INFLUENCE OF SILICON ON HEAVY METALS UPTAKE AND MOBILITY, YIELDS, AND QUALITY OF SELECTED LEAFY VEGETABLES IN KIAMBU COUNTY, KENYA By MOSES MWANGI NGUGI (BSc. Agricultural Education and Extension) A144/25561/2018 A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE AWARD OF MASTER OF SCIENCE DEGREE IN AGRONOMY IN THE SCHOOL OF AGRICULTURE ENTERPRISE DEVELOPMENT, KENYATTA UNIVERSITY OCTOBER, 2021. DECLARATION I declare that this thesis is my original work and has not been presented for a degree or any other award in any university. Signature………………………………DATE………………………. MOSES M. NGUGI REG NO: A144/25561/2018 Supervisors The research thesis has been submitted for review with our approval as university supervisors. Signature ………………………………. DATE…………………… Dr. Joseph P. Onyango Gweyi Department of Agricultural Science and Technology Kenyatta University Signature………………………………DATE………………………… Dr. Catherine W. Muui Department of Agricultural Science and Technology Kenyatta University DEDICATION I dedicate this thesis to the Great Kamindo family. ACKNOWLEDGEMENT I humbly thank the Almighty God for his mercy, opportunity, and strength, above all His faithfulness and unconditional love during my entire academic life. Psalms 106:1, Praise the LORD. Give thanks to the LORD, for he is good; his love endures forever. I am deeply grateful to my Supervisors, Dr. Joseph Gweyi and Dr. Catherine Muui, Kenyatta university, for their unwavering guidance, invaluable insights, profound belief in my abilities, constructive criticism, and scholarly comments throughout the research work and coursework. I owe them my deepest gratitude for their dedication and encouragement, without their ingenious suggestions and dedication, this thesis would have not materialized. I would also like to extend my deepest gratitude to the entire Agricultural science (AST) department, Kenyatta university for providing a conducive learning environment, farm, and laboratory facilities. I am deeply indebted to Karen Kaaria, Winnie Ntinyari, Mathew kariuki, Phobian Makoha, Michael Sakha, and Jane Karambu for their unparalleled knowledge, practical suggestion, and helpful advice during laboratory work. I extend my sincere thanks to Barnabas, Mbogo, and Peter for their help during greenhouse and field experiments. I also extend my appreciation to my fellow students, Jeff, Ochieng, Josette, Michael, Psiwa, and Dennis for their guidance, moral support, advice, and patience that cannot be underestimated, during my coursework and research. Finally, am extremely grateful to my lovely parents, brother, and sisters. I wholeheartedly appreciate that your encouragement and prayers proved monumental towards the success of this study. TABLE OF CONTENTS DECLARATION ......................................................................................................... ii DEDICATION .......................................................................................................... iii ACKNOWLEDGEMENT .......................................................................................... iv LIST OF TABLES .................................................................................................. viii LIST OF FIGURES .................................................................................................... ix LIST OF ACRONYMS AND ABBREVIATIONS ................................................... xi ABSTRACT .............................................................................................................. xii CHAPTER ONE: INTRODUCTION ......................................................................... 1 1.1 Background ........................................................................................................ 1 1.2 Statement of the problem ................................................................................... 3 1.3 Research objectives ............................................................................................ 4 1.3.1 General objective......................................................................................... 4 1.3.2 Specific objectives....................................................................................... 4 1.4 Hypotheses ......................................................................................................... 4 1.5 Significance of the study .................................................................................... 4 1.6 Conceptual Framework ...................................................................................... 6 CHAPTER TWO: LITERATURE REVIEW ............................................................. 7 2.1 Classification of leafy vegetables ...................................................................... 7 2.1.1 Spinach ........................................................................................................ 7 2.1.2 Kales ............................................................................................................ 8 2.1.3 Pigweed (Amaranth) ................................................................................... 8 2.2 Leafy vegetables utilization and production ...................................................... 9 2.3 Heavy metal contamination ............................................................................. 10 2.3.1 Heavy metal contamination on agricultural lands ..................................... 11 2.3.2 Lead ........................................................................................................... 12 2.3.3 Cadmium ................................................................................................... 14 2.4 Transfer of heavy metals to the plant tissues ................................................... 16 2.5 Mobility of heavy metals in plants .................................................................. 17 2.6 Silicon .............................................................................................................. 18 2.6.1 Role of silicon in plant growth .................................................................. 18 2.6.2 Influence of silicon on heavy metals uptake ............................................. 19 2.6.3 Ameliorative mechanism of silicon on heavy metal stress in plants......... 19 2.7 Permissible levels of lead and cadmium in soils and plant tissues .................. 20 CHAPTER THREE: MATERIALS AND METHODS ............................................ 22 3.1 Study area ........................................................................................................ 22 3.2 Research design ............................................................................................... 23 3.2.1 Field experiment ........................................................................................ 23 3.2.2 Greenhouse experiment ............................................................................. 23 3.2.3 Planting and agronomic practices ............................................................. 24 3.3 Data collection ................................................................................................. 24 3.3.1 Soil sampling ............................................................................................. 24 3.3.2 Plant sampling ........................................................................................... 24 3.3.3 Soil pH determination ............................................................................... 25 3.3.4 Determination of soil organic matter ........................................................ 25 3.3.5 Determination of soil phosphorous ........................................................... 26 3.3.6 Determination of lead and cadmium concentration in soil ....................... 26 3.3.7 Determination of lead and cadmium concentration in plant tissues.......... 26 3.3.8 Determination of lead and cadmium in irrigation water ........................... 27 3.3.9 Transfer factor ........................................................................................... 27 3.3.10 Mobility index ......................................................................................... 28 3.3.11 Plant growth tolerance indices ................................................................ 28 3.4 Statistical analysis ............................................................................................ 29 3.5 Ethical issues .................................................................................................... 29 CHAPTER FOUR: RESULTS AND DISCUSSIONS ............................................. 30 4.1 Soil characteristics ........................................................................................... 30 4.2 Effects of Cd and Pb on growth parameters of spinach, kale, and amaranths . 31 4.2.1 Root Length ............................................................................................... 31 4.2.2 Shoot length............................................................................................... 34 4.2.3 Root dry biomass ....................................................................................... 37 4.2.4 Shoot dry biomass ..................................................................................... 41 4.2.5 Leaf area .................................................................................................... 47 4.2.6 Growth tolerance index (GTI) ................................................................... 54 4.3 Concentrations of lead and cadmium in plant tissues ...................................... 59 4.3.1 Concentration of cadmium ........................................................................ 59 4.3.2 Concentration of lead ................................................................................ 63 4.4 Transfer factor of lead and cadmium to plant tissues ...................................... 66 4.4.1 Transfer factor of cadmium ....................................................................... 66 4.4.2 Transfer factor of lead ............................................................................... 69 4.5 Mobility index of lead and cadmium in plant tissues ...................................... 72 4.5.1 Translocation index of cadmium ............................................................... 72 4.5.2 Translocation index of lead ....................................................................... 75 4.6 Uptake of lead and cadmium by vegetables .................................................... 78 4.6.1 Cadmium uptake ....................................................................................... 78 4.6.2 Lead uptake ............................................................................................... 83 4.7 Relationship between growth parameters and concentrations of lead and cadmium in leafy vegetables .................................................................................. 86 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS ...................... 95 5.1 Conclusions ...................................................................................................... 95 5.2 Recommendations ............................................................................................ 96 REFERENCES .......................................................................................................... 97 APPENDICES ......................................................................................................... 118 LIST OF TABLES Table 4.1 Soil characteristics in greenhouse and field experiments .......................... 30 Table 4. 2 Stem dry weight of spinach, kale and amaranths in greenhouse experiment as ameliorated by silicon amendment, on lead and cadmium stress ...... 42 Table 4. 3 Spinach, kale, and cadmium leaf dry weight in greenhouse experiment grown on lead and cadmium stress alleviated by silicon application ........................ 45 Table 4. 4 Lead and cadmium effects on leaf area of spinach, kale, and amaranths in the field experiment ................................................................................................... 53 Table 4. 5 Biomass tolerance index of spinach, kale and amaranths to lead and cadmium as affected by silicon amendment in greenhouse experiment ................... 55 Table 4. 6 Shoot and root length tolerance index of spinach, kale and amaranths to lead and cadmium as affected by silicon amendment in greenhouse experiment ..... 57 Table 4. 7 Cadmium mobility index in spinach, kale, and amaranths in the greenhouse experiment as affected by Silicon amendment ....................................... 73 Table 4. 8 Cadmium mobility index in spinach, kale, and amaranths as affected by Silicon amendment in the field experiment ............................................................... 74 Table 4. 9 Lead mobility index in spinach, kale, and amaranths in the greenhouse experiment as affected by silicon application............................................................ 76 Table 4. 10 Cadmium mobility index in spinach, kale, and amaranths in the field experiment as affected by Silicon amendment .......................................................... 77 Table 4. 11 Person correlations coefficient between measured spinach growth parameters and lead and cadmium concentrations in soil and plant tissues .............. 87 Table 4. 12 Person correlations coefficient between measured kale growth parameters and lead and cadmium concentrations in soil and plant tissues .............. 89 Table 4. 13 Person correlations coefficient between measured amaranth’s growth parameters and lead and cadmium concentrations in soil and plant tissues .............. 91 LIST OF FIGURES Fig 1. 1 Conceptual Framework .................................................................................. 6 Fig 3. 1 Map showing the study site in Kiambu county ............................................ 22 Fig 3.2 Field experiment layout ................................................................................. 23 Fig 4.1 Root length of spinach (a), kale (b), and amaranth (c) in greenhouse experiment as affected by lead and cadmium stress, as alleviated by silicon amendment ................................................................................................................ 32 Fig 4.2 Root length of spinach (a), kale (b), and amaranth (c) in field experiments as affected by lead and cadmium stress, alleviated by silicon amendment ................... 