mix thoroughly. Take portions of this solution for required metal determinations.

2.4.3 Nitric Acid-Hydrochloric Acid Digestion Apparatus see 2.4.2

Reagents

a) Nitric acid, HNO3, conc.

b) Hydrochloric acid, 1 + 1

Procedure

a) Total HNO3/HCl: Transfer a measured volume of wellmixed, acid-preserved

sample appropriate for the expected metals concentrations to a flask or
beaker. Add 3 mL conc HNO3. Place flask or beaker on a hot plate and

cautiously evaporate to less than 5 mL , making certain that sample does not
boil and that no area of the bottom of the container is allowed to go dry. Cool
and add 5 mL conc HNO3. Cover container with a watch glass and return to

hot plate. Increase temperature of hot plate so that a gentle reflux action occurs. Continue heating, adding additional acid as necessary, until digestion is complete (generally indicated when the digestate is light in color or does not change in appearance with continued refluxing). Evaporate to less than 5 mL and cool. Add 10 mL 1 + 1 HCl and 15 mL water per 100 mL anticipated final volume. Heat for an additional 15 min to dissolve any precipitate or residue. Cool, wash down beaker walls and watch glass with water, and adjust to a predetermined volume based on the expected metals concentrations. If necessary (when particles are present), let settle overnight.

b) Recoverable HNO3/HCl: For this less rigorous digestion procedure, transfer

a measured volume of well-mixed, acid-preserved sample to a flask or
beaker. Add 2 mL 1 + 1 HNO3 and 10 mL 1 + 1 HCl and heat on a steam

bath or hot plate until volume has been reduced to near 25 mL, making certain sample does not boil. Cool and let settle overnight. Adjust volume to 100 mL and mix.