33 Fig 4.3 Shoot length of Spinach (a), kale (b), and amaranth (c) ameliorated by silicon application on lead and cadmium stress in the greenhouse experiment ........ 35 Fig 4.4 Spinach(a), kale(b), and amaranth(c) shoot length ameliorated by silicon application on lead and cadmium stress in field experiments ................................... 36 Fig 4.5 The root biomass spinach (a), kale (b), and amaranth (c) grown under lead and cadmium stress, as alleviated by silicon application in the greenhouse experiment ................................................................................................................. 38 Fig 4.6 The root biomass of spinach, kale, and amaranths grown under lead and cadmium stress, alleviated by silicon application in Field experiments.................... 40 Fig 4.7 Stem dry weight of Spinach (a), kale (b), and amaranths (c) in field experiments, ameliorated by silicon amendments when growing in lead and cadmium polluted soils .............................................................................................. 43 Fig 4.8 Spinach (a), kale (b) and amaranths (c) leaf dry weight in field experiments, ameliorated by silicon amendments when growing in lead and cadmium polluted soils ............................................................................................................................ 46 Fig 4. 9 Leaf area of spinach(a) kale(b) and amaranths(c)in the greenhouse experiment as affected by Lead and Cadmium ......................................................... 48 Fig 4. 10 The concentration of cadmium in spinach (a) kale (b) and amaranths (c) tissues as affected by silicon application in the greenhouse experiment. .................. 60 Fig 4.11 The concentration of cadmium in spinach (a) kale (b) and amaranths (c) tissues as affected by silicon amendment in field experiments ................................. 61 Fig 4.12 Lead concentrations in spinach (a) kale (b) and amaranths (c) as affected by silicon amendment in the greenhouse experiment. ................................................... 64 Fig 4.13 Lead concentrations in spinach (a) kale (b) and amaranths (c) as affected by silicon amendment in field experiments. ................................................................... 65 Fig 4.14 Cadmium transfer factor to spinach (a) kale (b) and amaranths (c) as ameliorated by silicon application in the greenhouse experiment ............................. 67 Fig 4.15 Cadmium transfer factor to spinach (a) kale (b) and amaranths (c) tissues as ameliorated by silicon application in the field experiments ...................................... 68 Fig 4. 16 Lead transfer factor in spinach(a) kale(b) and amaranths(c) as influenced by silicon application in greenhouse experiment ...................................................... 70 Fig 4. 17 Lead transfer factor in spinach (a) kale (b) and amaranths (c) as influenced by silicon application in the field experiment ........................................................... 71 Fig 4.18 Root uptake of cadmium by leafy vegetables as affected by silicon amendment in the greenhouse (a) and field experiments (b)..................................... 79 Fig 4.19 Shoot uptake of cadmium by spinach(a) kale(b) and amaranths(c)as affected by silicon amendment in greenhouse experiment ........................................ 80 Fig 4.20 Shoot uptake of cadmium by spinach, kale, and amaranths as affected by silicon amendment short rains,2019 (a) and long rains, 2020 (b) in the field experiments ................................................................................................................ 81 Fig 4. 21 Root uptake of lead alleviated by silicon amendment in spinach, kale and amaranths in greenhouse (a) and field experiments (b) ............................................. 83 Fig 4.22 Shoot uptake of lead as alleviated by silicon amendment in spinach(a) kale(b) and amaranths(c) in the greenhouse experiment ........................................... 84 Fig 4.23 Silicon alleviation on lead shoot uptake by spinach, kale, and amaranths in the short rains, 2019 (a) and long rains,2020 (b) of the field experiment ................. 85 LIST OF ACRONYMS AND ABBREVIATIONS AAS Atomic Absorption Spectrophotometer ANOVA Analysis of variance BAF Bioaccumulation factor Cd Cadmium CRD Completely Randomized Design FAO Food and Agriculture Organization of the United Nations KePHIS Kenya Plant Health Inspectorate Service Pb Lead PCD Programmed Cell Death RCBD Randomized Complete Block Design ROS Reactive Oxygen Species Si Silicon SSA Sub Sahara Africa TF Transfer Factor UN United Nations US EPA United States Environmental Protection Agency USA United States of America WHO World Health Organization ABSTRACT Heavy metal contamination and accumulation on agricultural soils pose a great risk due to toxicity, toxic materials of multiple sources, and of non-biodegradable nature. Lead and cadmium have phytotoxic and toxic effects on plants and animals. Leafy vegetables accumulate the metals resulting in enrichment along the food chain. Contamination in agricultural soils significantly reduces crop yields. Silicon has beneficial effects in enhancing plant tolerance to biotic and abiotic stresses. Silicon is known to reduce the uptake of metal ions by forming complex metal ions and altering soil pH. It also precipitates metal ions, compartmentalizes metal ions in cell vacuoles or cell walls, and hence reduced translocation to shoots, and promotes activities of antioxidant enzymes. However, information on its benefits has not been adequately explored. The study aimed to determine the amelioration of silicon on lead and cadmium uptake, mobility, and concentration in plant tissues and to evaluate the influence of lead and cadmium on the growth and biomass of leafy vegetables. The experiment was carried in two field experiments and a greenhouse experiment. The greenhouse experiment treatments were: lead (Pb), Si (silicon), Cadmium (Cd), Pb+Si, Cd+Si, and control. It was designed in Split plot CRD, with leafy vegetable species as the main plot and treatments as subplots. The field experiment was carried at the Kenyatta University research farm, and treatments included Si application and control. It was arranged in Randomized Complete Block Design (RCBD). Data collected was subjected to ANOVA using R software version 4.0.2 package and where there were significant differences, mean separation was done with Tukey at P< 0.05. Cadmium and lead significantly reduced leafy vegetable growth and production. Cadmium application reduced root biomass by 53.04%, 63.32%, and 70.20% in amaranths, spinach, and kale respectively. Lead reduced by 54.11%, 55.76%, and 60.72% in spinach, amaranths, and kale when compared with controls respectively. Lead and cadmium reduced above ground biomass with 25% in leafy vegetables. Silicon application enhanced leafy vegetable biomass tolerance by 30% when compared with soils spiked with lead and cadmium. Vegetables accumulated lead and cadmium beyond WHO concentration limits in plant tissues. Cadmium transfer factor and mobility were higher than for Lead. Lead and cadmium transfer and mobility index were less than one (<1). Results showed a strong negative correlation between cadmium concentrations and growth parameters (R2>0.8). Pearson correlation coefficient also indicated a strong positive correlation between cadmium concentrations in soils and plant tissues of leafy vegetables. Leafy vegetable uptake, translocate and accumulate metal ions in plant tissues. Silicon application enhanced leafy vegetable tolerance to lead and cadmium, and reduced transfer and mobility of metal ions. The study recommends silicon application on lead and cadmium contaminated soils to alleviate metal ions toxicity and reduced accumulation on edible plant tissues. CHAPTER ONE: INTRODUCTION 1.1 Background Vegetable production and consumption have the potential to create employment, provide nutrients, antioxidants, and phytochemicals that may help protect the population from non-communicable diseases and provide income in developing countries (Chagomoka et al., 2015; Munene et al., 2016 ). In Kenya, the horticultural sector is the leading source of foreign exchange revenue with the vegetable industry critical in achieving food security, improving the livelihood of small-scale farmers who produce 100% of the African vegetables and up to 70% of the exotic vegetables (Lans, et al., 2012). In 2014 it contributed 36% of the gross domestic value of horticulture in Kenya (Embassy of the Kingdom of Netherlands, 2017). Vegetable production in the world has intensified in the last two decades with approximately 60% (FAOSTAT, 2020). In Kenya, the production area, yields, and value of vegetables have increased by 26, 12, and 11 percent respectively (Embassy of the Kingdom of Netherlands, 2017). Fresh vegetable consumption in Kenya is on regular basis by nearly every household (Lans et al., 2012; Munene et al., 2016), with the purchase of fresh vegetables being second on expenditure priority and accounts for 25% of total expenditure. Nevertheless, vegetable consumption in many African countries including Kenya remains below the recommended level, negatively impacting the nutrition condition of the population (Lans et al., 2012). In Nairobi, the supply of fresh vegetables in the market remains low, especially in dry seasons elevated by the risk of climate change (Lans et al., 2012). Silicon is the second most abundant element on the earth’s crust (Zia-ur-Rehman et al., 2016), with beneficial effects in enhancing plant varieties tolerance to biotic and abiotic stress including salt stress, water stress, and high temperatures (Liang et al., 2015: Kaaria et al., 2021). Silicon is quasi-essential in plant growth. The plants absorb silicon from the soil in form of monosilicic acid (H4SiO4). Its distribution and amount in the soil are determined by parent rock material, climate, vegetation, and degree of weathering (Zheng and Zhang, 2012). Its crop removal from the soil is in large quantities especially in high-Si-accumulators crops (Tubana et al., 2016) hence intensive silicon fertilization has been used in countries including the USA, China, and South Africa to improve the productivity of crops including rice, sugarcane, and corns (Zheng et al., 2012). Heavy metals refer to materials with a specific gravity of greater than 4.0 or 5.0g/cm3, and bio-toxic at high concentrations (Ali et al., 2012). They compose the earth's crust and include substances such as mercury, lead, zinc, and copper among others. Heavy metal pollution in the soil refers to excessive deposition and accumulation of these toxic materials either from natural sources or anthropogenic activities. In recent years, due to global industrialization and urbanization, the levels of contamination by these pollutants have significantly increased (Su et al., 2014; Gweyi-Onyango and Osei- Kwarteng, 2011). Pollution on soils is mainly from sewage irrigation, improper deposition of industrial effluents, quarry activities, and the use of agrochemicals. High levels of metal ions accumulation pose a great environmental risk in newly industrialized countries such as China, due to the detrimental effect on human and animal health. In Africa’s major cities, heavy metals contamination in the neighborhood is a raising threat due to poor sewage disposal and open dumpsites. In Kenya, contamination of soils in the adjacent agricultural lands to the cities with heavy metals poses a great risk to healthy vegetable production (Kioko, 2015; Wambua et al., 2018). 1.2 Statement of the problem Heavy metal contamination and accumulation pose a great risk globally due to toxicity, multiple sources, non-biodegradable, and its accumulation property (Hu et al., 2017), with a significant proportion entering human bodies via contaminated food, drinking water, or air (Ali et al., 2012). The use of wastewater for irrigation production accounting for 50% of the vegetable supply to the urban areas in most Africa and Asia cities (Wuana and Okieimen, 2014). In Kenya urban farming is considered instrumental in achieving vegetable production demand but risked by the use of sewage water (Lans et al., 2012). Pollution of agricultural soil is an emerging risk significantly reducing crop productivity (Sharma and Dubey, 2005). In Kenya, vegetable consumption is below WHO health recommendations (Lans et al., 2012). Vegetables growing in contaminated and polluted environments accumulate heavy metals in tissues (Ali et al., 2012), resulting in enrichment along the food chain (Kumar et al., 2020). Their consumption, especially in urban areas, presents a high health risk to both humans and animals (Kiende, 2012). Heavy metals have detrimental effects on human health even in small quantities such as carcinogenic, toxicity to the nervous, renal, pulmonary system, and skin (Kalyani et al., 2018). Production of vegetable crops in polluted soils with metals ions results in higher concentrations in edible plant tissues, hence the need to reduce the transfer of the pollutants and improve crops productivity (Zama et al., 2018). Silicon alleviates environmental stress and plants' nutrient depletion (Guntzer et al., 2012). It is found in abundance in soil, however, most are not available to the plants(Guntzer et al., 2012). Its quantities in the soil are reducing in both tropics and temperate regions hence the need for exploration silicon fertilization (Zheng and Zhang, 2012). 1.3 Research objectives 1.3.1 General objective To contribute to enhanced performance and improved quality of selected vegetable by use silicon for amelioration of heavy metals uptake in Kiambu County. 1.3.2 Specific objectives i. To determine the effect of lead and cadmium on biomass and yield of spinach, kales, and amaranths. ii. To evaluate the transfer and concentration of heavy metals from soil to plant tissues of spinach, kales, and amaranths. iii. To assess the effects of silicon on lead and cadmium uptake and compartmentation in tissues of spinach, kales, and amaranths. 1.4 Hypotheses 1 There is a differential effect of lead and cadmium on biomass and the yield of spinach, kales, and amaranths. 2 There are differences in concentration and transfer of heavy metals from soil to plant tissues of spinach, kales, and amaranths. 3 Silicon amendment has ameliorative effect on the uptake and compartmentation of lead and cadmium in tissues of spinach, kales, and amaranths. 1.5 Significance of the study The information generated will enhance understanding on the influence of silicon in mitigating the transfer of lead and cadmium from the soil to edible tissues of vegetables. Pollution of agricultural land is a limiting factor in agricultural production in urban and peri-urban areas. Data from this research will help farmers in the determination of the inclusion of silicon in fertilizer formulations to reduce health risks due to heavy metals on edible plant tissues. The findings from this study will also be compared to the findings of other related studies for the benefit of the scientific pool of knowledge. 1.6 Conceptual Framework Human health: Safe vegetables Increased vegetable intake Vegetables: High vegetable yields Increased area of production Uptake and accumulation of heavy metals in edible plant tissues Reduced plant growth and biomass Human health effects Toxic effects on plants Silicon application Reduced uptake, concentration, transfer and transloc ation in plants Contaminated irrigation water Pesticides use Herbicides use Industrial effluents Agricultural soils contaminated Fig 1. 1 Conceptual Framework CHAPTER TWO: LITERATURE REVIEW 2.1 Classification of leafy vegetables Vegetables are commonly classified based on botanical families, edible parts of the plant, families, color, and food composition (Thompson et al., 2011). The edible parts of vegetables include root, stem, leaf, immature flower bud, and sprout (Alvino and Barbieri, 2015). Vegetables have high rates of respiration, water loss, and high perishability due to fast senescence on harvested vegetables (Alvino et al., 2015). Leafy vegetables are popularly referred to as greens and protherbs and are popularly grown in most parts of the world (Dhaliwal, 2017). They are generally defined as refers to vegetables cultivated for edible parts constituted of foliar structures (Alvino et al., 2015). Additionally, they are also referred to as greens if cooked before utilization as in many Brassicaceae and Chenopodiaceae, or as raw if consumed fresh such as in salads (Alvino et al., 2015). They can further be categorized as either cool or warm-season crops, grown as annuals, biennials, and perennials (Alvino et al., 2015). Most have shallow root systems mainly growing in soils with a pH of 6.0-6.8 (Alvino et al., 2015). They are quick-growing, highly perishable, grown in peri-urban areas. They provide vitamins, proteins, roughages, and minerals including iron, calcium, and phosphorous (Dhaliwal, 2017). Some of the leafy vegetables are spinach, kale, and amaranths. 2.1.1 Spinach Spinach, Spinacia oleraceae, belongs to the family Chenopodiaceae (Goosefoot family). Spinach is an annual leafy vegetable that preferably is grown in a cool climate. It requires temperatures ranging from 15oC to 20oC for optimal growth, though it has a cardinal temperature range of 10oC -32oC. Moreover, young plants can withstand low temperatures of -9oC. It requires well-drained fertile sandy loams or loam soils. It's native to Southwest Asia (Boriss and Kreith, 2006). Spinach consumption throughout the world is on the rise, consumed as both fresh and processed spinach. Spinach is rich in vitamins and minerals, growing in cool and humid regions grown for its leaves and used as a vegetable. It is produced in many countries in the world (Simko et al., 2014), with China being the world's largest exporter, seconded by the United States of America (Boriss et al., 2006). In Kenya, spinach production tons and land acreage has increased by 11% in the last decade with Kiambu and Nakuru counties being the highest producers (GoK, 2016). 2.1.2 Kales Kales, Brassica oleraceae, is locally known as sukumawiki and belongs to the brassica’s family. It is an annual and cool-season crop. It is a major source of vitamins, minerals, fiber, and calories mainly produced for local markets. It grows in areas of altitudes 800-2200m above sea level and requires fertile well-drained soils and a pH range of 5.5-7.5. Additionally requires an optimum temperature range of 16-21oC and well-distributed rainfall of 30-550 mm, which can be supplemented with irrigation. Globally, China is the highest producer of kales on exports, while the USA imports the highest amounts (Tridge, 2010). In Kenya, sukamwiki is the most popular leafy vegetable consumed nearly by every household and produced in all counties in the country (GoK, 2016). The area under kale production is estimated to be 32,347ha with Kiambu and Nakuru counties leading in its production (GoK, 2016). 2.1.3 Pigweed (Amaranth) Amaranth, Amaranthus spp, is an herbaceous annual crop that belongs to the family Amaranthaceae. There are 60 species of amaranths, but only a few are cultivated due to their weedy nature (Blodgett et al., 2007). It is native to tropical America but is currently distributed throughout the tropics. In Kenya, the commonly cultivated species are A. dubius, A. tricolor, A. caudatus, and A. cruentus. It is an indigenous vegetable mainly produced in the Western and Nyanza regions of the country (Shikuku, 2007; Abukutsa ??/). Amaranth is a critical source of nutrients and with the potential to alleviate malnutrition problems in developing countries. However, its consumption is rising but limited due to anti-nutritional factors such as oxalic acid (Shikuku, 2007).In Kenya, it is mostly utilized vegetables with an approximate production of 31,965 million tons annually (Lans et al., 2012). The grains of amaranths are utilized as protein sources, or as a leafy vegetables (Blodgett et al., 2007; Ngetich et al., 2017,2018). It grows at an altitude of 0-2400M and can be produced in both wet and dry seasons with a temperature range of temperatures of 22-30oC. It optimally grows in loam soils and has a pH range of 4.5 to 8.0 (Blodgett et al., 2007). The establishment in the seedbed can be through direct seeding or transplanting from the nursery. 2.2 Leafy vegetables utilization and production Vegetables are mainly consumed as sources of vitamins such as vitamins A, C, and E, and minerals including calcium. They are also important sources of dietary fiber and phytochemical compounds. Vegetable consumption is on the rise due to the health benefits against some chronic illnesses such as obesity and diabetes (Ülger et al., 2018). Vegetable production and consumption are on the rise globally with dieticians recommending high daily consumption of leafy vegetables per day (Dhaliwal, 2017). Globally FAO estimates one billion people are malnourished with 30% in 2010 living in Sub-Sahara Africa (Lans et al., 2012). Food security in Africa is greatly challenged hence high malnutrition levels, and the consumption of vegetables is consequently regarded as a privilege among the poor (Lans et al., 2012). WHO estimates only 10% of the global population meets the consumption minimum (CDCP, 2017). The daily intake of vegetables in Sub-Sahara is below the recommended rate (Lans et al., 2012). In Kenya, the utilization of vegetables is limited by poverty, negative socio- psychological perspectives, vegetables perishability, government policies, and health safety issues (RSA, 2015). Approximately 10% of the Kenyan population is food insecure with one-third being urban and peri-urban populations (Lans et al., 2012b). Production of vegetables is challenged by climate change, economic crisis, rapidly growing world populations, and prolonged droughts. The reduced production results in decreased demand against the supply hence higher pricing results in less utilization, micronutrient deficiencies, and a higher risk of non-communicable diseases (Lans et al., 2012). The consumption of vegetables in Sub-Sahara Africa is 27-114kg against the WHO/FAO recommended 146kg per person annually. In Kenya, despite most households spending 21-27% of the family budget on food, only 21% of the budget is utilized on vegetables, and with most households consuming sukumawiki (42%) at 13kg monthly (Lans et al., 2012) and spinach (42%) (Research Solutions Africa (RSA) Ltd, 2015). 2.3 Heavy metal contamination Metal ions pollutions in soil and water are mainly due to industrialization presenting a great environmental threat globally due to their toxicity (Afzal et al., 2014). Additionally, they have the potential to accumulate with less physical visibility hence posing as silent danger. Environmental pollution with metals including lead, arsenic, cadmium, and mercury to soil and water, pose a great health risk to human beings, plants, and animals (Sankhla et al., 2016). Globally, pollution with heavy metals accounts for two out of ten major environmental events (Sankhla et al., 2016). In many African and Asian countries farming using untreated wastewater for irrigation contribute to over 50% of the vegetables supplied to cities (Sankhla et al., 2016). Most metals including arsenic, lead, cadmium, and mercury are not essential for plant growth and lack any known physiological function. Some heavy metals such as cadmium have toxic effects on human health at low concentrations. Consumption of vegetables that have accumulated heavy metals due to growth on polluted lands is the common dietary intake (Jaishankar et al., 2014). The other methods of intake include geophagia among children, inhalation during spraying lead-based pesticides and paints, dermal or transcutaneous absorption, and drinking of contaminated water (Sankhla et al., 2016). 2.3.1 Heavy metal contamination on agricultural lands Agricultural soils are mainly polluted with metals and metalloids through the accumulation of emissions from industries, mines, leaded gasoline and paints, inorganic fertilizer use, poorly decomposed animal manure, human waste sludge, wastewater irrigation, fossils utilization, and atmospheric deposition (Sankhla et al., 2016). Wastewater effluents have relatively low concentrations, but long-term irrigation results in heavy metal accumulation (Sankhla et al., 2016). Soil acts as sequestration for metal ions interrupting, the soil-plant-animal or human cycle in which they exert their toxicity (Sankhla et al., 2016). Pollution of soils with heavy metals poses a great risk on agricultural soils, that’s difficult to remediate usually worsened by increased use of sewage irrigation water (Chibuike and Obiora, 2014). Heavy metals have a high potential for toxicity and are accumulated along the food chain (Sankhla et al., 2016). Additionally, they have adverse effects on soil ecology, agricultural product quality, and quantity, water quality, and health of living organisms (Chibuike and Obiora, 2014). Metals ions alter composition and characteristic of soil microorganism affecting soil biological and biochemical properties (Sankhla et al., 2016). The effects on biological and biochemical properties are determined by pH, clay content, and organic content in the soil (Sankhla et al., 2016). Heavy metals have phytotoxic effects leading to chlorosis, weak plant growth, decreased yields, low nutrients uptake, interference on plant metabolism, nitrogen fixation in legumes (Wambua et al., 2019), and delayed seed germination. The accumulation of metal ions depends on crop species and soil biochemical properties (Sankhla et al., 2016). 2.3.2 Lead Lead is a toxic heavy metal atomic with number 82, with higher density and abundant on earth crust (Wilson et al., 2015). It is toxic and non-nutrient to plant (Prasad et al., 2013), occurring naturally as galena (lead sulphide, PbS), lead carbonate (PbCO3), and lead suphate (PbSO4) (Wilson et al., 2015). In nature, lead pollution occurs from volcanic emissions and forest fires. Artificial sources include human activities especially emissions from industries, and the use of leaded gasoline in engines (Wilson et al., 2015). Other sources are mining and smelting, coal burning, lead-based paints among others (Sharma and Dubey, 2005). Lead is non-biodegradable hence it persists and is accumulated in soils(Winnie et al., 2018), water bodies, and sediments through deposition, leaching, and erosion and eventually becoming a global health issue (Prasad et al., 2013). 2.3.2 .1 Mechanism of lead toxicity in plants Leafy vegetables growing in lead-contaminated soils show toxicity symptoms including chlorosis, reduced root growth, and stunted growth. Lead phytotoxic effects are due to interference with hormones, membrane structure and permeability, osmotic balance, and inhibition of photosynthesis in a plant (Sharma and Dubey, 2005). Lead uptake from soil occurs through channels such as Ca-channel and inhibits plant absorption of calcium through blocking of the channels and competitive transport (Sharma and Dubey, 2005). Lead inhibits enzymatic activities such as sulphydyrl groups, reduces germination percentage and plant biomass. Inhibition of enzymatic activities alters metabolic processes such as chlorophyll synthesis, carbon (iv) oxide fixation, sugar metabolism, and protein hydrolysis. Lead also enhance some enzymatic activities as a result of immobilization of enzyme inhibitor, effector molecules, and altering of enzyme synthesis, such as hydrolytic and peroxidase enzymes. Lead also enhances the formation of reactive oxygen species causing oxidative stress causing lipid peroxidation (Sharma and Dubey, 2005). The reduction in growth is due to inhibited cell division, and chromosome morphology (Sharma and Dubey, 2005). Lead damages microtubules of mitotic spindles leading to c-mitosis due to blocking of cells in pro-metaphase. It also causes the leaking of potassium ions from root cells. It also increases the amount of abscisic acid, which induces stomatal closure. 2.3.2.2 Hazardous effects of lead on humans Lead enters the body as a result of inhaling polluted air (Prasad et al., 2013) or ingesting food contaminated by poor storage and handling or crop uptake from polluted soils (Wilson et al., 2015). Lead in the body gets accumulated in the bones and soft tissues leading to chronic toxicity and damage to vital body organs (Wilson et al., 2015). Lead poisoning effects are fatal and more severe in infants and young children (Wilson et al., 2015) and can lead to mental retardation (Prasad et al., 2013). Lead toxicity in the body is due to inhibition of calcium and zinc activities, enhancing oxidative reactions producing reactive oxygen species (ROS) thereby impairing DNA repair and inducing nucleic acid damage and peroxidation (Wilson et al., 2015). 2.3.3 Cadmium Cadmium (Cd), is a naturally occurring silvery-white heavy metal, with atomic number 48, in period 5, group 12 in the periodic table (Honey et al., 2015). Its concentration in the earth's crust is approximately 0.15 ppm (Honey et al., 2015), occurring in the form of greenockite (CdS), as a divalent cation (Bernhoft, 2013). Cd is mainly released as a by-product of zinc production (Parada et al., 2013). The natural source is mainly volcanic activities, with human activities being the use of fossil fuels, steel and iron production, quarrying, and the utilization of phosphate fertilizers (Honey et al., 2015). Additionally from paint pigments, cosmetics, and galvanizing steel barriers to nuclear fission (Bernhoft, 2013). It is also used in batteries, coatings, PVC stabilizers, and alloys in industries (Honey et al., 2015). Cd is widely spread in the environment and persists in soils for many years, leading to its accumulation. Crops growing in contaminated soils absorb it and eventually get concentrated along the food chain (Parada et al., 2013). 2.3.3.1 Mechanism of cadmium toxicity in plants Cadmium at low concentrations is highly toxic to plants. Phytotoxic symptoms of cadmium include stunted growth and chlorosis. Cadmium chlorosis is due to change in Fe: Zn ratio and inhibition of iron, calcium, magnesium, manganese, potassium, and phosphorous uptake by plants (Das et al., 1997). Cadmium inhibits the availability of plant nutrients and reduces the population of soil microbes due to its toxicity (Benavides et al., 2005). It also inhibits the transport of water, Ca, Mg, P, and K. Cadmium inhibit nitrate reductase enzyme activity in shoots, absorption of nitrate by plants, and nitrogen fixation in legumes. Cadmium reduces cell division in meristematic cells reducing plant growth and biomass (Ismael et al., 2019). It reduces the root length but increases the root diameter due to an increase in parenchyma cells and cortex tissue cells which alleviates salt and water stress in plants as a response to Cadmium stress. It reduces chlorophyll synthesis and alters chloroplast structure hence reducing the rate of photosynthesis. Inhibition in photosynthesis is also due to inhibition of enzymatic activities involved in carbon (iv) oxide fixation, promoting lipid peroxidation, and altering Nitrogen and Sulphur metabolism (Ismael et al., 2019). Cadmium reduces the viability and germination rate of seeds. It also inhibits pollen germination by altering endomembrane organelles, inhibiting endocytosis and exocytosis, and the formation of acidic vacuoles (Ismael et al., 2019). 2.3.3.2 Hazardous effects of cadmium in humans Cadmium is hazardous to humans and the environment at very small concentrations with effects on pregnancy, lactation, kidney, liver, pancreas, heart, and testis (Honey et al., 2015). Human exposure occurs through inhaling or ingestion of contaminated food especially leafy vegetables (Bernhoft, 2013), and significantly through cigarette smoking (Parada et al., 2013). It causes chronic toxicity in humans (Parada et al., 2013). Cd has carcinogenic effects, causes osteotoxicity, cardiovascular abnormalities (Bernhoft, 2013), renal disorders, hypertension, and diabetes (Honey et al., 2015). Additionally, it disrupts the endocrine system, male infertility, insulin resistance, and suppresses the immune system (Bernhoft, 2013). Its toxicity in the body is through causing epigenetic alteration in DNA expression, reduced tubule transport pathways inhibit heme-synthesis, and competitive interference with physiologic of zinc, and magnesium (Bernhoft, 2013). 2.4 Transfer of heavy metals to the plant tissues Transfer Factor (TF) is the ratio of the concentration in plant tissues to the respective concentration of the metal ions in the growth medium. Soil characteristics influence metals' mobility, bio-availability, and eco-toxicological risk (A.el-Amier et al., 2017). Soil properties that influence the uptake include the soil pH, organic matter, the amount of phosphorous, carbonates, and cation exchange capacity. Soil microbial activities mobilize the metal ions raising their bioavailability for plants (Tangahu et al., 2011). The bioavailability of lead has a negative correlation with soil pH. In acidic soils, lead exists in the aqueous form Pb(H2O6)2+, while in basic soil it exists as aqueous hydroxyl ions (OH-) (Kumar et al., 2020). Most heavy metals dissolve easily with a reduction in soil redox potential and hence usually accumulate in poorly drained soils. High amounts of clay in the soil promote metal adsorption through ion exchange capacity and specific adsorption mechanisms. Soil organic carbon influences microbial activities and solubility of metals and bioavailability of the ions to plants (Kumar et al., 2020). Plants have differential uptake of metals ions from the soil, with some being hyper- accumulators, and can be effectively used for phytoremediation in polluted agricultural lands. The plant uptake and translocate metals from the soil through the use of proton pumps, co and anti- transporters, and channels (Tangahu et al., 2011). Lead uptake is non-selective and depends on low-affinity cation transporters and is independent of H+/ATPase pumps, and commonly competing for calcium channels for uptake (Kumar et al., 2020). 2.5 Mobility of heavy metals in plants The translocation index or the mobility index refers to the ratio of the shoot concentration to the respective concentration on the roots. The amount of metals translocated varies depending on the crop species, with some transporting less to the leaves but much to the stems (Mehes-Smith et al., 2014). The translocation of metal ions from roots to the shoots is regulated and hence most crops do not accumulate higher than the metabolic needs(Tangahu et al., 2011). The transport of metals to the shoot is associated with evapotranspiration (Kumar et al., 2020). A significant amount of heavy metals absorbed by plants is accumulated in the roots and lower amounts are transported to the above-ground biomass (Sharma and Dubey, 2005). The apoplast transport of heavy metals including lead is limited due to the nature of lead binding with carboxyl, galacturonic, and glucuronic acid. Apoplast transport leads to metals accumulating on root endodermis providing a partial barrier on translocation to the shoot (Kumar et al., 2020). The heavy metals also bind to ion exchangeable sites and extracellular precipitation deposition on cell walls. Metals such as lead at higher concentrations damage the cells, and the semi-permeability function of cell membranes and tonoplast, and hence some metals may be transported via symplast (Sharma and Dubey, 2005). 2.6 Silicon Silicon (Si), which has an atomic number of 14, is naturally found as alkoxysilane compounds, sodium silicate, and tetraethylorthosilicate (Permatasari et al., 2017). Its second most abundant element constituting 28% of the earth's crust and is regarded as a beneficial and not an essential element hence not always present in nutrient solutions (Pozo et al., 2015). The amount of silicon absorbed by plants is dependent on the amount available in soil (Pozo et al., 2015), and it does not harm the plant even if absorbed in excessive amounts (Jarosz et al., 2014). The plant absorbs silicon in monosilic form Si(OH)4 or orthosilicic acid (H4SiO4) but is mostly found in an insoluble form hence not available for plants (Pozo et al., 2015). Its quantity in plant tissues is equivalent to macronutrients including calcium, magnesium, and phosphorous amounts. 2.6.1 Role of silicon in plant growth Silicon is beneficial to crops with the ability to improve yields and resistance to biotic and abiotic stress (Jarosz and SciPol, 2014). Silicon enhances biotic stress tolerance through altering host-pathogen recognition and stimulation of defense mechanisms via changing the makeup of herbivore-induced plant volatiles (HIPV) (Deshmukh et al., 2017). It enhances enzymatic activities, photosynthesis, chlorophyll synthesis, and tolerance to abiotic stress including salinity and metals ions stress (Emamverdian et al., 2018) 2.6.2 Influence of silicon on heavy metals uptake Silicon stimulates heavy metals to stress through reduction of uptake and formation of complex silicon compounds with metal ions (Emamverdian et al., 2018). It forms silicates and oxides of metal ions reducing their availability for uptake by plants (Bhat et al., 2019). Biosolids silicon compounds raise the soil pH promoting silicon absorption (Ngugi et al., 2021). The rise in soil pH reduces availability and stimulates the immobilization of metal ions such as cadmium (Emamverdian et al., 2018). Silicon regulates the activities of metal transporters in plants (Bhat et al., 2019). 2.6.3 Ameliorative mechanism of silicon on heavy metal stress in plants Silicon promotes tolerance through altering the structure of the cell wall via transport control, promoting activities of antioxidant enzymes, and the complexation of metal ions (Emamverdian et al., 2018). The antioxidant enzymes scavenge ROS enhancing plant tolerance against oxidative stress which has negative morphological, biochemical, and physiological effects on the plant. It also enhances non-enzyme antioxidant activities which reduce transport in plants. Silicon protects the plant also via the accumulation of polysialic acid in plant cells (Emamverdian et al., 2018). Silicon application reduces lipid peroxidation and increases plant biomass (Bhat et al., 2019). Silicon reduces the apoplastic translocation of metals to shoots through the reduction of free metal ions in the plant tissues. It enhances apoplastic barriers in roots: exodermis, epiblema, and endodermis reducing metals ions translocation (Emamverdian et al., 2018). Silicon also reduces the symplastic transport of metal ions (Bhat et al., 2019). Silicon forms complex metal ions and precipitates metal ions as cofactors. It chelates heavy metals and compartmentalizes excess metals into the cell vacuoles or cell walls (Bhat et al., 2019). Compartmentation results in more accumulation of metal ions in roots and hence less ions are translocated to edible above-ground biomass. Accumulation of silicon on leaves reduces cuticle transpiration, reducing evapotranspiration, which results in less translocation of metal ions such as cadmium (Emamverdian et al., 2018). Silicon application changes the plant structure including promoting the shoot height and root length, leaf number, and leaf area, hence enhancing plant tolerance to metal stress (Bhat et al., 2019) 2.7 Permissible levels of lead and cadmium in soils and plant tissues The permissible amounts change depending on the country or the regulating body. The permissible levels of cadmium in soils for agriculture are 0.03mg/kg and in-plant, tissues are 0.002mg/kg as per WHO guidelines. United States of America Environment Protection Agency (USA-EPA) allowable limits are 0.01mg/kg in agricultural soils and 0.2mg/kg in vegetable dry weight. Kenya National Environment Management Authority (NEMA) and Kenya Bureau of Standards (KEBS) allowable limits for cadmium concentrations in plant tissues is 0.05-0.2mg/kg while concentrations in agricultural soils were not given (Kinuthia et al., 2020). WHO allowable level of lead is 85mg/kg in soils and 2mg/kg in plant tissues (Fortin, 2009). USA-EPA sets lead limits at 200mg/kg in agricultural soils and 0.3mg/kg in vegetables. Kenya NEMA and KEBS allowable limits of lead is 0.3mg/kg in vegetables while in agricultural soils it was not given (Kinuthia et al., 2020). USA- EPA limits lead and cadmium concentrations in irrigation water at 5mg/l and 0.01mg/l respectively (Kinuthia et al., 2020). Agricultural production of crops beyond these permissible levels of lead and cadmium poses a great human health risk (FAO/WHO, 1995). CHAPTER THREE: MATERIALS AND METHODS 3.1 Study area The study was carried out at Kenyatta University School of Agriculture and Enterprise Development Research Farm, situated in Kiambu County about 18km along Nairobi- Thika Road, North-East of Nairobi Central Business District (Fig 3.1). It lies on coordinates 1°10'50.0"S, 36°55'41.0"E (Latitude: -1.180568; Longitude: 36.928042). It is 1608m Above-Sea Level. The area is relatively warm with relative humidity between 74%and 62%. The rainfall pattern is bimodal, with long rains occurring between March-May and shorts rains in October-December, with an average rainfall of 846 mm (Jaetzold and Schmidt, 1983). The mean annual temperature is 19oC and is generally characterized by high day temperatures, hot and sunny conditions. The soils are Orange Brown Gritty Loam (Scott et al., 1963). Fig 3. 1 Map showing the study site in Kiambu county 3.2 Research design 3.2.1 Field experiment A seedbed measuring 18m x 9m was prepared with a total of 18 plots each measuring 2m x 2m separated by 0.5m within the block and 1m between the blocks (Fig 3.2). The two treatments (without Silicon, and with Silicon) were randomly assigned to kales, spinach, and amaranths in a RCBD arrangement. Fig 3.2 Field experiment layout 3.2.2 Greenhouse experiment The 6 treatments: Lead (Pb), Cadmium (Cd), Cadmium and Silicon (Cd+ Si), Lead and Silicon (Pb+ Si), Silicon (Si), and Control were assigned to kales, spinach, and amaranths randomly. The experimental layout was a Completely Randomized Design (CRD) with a split-plot arrangement. The vegetable species constituted the main plot while the Cd, Pb, Pb+ Si, Cd+Si, Si, and Control represented the sub-plot. The soils used to fill the pots were analyzed and homogenized. The soils were spiked with Lead and Cadmium, with Lead Nitrate Pb (NO3)2, and Cadmium Nitrate Cd (NO3)2 as the sources respectively. 3.2.3 Planting and agronomic practices Land preparation was done 3 weeks before transplanting, to expose the soil-borne pests to predators, improve soil aeration, root penetration, and water infiltration. Harrowing was done to attain fine tilth. Certified vegetable seeds were obtained from KePHIS, planted in the nursery, and transplanted after 4 weeks. Spinach, Ford hook giant variety, seedlings were transplanted and established in the seedbed at a spacing of 35cm x 20cm. Kale, Holland variety, seedlings were transplanted at the spacing of 45cm x 30cm, while amaranths, Dubia giant variety, was established at 45cm x20cm spacing. Triple superphosphate (TSP) fertilizer was applied as a phosphorous source during transplanting. The vegetable varieties selected were high-yielding, drought-tolerant, and had resistant to pest and disease attacks. Routine management practices such as weeding, pest and disease control, and watering were done as required for all the treatments. 3.3 Data collection 3.3.1 Soil sampling The soil samples from the field were collected as described by Okalebo et al., (2002). The soil cores series were collected at 0-20cm and 21-40cm using the Transverse method. The composite sample was obtained by mixing, drying, grinding, and sieving the soil cores per depth, and used for analysis. 3.3.2 Plant sampling The vegetable samples were harvested at an interval of 10 days, after 35days post- transplanting. The whole plant was uprooted and washed with fresh water to remove adhering dirt and later with distilled water. The vegetable samples were portioned into roots, stems, and leaves, and data were recorded on parameters such as root length, root length, fresh weight, and dry weights. The portioned plant samples were oven-dried at 60°C until there was no change in weight, and later dry weight was recorded. The dry samples were ground and stored in labeled zip lock polythene bags for analysis. 3.3.3 Soil pH determination The pH of soil samples was determined electrometrically both in water (pH water) and in 0.01 M CaCl2 (pH CaCl2) at a (1:2) soil: solution ratio (weight/volume) as outlined by Okalebo et al., (2002). About 10g of air-dry soil samples were added to 20ml of distilled water and the mixture was shaken at 260 reciprocations per minute for 10 minutes and allowed to settle for 30 minutes. The pH of the soil suspension was recorded thereafter, using a pH meter (Model SG78) on a glass electrode. 3.3.4 Determination of soil organic matter The colorimetric method was used where 0.3g of the ground soil samples were added into a 100ml digestion tube and 2ml of distilled water was added. 10ml of 5% K2Cr2O7 solution was added to wet the soils, and 5ml H2SO4. The samples were later digested for 30 minutes at 150°C, cooled, and 50ml 0.4% BaCl2 added. The absorbance of the samples was recorded at 600nm. The percentage of organic soil carbon in samples was quantified using Equation I (Okalebo et al., 2002). .............. ............ %= (..-..)*0.10w (I) Where, a= concentration of Cr3+ in the sample, b= concentration of Cr3+ in the sample, and w= weight of soil sample. 3.3.5 Determination of soil phosphorous Phosphorous was determined using Bray 3 method on the acid soils, 2.5g of sieved (2mm) dry soil was added into 50ml Bray 2 extracting solution, 0.03N NH4F, and 0.1N HCl, shaken for 5 minutes, and later filtered. 20ml of distilled water and 5ml 0.8M H3BO3 were added, and 10ml of ascorbic acid reagent. The intensity of the blue color was measured at 880nm using a colorimeter. The quantity of phosphorous in the soil was determined using Equation II (Okalebo et al., 2002). .......................... (..../....)= (a-b)*v*f*10001000*w (II) Where, a= concentration in the solution, b- concentration of the blank, v= final volume of the digestion process, w= weight of the sample used and f= the dilution factor. 3.3.6 Determination of lead and cadmium concentration in soil Atomic absorption spectrometry (AAS) technique was used where one gram of homogenized dry soil was digested by the addition of 150ml HCl and 5ml HNO3. The samples were put on a sandy bath for one hour, and 5ml HCl and 50ml deionized water were added after cooling the solution (Al-Hamzawi and Al-Gharabi, 2019). The solution was filtered, and filtrate absorbance was determined using the AAS calibrated for lead and cadmium at 283.31 nm and 228.80 nm wavelengths respectively. The actual concentrations were computed using Equation II (Okalebo et al., 2002). 3.3.7 Determination of lead and cadmium concentration in plant tissues The AAS technique was used where, 0.3g of the plant sample was digested with 2.5ml of digestion mixture, Selenium-Sulphuric acid mixture, for two hours at room temperature. The sample digest was heated at 110°C for 1 hour, cooled and 30% H2O2 added, and later heated at 330°C until the digest became colorless or light-yellow color. The contents were topped to a 50ml volumetric flask mark with deionized water. The sample absorbance was determined using the AAS calibrated for lead and cadmium at 283.31 nm and 228.80 nm wavelengths respectively. The concentrations were determined using Equation II (Okalebo et al., 2002). 3.3.8 Determination of lead and cadmium in irrigation water Water samples were collected as outlined by the international center for agricultural research in the dry areas (ICARDA) (Nicholson, 1984). The water samples were collected biweekly and composite samples were stored at 4°C. The water was sieved and the bio-solids digested and the amount of lead and cadmium was determined by the use of the AAS technique (Okalebo et al., 2002). The amount of lead and cadmium in the irrigation water was determined using Equations II and III (Smith, 1983). .......................... (..../..)=(a-b) (III) Where a and b are sample concentration and blank concentration respectively 3.3.9 Transfer factor The transfer factor (TF) index, was computed as a ratio of the lead and cadmium concentrations in the plant tissues to their respective concentration in the soils, using Equation IV. It was used to quantify the amount of lead and cadmium that the plants absorbed from the growth medium (Amin et al., 2018). ................ ............ (....)= ............(..../....) C soil(mg/kg) (IV) Where C plant and C soil is the concentration. 3.3.10 Mobility index The translocation factor or the mobility index was computed as the ratio of lead and cadmium in the root to the stem and leaf concentration. It was used as a measure of the amount of metal ions translocated by the plant to above-ground biomass, from the roots. It was calculated using Equation V (Amin et al., 2018). ................ .......... = ....h......(..../....) C root(mg/kg) (V) Where C shoot and C root is the concentration 3.3.11 Plant growth tolerance indices The growth tolerance indices of the plant were measured, in Equations VI, VII, and VIII, to determine the vegetable species, that could grow and thrives in soils polluted with heavy metals (Amin et al., 2018). ....= .... ......*100 VI ....= .... ......*100 VII ......= ...... ........*100 VIII Where Tr= Root tolerance, Ts= Shoot tolerance, Ttp= Total plant tolerance, Mrc= Means value of control root dry weight, Msc= Mean value of Shoot dry weight, and Mtpc = Mean value of Total plant dry weight. 3.4 Statistical analysis Data collected from the field was arranged and compiled for statistical analysis using Microsoft suit-excel. Analysis of variance (ANOVA) was performed using the R- software version 4.0.2 package. Means were separated using the Tukey LSD test at a 5% significance level. Association between variables was determined by regression analyses. 3.5 Ethical issues The introduction and accumulation of metal ions on agricultural lands pose a great risk for safe and healthy food production in Kenya (Kioko, 2015). The uptake and accumulation of metal ions by vegetables growing on polluted soils, and their enrichment on the food chain (Ali et al., 2012). Lead and cadmium pose great risks due to toxicity, non-biodegradable, and bioaccumulation nature (Hu et al., 2017). During the research, the field site selection was done after screening for previously contaminated agricultural lands to avoid the introduction of toxic lead and cadmium hence zero spikings of soils in the field (Ole-Holm et al., 2002). The greenhouse experiment was designed in pot experiments and at the end of experiments, contaminated materials were disposed of as outlined by the European Union guidelines for the soils (Stals et al., 2010) and contaminated plant biomass (Onwughara et al., 2010). The research was also registered with Kenya National Commission for Science, Technology & Innovation (NACOSTI), to ensure conformity of research with national research policies, appendix 9. CHAPTER FOUR: RESULTS AND DISCUSSIONS 4.1 Soil characteristics The field experiment soils had significant levels of lead and cadmium. Lead levels in field experiment sites were 86.70 mg/kg of soil (Table 4.1). The lead levels were higher than allowable limits in agricultural soils of 85mg/kg (Fortin, 2009). The Cadmium levels were 0.5mg/kg of soil against the WHO allowable limits of 0.3 mg/kg of soil and 0.01 mg/kg limits by USA-EPA (Kinuthia et al., 2020). The levels of cadmium and lead in greenhouse experiments soils spiked with Cadmium Nitrate and Lead Nitrate resulted in cadmium concentrations of 2.5mg/kg and 89.90 mg/kg respectively (Table 4.1). Table 4.1 Soil characteristics in greenhouse and field experiments Parameter Greenhouse Field experiment pH(water) 5.31 5.37 pH(CaCl2) 4.52 4.87 Organic Carbon (%) 0.60 0.80 Phosphorous (mg kg-1) 25.67 29.85 Lead (mg kg-1) Nd 86.57 Cadmium (mg kg-1) Nd 0.50 Lead- Spiked (mg kg-1) 89.90 * Cadmium-spiked (mg kg-1) 2.50 * Pb-Irrigation water(mg/l) Nd 4.00 Cd- Irrigation water (mg/l) Nd 0.02 Nd- not detected, *- not applicable The pH of greenhouse and field experiment soils was low (Table 4.1). Levels of lead and cadmium in greenhouse irrigation water were not detected. The field experiment irrigation water average levels of lead and cadmium were 4.0 mg/l and 0.02mg/l respectively (Table 4.1). The levels were within the allowable USA-EPA limits of 5mg/l and 0.01mg/l for lead and cadmium respectively (Kinuthia et al., 2020). The amount of phosphorous and organic carbon was low (Okalebo et al., 2002). 4.2 Effects of Cd and Pb on growth parameters of spinach, kale, and amaranths 4.2.1 Root Length The treatments had significant (P<0.001) effects on the root length of vegetables. The application of silicon resulted in the highest root length while cadmium had the lowest root length in spinach and kale. Cadmium application decreased root length by 51.16%, 37.81%, and 22.26% in spinach, kale, and amaranths (Fig 4.1). Lead treatment inhibited root length by 53.58%, 28.86%, and 14.91% in spinach, kale, and amaranths when compared with control (Fig 4.1). (a) (b) (c) Fig 4.1 Root length of spinach (a), kale (b), and amaranth (c) in greenhouse experiment as affected by lead and cadmium stress, alleviated by silicon amendment Silicon application increased the root length of spinach, kale, and amaranths by 23%, 20%, and 17% respectively when compared with control (Fig 4.2). (b) (a) (c) Fig 4.2 Root length of spinach (a), kale (b), and amaranth (c) in field experiments as affected by lead and cadmium stress, alleviated by silicon amendment The inhibited root growth in vegetables by cadmium is because cadmium reduces the uptake of essential nutrients such as phosphorous, required for proper root growth. Cadmium also inhibits cell division in meristematic cells at root tips. It also causes root necrosis and affects plant biochemical activities (Alia et al., 2015). Lead affects metabolism and respiration in crops through inhibiting enzymatic activities (Feleafel and Mirdad, 2013). Silicon application enhances root growth in plants growing in soils contaminated with lead and cadmium due to that it reduces uptake of metal ions by forming complex metal ions not available to plants. Silicon also precipitates the metal ions and promotes the activities of antioxidant enzymes, which scavenge for ROS (Haddad et al., 2018). The results on inhibited root length in spinach were in agreement with a similar study on spinach (Alia et al., 2015). Pb and Cd inhibition has also been shown by related studies on other crops such as purslane seedlings (Naz et al., 2013), cucumber (An et al., 2004), and peas (Devi et al., 2007). 4.2.2 Shoot length The treatments had significant (P<0.05) effects on the shoot length of vegetables. Silicon treatment resulted in the highest shoot length among the vegetables. Cadmium application decreased shoot length when compared with control by 4.95%, 23.68%, and 19.27% in spinach, kale, and amaranths (Fig 4.3). Lead treatment reduced shoot length significantly in kale and amaranths by 9% and 12% when compared with control (Fig 4.3). (a) (c) (b) Fig 4.3 Shoot length of Spinach (a), kale (b), and amaranth (c) ameliorated by silicon application on lead and cadmium stress in the greenhouse experiment Silicon application resulted in a significant increase in the shoot length in the field experiment. The application increased the shoot length 23%, 25%, and 13% in spinach, kale, and amaranths when compared to the control (Fig 4.4) (a) (b) (c) Fig 4.4 Spinach(a), kale(b), and amaranth(c) shoot length ameliorated by silicon application on lead and cadmium stress in field experiments Lead treatment induced water stress in vegetables and inhibited cell division at shoot apical meristem cells (Feleafel and Mirdad, 2013). Cadmium affects metabolism by inhibiting the activities of the nitrate reductase enzyme and also reduces nitrogen uptake by plants. Cadmium also limits the uptake of zinc, reduces photosynthesis by degrading chloroplast, chlorosis, and damage to cells (Awan, 2017). Silicon enhanced shoot growth by increased nitrogen uptake and root expression of nitrate transporter gene, detoxification of ROS, and increased photosynthetic activity of the crop (Haddad et al., 2018). The results of inhibited shoot growth were in agreement with a similar study on spinach and amaranths (Ahada et al., 2015), leafy vegetables (Feleafel and Mirdad, 2013). Inhibited growth has also been reported on other crops such as maize (Awan 2017), sunflower (Alaboudi et al., 2018) tomato (Dong et al., 2005), Miscanthus spp (Guo et al., 2016), and Chickpea (Ullah et al., 2020). Enhanced shoot length due to silicon treatment on lead and cadmium soils was reported also in lettuce, melon, cucumber, and strawberries (Artyszak, 2018). 4.2.3 Root dry biomass Results showed that treatments had significant (P<0.001) decrease on the root biomass. Cadmium application reduced root dry weight significantly when compared with control by 53.04%, 63.32%, and 70.2% in amaranths, spinach, and kale (Fig4.5). Lead treatment reduced the biomass by 54%, 55%, and 60% in spinach, amaranths, and kale respectively (Fig 4.5). (a) (b) (c) Fig 4.5 The root biomass spinach (a), kale (b), and amaranth (c) grown under lead and cadmium stress, as alleviated by silicon application in the greenhouse experiment Silicon application resulted in a significant increase on root dry weight. Silicon enhanced root biomass by 30%, 31%, and 37% in spinach, kale, and amaranths respectively when compared with control (Fig 4.6). 0 5 10 15 20 25 30 35 40 45 50 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 Spinach kale Amaranths Spinach kale Amaranths Short rains (2019) Long rains (2020) Root dry weight (g) Days after treatments Control Si Fig 4.6 The root biomass of spinach, kale, and amaranths grown under lead and cadmium stress, alleviated by silicon application in Field experiments The reduced root biomass is due to lead causing a reduced root growth rate and the branching pattern. Lead inhibits the growth of primary and lateral roots, by inhibiting cell division at the apical meristems by damaging microtubules (Sharma and Dubey, 2005). It also causes root necrosis (Ali and Nas, 2018). Lead and cadmium are more accumulated in roots than in shoots, hence causing inhibition on root growth (Sharma and Dubey, 2005). The reduced root biomass by lead and cadmium was in agreement with a similar study on spinach, radish, and okra (Singh and Aggarwal, 2006). It was also observed in Leucaena leucocephala (Shafiq et al., 2010), Pisum Sativum, in Zea mays (Ali and Nas, 2018), chicken peas (Ullah et al., 2020), and eggplant (Yilmaz et al., 2009). 4.2.4 Shoot dry biomass 4.2.4.1 Stem dry weight Treatments had significant (P<0.001) effects on the stem dry weight of vegetables. Cadmium application decreased stem dry weight by 49.59%, 54.75%, and 41.82% on spinach, kale, and amaranths when compared with controls respectively (Table 4.2). Treatment with lead reduced stem dry weight when compared with control by 31%, 44%, and 43% in spinach, kale, and amaranths. Silicon application resulted in the highest stem weight in vegetables. Table 4. 2 Stem dry weight of spinach, kale and amaranths in greenhouse experiment as ameliorated by silicon amendment, on lead and cadmium stress Vegetable Treatment Days after treatment 15 25 35 45 Spinach Pb 0.327c 0.387d 1.873c 2.433b Cd 0.327c 0.840c 1.203d 1.297c Pb +Si 0.490b 1.020c 2.190bc 2.390b Cd +Si 0.463b 0.950c 2.057c 2.123b Control 0.557b 1.487b 2.577b 2.653b Si 0.870a 2.043a 3.603a 4.197a P* *** *** *** *** Kale Pb 0.190b 0.370c 1.490c 1.647bc Cd 0.307b 0.300c 1.187c 1.217c Pb +Si 0.313b 0.417c 1.760bc 2.023bc Cd +Si 0.233b 0.330c 1.443c 1.607bc Control 0.427b 1.217b 2.413b 2.597b Si 1.040a 2.003a 3.783a 5.020a P* *** *** *** *** Amaranths Pb 0.293b 1.157cd 2.910c 3.260c Cd 0.407b 0.857d 2.787c 3.743bc Pb +Si 0.657ab 1.763bc 3.063c 4.733bc Cd +Si 0.570ab 1.567cd 3.773b 5.087bc Control 0.933ab 2.413b 4.267b 5.783b Si 1.533a 3.390a 5.870a 7.930a P* *** *** *** *** Means followed by different superscript letters (down the column within the same vegetable species) differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant. Silicon application on soils contaminated with lead and cadmium significantly increased the stem dry weight of vegetables. Application of silicon increased the stem dry weight by 33%, 21%, and 26% in spinach, kale, and amaranths when compared with control (Fig 4.7) 0 10 20 30 40 50 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 Spinach kale Amaranths Spinach kale Amaranths Short rains (2019) Long rains (2020) Dry weight (g) Days after treatment Control Si Fig 4.7 Stem dry weight of Spinach (a), kale (b), and amaranths (c) in field experiments, ameliorated by silicon amendments when growing in lead and cadmium polluted soils Cadmium inhibits metabolism in the shoots by inhibiting the activity of the nitrate reductase enzyme. It also inhibits the uptake of calcium, magnesium, manganese, nitrogen, and potassium. It also reduces the translocation of iron from the roots, which causes its deficiency in the shoot (Piršelová et al., 2016). Lead stimulates the formation of ROS, leading to oxidative stress leading to peroxidation. Silicon absorbed by plants is deposited on plant cell walls as silica gel, thereby strengthening the stem. It reduces lodging in plants and exposure to light hence a higher rate of photosynthesis leading to higher biomass. Results were in agreement with similar studies on Arugula (Cannata et al., 2013), chicken peas (Ullah et al., 2020), and eggplant (Yilmaz et al., 2009). 4.2.4.2 Leaf dry weight Results showed significant (P<0.001) effects on the leaf dry weight due to treatments. Cadmium application reduced leaf dry weight in spinach, kale, and amaranths by 52.90%, 63.05%, and 47.24% when compared with control (Table 4.3). Cadmium inhibited the leaf dry weight than lead application. Treatment with lead decreased dry weight by 41%, 45%, and 46% in spinach, kale, and amaranths. Table 4. 3 Spinach, kale, and cadmium leaf dry weight in greenhouse experiment grown on lead and cadmium stress alleviated by silicon application Vegetable Treatment Days after treatment 15 25 35 45 Spinach Pb 1.013b 1.460cd 2.633c 4.930cd Cd 0.940b 1.187d 2.777c 3.140d Pb +Si 1.487b 1.940c 3.503bc 6.673bc Cd +Si 1.503b 2.580b 4.080bc 6.070c Control 1.457b 2.560b 4.633ab 8.430ab Si 2.230a 4.423a 5.960a 9.220a P-value *** *** *** *** Kale Pb 0.477d 1.157d 1.977cd 4.727cd Cd 0.547cd 0.843d 1.947d 2.333e Pb +Si 0.957bc 1.837c 3.97abc 6.127bc Cd +Si 1.347b 1.867c 3.233bcd 3.687de Control 1.253b 2.480b 4.750ab 6.860ab Si 1.880a 3.357a 5.840a 7.913a P-value *** *** *** *** Amaranths Pb 0.833b 1.340cd 2.187c 2.717b Cd 0.610b 1.077d 2.623c 2.900b Pb +Si 1.270ab 1.687bc 2.970bc 3.360b Cd +Si 0.650b 2.220b 3.107bc 3.357b Control 1.563a 2.160b 3.993ab 5.697a Si 1.830a 3.493a 4.540a 6.950a P-value *** *** *** *** Means followed by different superscript letters (down the column within the same vegetable species) differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant. Silicon application had significant alleviation on the leaf dry weight of vegetables. The leaf dry weight of spinach, kale, and amaranths increased by 41%, 42%, and 45% when compared with control in the field (Fig 4.8). 0 10 20 30 40 50 60 70 80 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 15 25 25 45 Spinach kale Amaranths Spinach kale Amaranths Short rains (2019) Long rains (2020) Leaf dry weight (g) Days after treatment Control Si Fig 4.8 Spinach (a), kale (b) and amaranths (c) leaf dry weight in field experiments, ameliorated by silicon amendments when growing in lead and cadmium polluted soils Lead reduces the rate of photosynthesis in plants through the destruction of the chloroplast and the synthesis of chlorophyll. Lead increases the concentration of abscisic acid, which induces stomatal closure which negatively affects carbon iv oxide fixation (Ali and Nas, 2018). Lead also affects respiration by inhibiting enzyme activities. Cadmium affects respiration, photosynthesis, and enzymatic reactions (Ullah et al., 2020). It also reduces the mobility of iron from roots hence deficiency in the leaves (Piršelová et al., 2016). Results on decreased leaf biomass were in agreement with similar studies on maize (Nosalewicz et al., 2008), eggplant (Yilmaz et al., 2009), and tomato (Ali and Nas, 2018). 4.2.5 Leaf area Treatments had significant (P<0.01) effects on the leaf area of vegetables. The application of silicon resulted in the highest leaf area in all the vegetables. Cadmium application reduced the leaf area more than lead (Fig 4.9). It reduced the leaf area by 31%, 36%, and 41% in spinach, kale, and amaranths respectively when compared with controls. Treatment with lead decreased the leaf area by 36.16%, 35.69%, and 32.85% when compared with controls in spinach kale and amaranths (Fig 4.9). (c) (b) (a) Fig 4. 9 Leaf area of spinach(a) kale(b) and amaranths(c)in the greenhouse experiment as affected by Lead and Cadmium Silicon application on soils contaminated with lead and cadmium had a significant effect on the leaf area of the vegetables. Treatment with silicon increased leaf area by 27%, 31%, and 30% when compared with controls in spinach, kale, and amaranths (Table 4.4) Vegetable Treatment Short rains (2019) Long rains (2020) Days after treatment Days after treatment 15 25 35 45 15 25 35 45 Spinach Si neg 557 1274 2533 3629 613 1335 2599 3694 Si post 824 2067 3514 4624 885 2129 3564 4680 t value -8.96 -23.14 -29.63 -66.42 -8.49 -24.11 -28.06 -67.28 P value *** *** *** *** *** *** *** *** Kale Si neg 382 1070 2338 3096 438 1130 2405 3158 Si post 686 1730 3088 4494 746 1785 3138 4558 t value -13.41 -25.28 -30.95 -39.73 -13.91 -22.89 -25.68 -36.21 P value *** *** *** *** *** *** *** *** Amaranths Si neg 458 833 1968 2802 514 892 2023 2866 Si post 665 1403 2670 4026 730 1468 2734 4080 t value -11.63 -10.86 -37.5 -37.49 -12.6 -10.75 -33.95 -37.98 P value *** *** *** *** *** *** *** *** Table 4. 4 Lead and cadmium effects on leaf area of spinach, kale, and amaranths in the field experiment Means within the same differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant. Lead causes nitrogen deficiency in the plant by inhibiting its uptake reducing growth in leafy vegetables (Sharma and Dubey, 2005). Lead also cause mitotic irregularities, which affect cell division and leaf growth. Lead also reduce leaf area by reducing the rate of photosynthesis by reduced chlorophyll and chloroplast synthesis, inducing stomatal closure and Carbon (iv) Oxide fixation in the leaf. Cadmium inhibits photosynthesis through decreasing stomata count per unit area and induced stomata closures due to its competition with calcium (Piršelová et al., 2016) Decreased leaf area by lead and cadmium was in agreement with the results of similar studies on maize (Nosalewicz et al., 2008), and eggplant (Yilmaz et al., 2009). It was also reported in soybean (Ali and Nas, 2018), Bambara nut (Oladele et al., 2017), and sunflower (Azevedo et al., 2005). 4.2.6 Growth tolerance index (GTI) Treatments had significant(P<0.001) effects on the growth tolerance index of vegetables. Spinach had the highest above ground biomass tolerance to lead and cadmium, followed by amaranths and kale in that order. Amaranths had the highest root biomass tolerance, followed by spinach and kale (Table 4.5). Spinach and kales showed higher biomass tolerance for lead than amaranths. The application of silicon enhanced the leafy vegetable root biomass tolerance for lead and cadmium by 25.65% and 28.52%. Silicon application also enhanced leafy vegetable shoot biomass tolerance by 22.46% and 26.48% for lead and cadmium respectively (Table 4.5) Table 4. 5 Biomass tolerance index of spinach, kale and amaranths to lead and cadmium as affected by silicon amendment in the greenhouse experiment Vegetable Treatment Above Ground Root Days after treatment Days after treatment 15 25 35 45 15 25 35 45 Spinach Pb 66.57cd 45.63d 62.51bc 66.44b 20.00b 34.94b 62.76c 78.50cd Cd 62.92d 50.08d 55.20c 40.03c 29.77b 37.55b 50.41c 51.70d Pb +Si 88.20bc 73.14c 78.96bc 81.78b 23.25b 45.35b 94.32b 95.20b Cd +Si 91.03b 87.23b 85.11b 73.93b 33.02b 61.71b 77.57bc 92.40bc Si 154.00a 159.79a 132.64a 12106a 154.41a 175.46a 172.43a 220.90a P value *** *** *** *** *** *** *** *** Kale Pb 39.68d 41.29c 48.40c 67.39c 12.90b 21.25c 57.45c 58.52c Cd 50.79cd 30.93c 43.74c 37.54d 9.360b 35.69bc 37.13c 39.67c Pb +Si 75.60bc 60.95b 79.99b 86.18b 18.71b 52.69b 95.93ab 98.01ab Cd +Si 90.71b 59.42b 65.29bc 55.97cd 22.58b 45.89bc 86.00bc 78.73bc Si 173.81a 144.98a 134.35a 136.76a 102.58a 154.10a 1.580a 141.03a P value *** *** *** *** *** *** *** *** Amaranths Pb 45.12b 54.60cd 61.70c 52.06b 49.60b 72.70b 43.82c 49.25d Cd 48.86b 41.88d 61.99c 55.53b 62.40b 73.20b 43.61c 43.80d Cd +Si 48.86b 82.80b 83.29b 73.55b 60.80b 93.70b 72.89b 96.28b Pb +Si 77.16b 75.44bc 73.04bc 70.50b 38.80b 96.30b 57.17bc 79.03c Si 134.69a 1.50.52a 126.03a 129.62a 169.61a 275.60a 149.14a 184.73a P value ** *** *** *** *** *** *** *** Means followed by different superscript letters (down the column within the same vegetable species) differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant. Treatments had a significant decrease on root and shoot length of vegetables except on shoot length of spinach and 15 days after treatment shoot length of kale. Spinach and kale had a higher shoot and root tolerance of cadmium than lead (Table 4.6). Amaranths had the lowest tolerance to cadmium (Table 4.6). Silicon application enhanced vegetable root length tolerance by 10% and 23% for lead and cadmium respectively. Table 4. 6 Shoot and root length tolerance index of spinach, kale and amaranths to lead and cadmium as affected by silicon amendment in the greenhouse experiment Vegetable Treatment Shoot length Root length Days after treatment Days after treatment 15 25 35 45 15 25 35 45 Spinach Pb 92.00a 94.60a 81.00a 82.21a 50.00b 44.44c 46.15c 46.67b Cd 92.00a 98.20a 90.00a 91.83a 58.33b 48.89bc 46.15c 46.67b Pb +Si 94.00a 96.10a 89.00a 91.35a 62.50b 71.11b 67.69b 54.67b Cd +Si 86.00a 90.00a 92.00a 90.38a 83.33ab 64.44bc 69.23b 65.33b Si 108.00a 132.10a 100.0a 99.04a 133.33a 100.00a 101.54a 94.67a P value ns ns ns ns ** *** *** *** Kale Pb 90.70a 94.60ab 89.02ab 89.41ab 90.32b 67.39b 67.80b 67.69b Cd 99.00a 99.00ab 86.59ab 91.76ab 67.74b 58.70b 61.02b 63.08b Pb +Si 97.70a 91.10b 90.85ab 92.94ab 54.84b 60.87b 81.36b 75.38b Cd +Si 88.40a 80.40b 71.95b 71.76b 93.55b 80.43b 83.05b 81.54b Si 137.20a 137.50a 125.61a 124.71a 170.97a 143.48a 137.29a 129.23a P value ns ** * * *** *** *** *** Amaranths Pb 91.20b 81.20ab 80.00b 83.25c 77.55b 75.00b 74.36b 74.39b Cd 85.00b 69.93b 73.51b 80.79c 75.51b 83.93b 76.92b 75.61b Pb +Si 95.00b 80.45ab 90.81b 93.94b 71.43b 94.64b 82.05b 80.49b Cd +Si 98.70ab 73.68b 83.24b 76.85c 67.35b 92.86b 78.21b 81.71b Si 138.70a 111.28a 114.05a 126.6a 136.73a 146.43a 134.62a 137.80a P value ** * *** *** *** *** *** *** Means followed by different superscript letters (down the column within the same vegetable species) differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant. The shoot had a higher tolerance to heavy metals, resulting from the lower transfer of metal ions from the shoots than the roots. The higher concentrations in the roots inhibited growth and decreased biomass than in shoots (Tangahu et al., 2011). Vegetables had a higher tolerance to lead and cadmium (Table 4.5, Table 4.6). The high tolerance of lead is due to the lower mobility of Lead and Cadmium. Cadmium also had a higher transfer factor than Lead. Spinach had the highest tolerance, followed by kale and amaranths in that order. The tolerance difference is due to the differential accumulation of lead and cadmium (Tangahu et al., 2011). Spinach had high tolerance index than kale, as spinach may compartmentalize the metal ions in cell walls and cell vacuoles hence causing less toxicity (Emamverdian et al., 2018). Silicon enhanced tolerance of vegetables by reducing uptake and ameliorating metal ions toxicity. Silicon forms complex compounds with metal ions, silicates, and oxides of metal ions and raises the soil pH, reducing availability and uptake of metal ions (Bhat et al., 2019; Emamverdian et al., 2018). Silicon ameliorates the toxicity of metal ions by reducing apoplastic and symplast transport from the roots (Bhat et al., 2019). Silicon decreased the rate of evapotranspiration in plants (Emamverdian et al., 2018). Amendment with silicon has also been reported to increase shoot and root length, leaf number, and area, which increases the plant tolerance to metal ions stress (Bhat et al., 2019). Silicon stimulates antioxidant enzymes, which scavenge for ROS enhancing the tolerance of vegetables against oxidative stress (Emamverdian et al., 2018). It also enhances non-enzyme antioxidant activities in plants. Silicon also increases the biomass of the plant by reducing lipid peroxidation (Bhat et al., 2019). The levels of lead tolerance index were in agreement with a similar study on Salix Integra (Wang et al., 2014), cowpeas (Shedeed et al., 2018), and maize (Christophe, 2012). 4.3 Concentrations of lead and cadmium in plant tissues 4.3.1 Concentration of cadmium Results showed treatments had significant (P<0.001) effects on concentrations of cadmium in plant tissues. Cadmium application on soils resulted in the highest plant tissue concentration. The cadmium concentrations in plant tissues were highest in roots, followed by stem and leaves in that order (Fig 4.10). Silicon application on soils spiked with cadmium resulted in reduced concentration by 20% in roots. It reduced stem concentrations with 21% in spinach and amaranths and with 32.09% in kale. Silicon decreased leaf concentrations by 28% in spinach and amaranths and 46.15% in kale (Fig 4.10). The application of silicon in field experiments had significant (P<0.01) effects on cadmium concentration in roots, stems, and leaf. Silicon application resulted in the lowest concentrations in the tissues of vegetables (Fig 4.11). Silicon reduced root, stem, and leaf cadmium concentrations by 20% in spinach. It decreased stem concentration by 31.63% and 33.29% in kale and amaranths. Treatment amendment, reduced cadmium concentrations in kale and amaranths by 39% and 32% respectively (Fig 4.11). (a) (b) (c) Fig 4. 10 The concentration of cadmium in spinach (a) kale (b) and amaranths (c) tissues as affected by silicon application in the greenhouse experiment. The dashed lines denote the cadmium allowable limit of 0.2 mg kg-1 in vegetables (a) (b) (c) Fig 4.11 The concentration of cadmium in spinach (a) kale (b) and amaranths (c) tissues as affected by silicon amendment in field experiments The concentrations of cadmium and lead in root, stem, and leaf increased as the vegetables continued to grow in contaminated soils (Fig 4.10, Fig 4.11). The concentrations in plant tissues increased with an increase with cadmium in soil concentrations hence higher concentrations in spiked soils (Page et al., 1987). The concentrations of cadmium in plant tissues were higher than the allowed limits by WHO but within the limits allowed in Kenya by KEBS and NEMA (Kinuthia et al., 2020). The higher concentration in roots than other plant tissues is because plants do not translocate all cadmium absorbed by the plant to the above-ground biomass. The high root concentration is also due to roots having a higher transfer factor of cadmium than other plant tissues (Sharma and Dubey, 2005). Silicon application reduced the concentrations of cadmium in plant tissues (Fig 4.10, Fig 4.11). Silicon enhances apoplastic barriers in roots such as endodermis and epiplema, reducing translocation to the shoot (Emamverdian et al., 2018). It also thickens the leaf cuticle reducing evapotranspiration, hence reduces cadmium uptake and transport in the plant (Emamverdian et al., 2018). Silicon has also been reported to reduce the symplastic transport of metal ions (Bhat et al., 2019). Higher concentrations of cadmium in plant tissues of spinach growing on spiked soils were reported by a similar study on spinach (Bui et al., 2016). Amaranths had a lower concentration of cadmium which is in agreement with a similar study that showed vegetables of the Brassicaceae family had a greater transfer factor of cadmium when compared with other vegetable species (Bui et al., 2016). 4.3.2 Concentration of lead Treatments had significant (P<0.001) effects on lead concentration measured in plant tissues. Treatment with lead resulted in the highest concentration in roots, stem, and leaf. The concentration of lead was highest in the root, followed by stem and leaf in that order in vegetables (Fig 4.12). Silicon application on soils spiked with lead, reduced the root and stem concentrations by 27%, in vegetables. It also reduced lead concentration by 51.25%, 53.76%, and 47.61% in spinach, kale, and amaranths. (a) (b) (c) Fig 4.12 Lead concentrations in spinach (a) kale (b) and amaranths (c) as affected by silicon amendment in the greenhouse experiment. The dashed lines denote the lead allowable limit of 2mg kg-1 in vegetables. Treatments had significant (P<0.001) effects on the concentration of lead in the field experiment. Silicon application on lead-contaminated soils resulted in the least concentrations on plant tissues. The roots concentration was highest, followed by stem and leaves in that order (Fig 4.13). The application of silicon reduced the root concentration by 28% and 30% in leaves and stems respectively of vegetables. (a) (b) (c) Fig 4.13 Lead concentrations in spinach (a) kale (b) and amaranths (c) as affected by silicon amendment in field experiments. The dashed lines indicate the lead allowable limit of 2mg kg-1 in vegetables. Spiking of soils with lead results in an increase in the concentration of the element in plant tissues: an increase in soil concentration raised the transfer factor of the lead to plant tissues. The root lead concentration was higher than other plant tissues due to the high transfer factor and less mobility to the shoot. Lead treatment with no silicon resulted in higher lead concentration in edible tissues of vegetables (Fig 4.12). Silicon application on lead spiked soils reduced the lead concentration to within the WHO limits (Fortin, 2009) but beyond allowed limits by US-EPA and KEBS/NEMA in Kenya (Kinuthia et al., 2020). Application of silicon in the field experiment (Fig 4.13) reduced lead concentration in edible tissues but exceeded the allowable limits by WHO, US-EPA, and KEBS/NEMA. Silicon application reduced concentrations in roots, stems, and leaves. It reduced the uptake of lead by plants by forming complex compounds with metal ions reducing the amount available for plant uptake. It also regulates the activities of metal transporters in plants. Silicon reduces symplastic and apoplastic transport (Bhat et al., 2019). Apoplastic transport is reduced by silicon thickening apoplastic barriers in the plant (Emamverdian et al., 2018). 4.4 Transfer factor of lead and cadmium to plant tissues 4.4.1 Transfer factor of cadmium Treatments had significant (P<0.001) effects on the transfer of cadmium to plant tissues. Cadmium transfer was highest in the roots, followed by stem and leaves in that order (Fig 4.14, Fig 4.15). Cadmium treatment resulted in the highest transfer to plant tissues in vegetables. Silicon amendment reduced the transfer of cadmium by 20% to roots and stems, and 27.20% to leaves in spinach. Silicon application reduced transfer in roots, stems, and leaves of kale by 25.97%, 32.09%, and 46.14% respectively. Silicon treatment decreased transfer by 21.82%, 22.77%, and 29.86% to roots, stems, and leaves of amaranth respectively (a) (b) (c) Fig 4.14 Cadmium transfer factor to spinach (a) kale (b) and amaranths (c) as ameliorated by silicon application in the greenhouse experiment (c) (a) (b) Fig 4.15 Cadmium transfer factor to spinach (a) kale (b) and amaranths (c) tissues as ameliorated by silicon application in the field experiments The transfer factor of cadmium to plant tissues was influenced by bioavailability and the availability of cadmium in soils (El-Amier et al., 2017). The low soil pH in greenhouse and field experiment soils and low amount of organic matter increased the transfer factor to plant tissues. The difference in cadmium transfer among the vegetable species was due to the differential uptake of metal ions by plants (Tangahu et al., 2011). The lower transfer of cadmium to the shoots than the roots of vegetables is due to the difference in cadmium mobility index among the vegetable species (Mehes-Smith et al., 2014). Silicon reduces the transfer of metal ions in plant tissues. The element reduces the uptake, by forming a complex silicon metal ions compound, that is not available for plant absorption (Emamverdian et al., 2018). Silicon changes the soil pH, stimulating cadmium immobilization, and also regulates the activities of metal transporters (Bhat et al., 2019; Emamverdian et al., 2018). Cadmium transfer factor of less than one (TF Cd<1) was in agreement with a similar study on leafy vegetables (Zhang et al., 2014) and Spinach (Hossain et al., 1970). However, a similar study had reported a transfer factor of more than one in amaranths (Jolly et al., 2013). The higher transfer factor of cadmium was also reported in a similar study on spinach (Pal et al., 2017) 4.4.2 Transfer factor of lead Results showed treatments had significant (P<0.001) effects on lead transfer to plant tissues. An increase in lead soil concentrations resulted in an increased transfer factor to vegetable tissues. The transfer was least in leaves, followed by stem and roots in that order (Fig 4.16, Fig 4.17). Silicon application decreased lead to transfer to roots and stems by 20% in vegetables. Silicon amendment reduced transfer to leaves by 51.26%, 5353.77%, and 47.62% in spinach, kale, and amaranths. (a) (b) (c) Fig 4. 16 Lead transfer factor in spinach(a) kale(b) and amaranths(c) as influenced by silicon application in greenhouse experiment (c) (a) (b) Fig 4. 17 Lead transfer factor in spinach (a) kale (b) and amaranths (c) as influenced by silicon application in the field experiment The acid soil pH and low soil organic carbon influenced the availability and transfer of lead. In acid, soils lead exists in aqueous form Pb(H2O6)+2 which is more available for the plants' uptake (Kumar et al., 2020). The transfer of lead was higher in roots and shoots due to a lower translocation index of lead from root to above-ground biomass. The difference in the transfer factor of lead among vegetables is due to the differential ability of crops to uptake and accumulate metal ions (Tangahu et al., 2011). Higher shoot transfer to shoots of leafy vegetables growing on lead spiked soils is due to high roots lead concentration, which damages the membrane permeability and roots apoplastic barrier resulting in higher translocation index to the shoots (Sharma and Dubey, 2005). Silicon alters the pH of the soil, reducing lead available for uptake by plants (Emamverdian et al., 2018). The rise in soil pH caused the precipitation of lead ions in soils (Kumar et al., 2020). It also forms silicates and oxides reducing the availability of metal ions for plant absorption (Bhat et al., 2019). Lead transfer factor of less than one (TF pb<1) was in agreement with a similar study on spinach and amaranths (Jolly et al., 2013), leafy vegetables (Roba et al., 2015), and spinach (Pal et al., 2017). 4.5 Mobility index of lead and cadmium in plant tissues 4.5.1 Translocation index of cadmium Results showed treatments had significant (P<0.001) effects on lead mobility to stem and leaves of vegetables. The translocation index was less than one (TF Cd<1). Cadmium treatment resulted in the highest mobility to stem and leaves (Table 4.7). Silicon application reduced the cadmium mobility to the leaves by 10% in the vegetables. Vegetable Treatment Stem mobility Leaf mobility Days after treatment Days after treatment 25 35 45 25 35 45 Spinach Si 0.5678c 0.7102b 0.6074b 0.3178c 0.3592c 0.3608b Control 0.6142c 0.4923c 0.8750a 0.1455d 0.1066d 0.1048c Cd +Si 0.7671b 0.8959a 0.8868a 0.6429b 0.6892b 0.7294a Cd 0.8958a 0.8244a 0.8588a 0.7529a 0.7710a 0.7510a P-Value *** *** *** *** *** *** Kale Si 0.5322b 0.5275b 0.6657b 0.4007b 0.4099b 0.4362c Control 0.3672c 0.4145c 0.5248c 0.1331c 0.1205c 0.1345d Cd +Si 0.7621a 0.7448a 0.8128a 0.4396b 0.4625b 0.6008b Cd 0.8559a 0.8003a 0.8778a 0.6983a 0.6526a 0.7508a P-Value *** *** *** *** *** *** Amaranths Si 0.5750b 0.7396a 0.6442b 0.3462c 0.3673c 0.3575c Control 0.6064b 0.5709b 0.4983c 0.1284d 0.1236d 0.1038d Cd +Si 0.8137a 0.8476a 0.9233a 0.6656b 0.6208b 0.6724b Cd 0.8638a 0.8816a 0.8807a 0.7298a 0.7332a 0.7202a P-Value *** *** *** *** *** *** Table 4. 7 Cadmium mobility index in spinach, kale, and amaranths in the greenhouse experiment as affected by Silicon amendment Means followed by different superscript letters (down the column within the same vegetable species) differ significantly at ***p<0.001 Table 4. 8 Cadmium mobility index in spinach, kale, and amaranths as affected by Silicon amendment in the field experiment Short rains (2019) Long rains (2020) vegetable Treatment Stem Leaf Stem Leaf Days after treatment Days after treatment Days after treatment Days after treatment 25 35 45 25 35 45 25 35 45 25 35 45 Spinach Si Post 0.8117 0.8823 0.9293 0.703 0.7651 0.8495 0.9104 0.9473 0.9044 0.3739 0.4481 0.5578 Si neg 0.9582 0.9356 0.7926 0.8744 0.8506 0.7901 0.9803 0.9511 0.9035 0.3577 0.4203 0.4523 t value 9.65 26.84 -13.22 12.69 7.2 -7.65 0.089 0.21 -0.03 -1.95 -0.62 -5.77 P value *** *** ** *** ** ** * * ns * * ** Kale Si Post 0.6904 0.6397 0.618 0.2544 0.4081 0.4499 0.4354 0.5854 0.6185 0.2712 0.2481 0.4052 Si neg 0.6228 0.5796 0.6511 0.251 0.4482 0.4861 0.5189 0.657 0.5885 0.2934 0.3792 0.4531 t value -17.27 -159.5 280.15 -2.44 146.16 207.02 2.44 3.97 0.48 0.38 6.31 1.1 P value ** *** *** ns *** *** ns * ns ns * * Amaranths Si Post 0.5502 0.5816 0.5741 0.3019 0.3224 0.3845 0.5804 0.5883 0.5925 0.3223 0.3595 0.436 Si neg 0.5826 0.6367 0.6404 0.2434 0.4408 0.4733 0.6193 0.6382 0.6329 0.29 0.3637 0.4146 t value 5.89 263.21 539.19 -252.5 557.2 527.98 0.92 2.42 0.86 -1.08 0.1 -1.11 P value * *** *** *** *** *** * ns ns * ns ns Means within the same column differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant Silicon application resulted in reduced cadmium mobility in the field experiment (Table 4.8). The cadmium mobility index was less than one (Cd TF<1) in the field experiment. Spinach had higher cadmium mobility, followed by kale and amaranths in that order. The application of cadmium resulted in a higher transfer factor to the roots, raising root cadmium concentrations. High concentrations in roots damaged apoplastic barriers in roots resulting in higher mobility to the shoot. Silicon treatment enhanced apoplastic barriers in the roots of vegetables, resulting in lower mobility to the shoot. It also forms and precipitates complex metal ions as cofactors (Bhat et al., 2019). Silicon stimulates the thickening of cuticle reducing the rate of evapotranspiration in vegetables, resulting in reduced cadmium mobility in vegetables (Kumar et al., 2020). It also compartmentalizes cadmium into cell vacuoles and cell walls in roots reducing mobility to the shoots (Bhat et al., 2019). 4.5.2 Translocation index of lead Results showed significant (P<0.001) alleviation in the mobility of lead in leaves and stems. Spiking soils with lead resulted in the highest translocation index to the shoots. The translocation index was less than one (TF pb<1) in all the treatments (Table 4.10). Silicon application reduced lead mobility to the shoot. It reduced the lead mobility to the leaves by 30%. Table 4. 9 Lead mobility index in spinach, kale, and amaranths in the greenhouse experiment as affected by silicon application Vegetable Treatment Stem mobility Leaf mobility Days after treatment Days after treatment 25 35 45 25 35 45 Spinach Control 0.15406c 0.0575c 0.02874c 0.1200c 0.2009c 0.1077d Si 0.1680b 0.0731b 0.3606b 0.3858b 0.0731c 0.4318c Pb +Si 0.7045a 0.8465a 0.9107a 0.4182b 0.4624b 0.5525b Pb 0.8841a 0.8976a 0.8199a 0.7434a 0.7277a 0.7106a P value *** *** *** *** *** *** Kale Control 0.1286c 0.1488b 0.1319d 0.1063c 0.1051c 0.0935d Si 0.1717c 0.0824c 0.3479c 0.6153a 0.0555d 0.4273c Pb +Si 0.6463b 0.8188a 0.9389a 0.336b 0.4342b 0.5275b Pb 0.824a 0.8156a 0.8671b 0.7094a 0.6728a 0.6955a P value *** *** *** *** *** *** Amaranths Control 0.2078d 0.1446b 0.1539d 0.1387c 0.1119c 0.1148d Si 0.3994c 0.0964c 0.3615c 0.3898b 0.0704c 0.5006c Pb +Si 0.6725b 0.8597a 0.9408a 0.4606b 0.4758b 0.5551b Pb 0.8598a 0.8338a 0.7658b 0.6964a 0.6985a 0.6382a P value *** *** *** *** *** *** Means followed by the same letter within the same column (down the column within the same vegetable species) are not significantly different at p<0.001 The mobility index of lead was less than one (TT Pb<1) in the field experiment. The application of silicon reduced lead translocation to the stem and leaves in spinach and kale, but it did not reduce mobility in amaranths (Table 4.12). Silicon application reduced lead mobility to the leaves of spinach and kale by 10%. Table 4. 10 Cadmium mobility index in spinach, kale, and amaranths in the field experiment as affected by Silicon amendment Short rains (2019) Long rains (2020) Vegetable Treatment Stem Mobility Leaf Mobility Stem Mobility Leaf Mobility Days after treatment Days after treatment Days after treatment Days after treatment 25 35 45 25 35 45 25 35 45 25 35 45 Spinach Si post 0.8117 0.8823 0.9293 0.703 0.7651 0.8495 0.8511 0.9267 0.9112 0.7161 0.7842 0.8189 Si neg 0.9582 0.9356 0.7926 0.8744 0.8506 0.7901 0.9398 0.945 0.8403 0.8614 0.8508 0.8266 t value 9.65 26.84 -13.22 12.69 7.2 -7.65 2.78 0.8 -3.73 10.25 5.08 1.1 P* *** *** ** *** ** ** * ns ns *** ** ns Kale Si post 0.8693 0.9135 0.8885 0.7424 0.7748 0.8661 0.8971 0.914 0.855 0.7312 0.7774 0.8358 Si neg 0.912 1.01 0.8998 0.8734 0.922 0.8718 0.9526 1.0378 0.8793 0.8837 0.9092 0.8671 t value 2.71 2.94 1.07 4.27 10.03 0.45 3.59 4.87 0.93 5.61 9.03 5.46 P* ns * ns * *** ns * * ns ** *** ** Amaranths Si post 0.8803 0.884 0.9067 0.7958 0.8126 0.8385 0.867 0.8849 0.8958 0.76 0.8328 0.8395 Si neg 0.9193 0.827 0.8642 0.8393 0.762 0.7758 0.9358 0.8153 0.8498 0.8412 0.7571 0.759 t value 3.8 -7.58 -3.93 3.01 -7.73 -8.44 4.5 -2.62 -2.4 4.43 -2.97 -3.96 P* * ** * * ** *** ** ns ns ** * * Means within the same column differ significantly at ***p<0.001, **<0.01, *<0.05, ns=not significant The lead application resulted in a higher transfer factor and concentrations in the roots. High lead concentrations damaged membrane structure and permeability and also destroyed apoplastic barriers in roots resulting in higher translocation index to the shoot (Sharma and Dubey, 2005). The damage to the semi-permeability function of cell membranes and tonoplast results in symplast transport of metal ions (Sharma and Dubey, 2005). Silicon alters the structure of the cell wall by transport control and reduces lead mobility in the plant (Emamverdian et al., 2018). Lead apoplast transport is reduced by lead binding with carboxyl, galacturonic, and glucuronic acid. It results in metal ion accumulation on root endodermis thus acting as a partial apoplast translocation barrier to the shoots (Kumar et al., 2020). Silicon compartmentalizes excess metal ions into cell vacuole and cell walls, leading to higher accumulation in roots and less translocation into the shoots (Bhat et al., 2019). 4.6 Uptake of lead and cadmium by vegetables 4.6.1 Cadmium uptake Results showed treatments had significant (P<0.001) effects on cadmium uptake by leafy vegetables. Cadmium treatment in the greenhouse experiment resulted in the highest uptake (Fig 4.18a). Application of silicon reduced cadmium uptake with 33% in spinach and kales, and with 44% in amaranths (Fig 4.18a). Silicon application on field soils polluted with cadmium decreased the uptake (Fig 4.18b). Cadmium uptake was higher in field experiment short rains, 2019 than long rains, 2020 (Fig 4.18b). Silicon addition reduced cadmium uptake with root uptake with 24% in amaranths, 30% in kale, and 41% in spinach. Amaranths had higher uptake, followed by spinach 0 0.2 0.4 0.6 0.8 1 Spinach Kale Amaranth Root uptake (kg ha - 1 ) Control Si Cd +Si Cd 0 0.0001 0.0002 0.0003 0.0004 Spiach Kale Amaranths Spiach Kale Amaranths Short rains,2019 Long rains,2020 Root uptake (kg ha - 1 ) Si Control and kale in that order in both the greenhouse experiment and field experiment (Fig 4.18 (a), (b)). (b) (a) Fig 4.18 Root uptake of cadmium by leafy vegetables as affected by silicon amendment in the greenhouse (a) and field experiments (b) There were significant (P<0.001) effects of treatment on shoot uptake of cadmium. Soils spiked with cadmium resulted in the highest shoot uptake (Fig 4.19). The application of silicon decreased uptake by 32% in spinach in the greenhouse experiment. Shoot uptake (kg ha - 1 ) Shoot uptake (kg ha - 1 ) Shoot uptake (kg ha - 1 ) Leafy vegetables cadmium shoot uptake was higher in the short rains, 2019 than in long rains, 2020 in the field experiment. Silicon application reduced cadmium shoot uptake by 21.42% in spinach and 10% in kale and amaranths when compared with control (Fig 4.20). (a) (c) (b) Fig 4.19 Shoot uptake of cadmium by spinach(a) kale(b) and amaranths(c)as affected by silicon amendment in greenhouse experiment (a) (b) Fig 4.20 Shoot uptake of cadmium by spinach, kale, and amaranths as affected by silicon amendment short rains,2019 (a) and long rains, 2020 (b) in the field experiments Cadmium uptake was higher in roots than in shoots due to higher concentrations as a result of low translocation index and accumulation of cadmium on the cell wall and cell vacuole (Pereira et al., 2018). The Uptake and translocation of cadmium are dependent on the leafy vegetables species and genotype (Zhao et al., 2020). Silicon deposition on cell wall lignin as silica gel (SiO2.nH2O) reduced the translocation of cadmium to the shoots (Sa, 2013). It also raised soil pH reducing the Phyto- availability of cadmium to plant (Zhao et al., 2020). Cadmium spiking in soils altered it's in soils as Acid Cd>Residual Cd> Oxidable Cd (Zhao et al., 2020). Acid soluble Cd includes exchangeable cd and carbonate bound Cd, reducible Cd includes iron-manganese oxide Cd, Oxidable Cd includes organic Cd. Residual Cd forms as stable compounds with primary and secondary minerals. Exchangeable Cd is adsorbed to soil and humus and is affected by environmental conditions and is readily absorbed by plants (Zhao et al., 2020). Silicon application reduced the amount of acid-soluble and reducible Cd and increased oxidable and residual Cd reducing cadmium bioavailability (Zhao et al., 2020). Reduction of cadmium uptake by silicon treatments was in agreement with similar uptake studies on paddy rice (Zhao et al., 2020), amaranths (Lu et al., 2017), cotton (Sa, 2013), maize, sugarcane, arabidopsis, peanuts, and tobacco (LU et al., 2018). It has also been reported in alfalfa and cucumber (Kabir et al., 2016), strawberries (Treder and Cieslinski, 2005), and Pfaffia glomerata (Pereira et al., 2018). However, the application of silicon had decreased roots uptake by half-fold in Pfaffia glomerata (Pereira et al., 2018). 0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 spinach kale amaranths Root uptake (kg ha - 1 ) Control Si Pb+si Pb 0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 spinach kale amaranths Root uptake (kg ha - 1 ) Control Si Pb+si Pb 4.6.2 Lead uptake Results showed treatments had significant (P<0.001) effects on the root lead uptake by leafy vegetables. Spiked soils with lead resulted in the highest root lead uptake while the control had the least in the greenhouse experiment (Fig 4.21a). The application of silicon decreased lead uptake by 20% in leafy vegetables. In the field experiment, amaranths showed the highest lead uptake followed by kale (Fig 4.21b). Silicon application in the field reduced root lead uptake with 43%, 45%, and 28% in spinach, kale, and amaranths respectively. (b) (a) Fig 4. 21 Root uptake of lead alleviated by silicon amendment in spinach, kale and amaranths in greenhouse (a) and field experiments (b) Shoot uptake (kg ha - 1 ) Shoot uptake (kg ha - 1 ) Shoot uptake (kg ha - 1 ) (a) (c) (b) Fig 4.22 Shoot uptake of lead as alleviated by silicon amendment in spinach(a) kale(b) and amaranths(c) in the greenhouse experiment (a) (b) Fig 4.23 Silicon alleviation on lead shoot uptake by spinach, kale, and amaranths in the short rains, 2019 (a) and long rains,2020 (b) of the field experiment The greenhouse and field experiment soils had low pH and organic carbon, hence more lead availability and uptake by leafy vegetables. In low soil pH lead ions exist in aqueous form Pb(H2O6)+2 that is readily absorbed by crops. Leafy vegetable species had differential lead transfer factors hence differences in uptake (Fahr et al., 2013). Similar studies have shown some leafy vegetables have higher lead uptake than other vegetables (Feleafel and Mirdad, 2013). The vegetables on lead treatment synthesis and deposit callose, which act as a barrier thus reducing lead uptake (Fahr et al., 2013). However, high lead concentrations damage the cells and the semipermeable function of the plasma membrane, allowing symplast transport (Sharma and Dubey, 2005). The apoplast and symplast transport caused higher shoot lead uptake in lead treatment. Silicon reduced lead uptake by forming complex silicon compounds with metal ions. It formed silicates and oxides reducing the availability of lead for uptake by vegetables (Bhat et al., 2019). Silicon also raised the soil pH reducing the lead availability. Silicon reduced shoot uptake by decreasing the apoplastic and symplastic transport of lead (Bhat et al., 2019). It also stimulated compartmentation of lead into the cell vacuoles and cell wall, hence higher accumulation in roots than shoots. The results were in agreement with similar studies on silicon ameliorated lead uptake on rice (Liu et al., 2015). 4.7 Relationship between growth parameters and concentrations of lead and cadmium in leafy vegetables Pearson correlation indicated lead concentrations in plant tissues had negative relationships with plant growth parameters in spinach (Table 4.13). The results showed lead concentrations had a strong negative correlation with root length, leaf dry weight, and leaf area. It also indicated moderate negative linear relationships between stem and root dry weight (Ratner, 2009). There were strong negative relationships between measured spinach growth parameters with lead and cadmium concentrations in soil. Table 4. 11 Person correlations coefficient between measured spinach growth parameters and lead and cadmium concentrations in soil and plant tissues Shoot length Root length Root dry Leaf dry Stem dry leaf area Shoot length 1 Root length 0.23 1 Root dry 0.25 0.58** 1 Leaf dry 0.26 0.86*** 0.74*** 1 Stem dry 0.31 0.68** 0.89*** 0.81*** 1 Shoot dry 0.29 0.84*** 0.81*** 0.98*** 0.9*** Leaf area 0.37 0.73*** 0.60** 0.74*** 0.67** 1 Stem Pb -0.33 -0.96*** -0.63* -0.91*** -0.70** -0.84*** Root Pb -0.33 -0.97*** -0.56* -0.92*** -0.63* -0.81*** Leaf Pb -0.35 -0.94*** -0.56* -0.92*** -0.57* -0.77** Stem Cd -0.22 -0.41 -0.49 -0.59* -0.79*** -0.09 Root Cd -0.22 -0.41 -0.5 -0.80* -0.80*** -0.10 Leaf Cd -0.21 -0.42 -0.52 -0.61* -0.81*** -0.12 Pb soil -0.28 -0.97*** -0.49 -0.86*** -0.65* -0.82*** Cd soil -0.17 -0.90*** -0.69** -0.88*** -0.80** -0.71** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Pearson correlation coefficient indicated strong positive linear relationships between measured kale growth parameters (Table 4.14). Cadmium and lead concentrations had a negative linear relationship with growth. There was a significant (P<0.001) strong negative linear relationship between kale root length, leaf area, root, leaf, and stem dry weight with lead and cadmium concentrations (Ratner, 2009). The Pearson correlation coefficient indicated a moderate linear relationship between cadmium concentrations and kale shoot length Table 4. 12 Person correlations coefficient between measured kale growth parameters and lead and cadmium concentrations in soil and plant tissues Shoot length Root length Root dry Leaf dry Stem dry Leaf area Shoot length 1 Root length 0.60*** 1 Root dry 0.40 0.72*** 1 Leaf dry 0.61*** 0.80*** 0.83*** 1 Stem dry 0.72*** 0.88*** 0.78*** 0.80*** 1 Leaf area 0.45* 0.85*** 0.89*** 0.82*** 0.81*** 1 Stem Pb 0.14 0.05 -0.22 -0.23 -0.22 -0.30 Root Pb 0.09 -0.001 -0.28 -0.29 -0.15 -0.36 Leaf Pb 0.002 -0.15 -0.41 -0.43 -0.0006 -0.51 Stem Cd -0.56 -0.84*** -0.87*** -0.98*** -0.79*** -0.89*** Root Cd -0.58* -0.84*** -0.86*** -0.98*** -0.80*** -0.89*** Leaf Cd -0.52 -0.83*** -0.85*** -0.97*** -0.77*** -0.91*** Pb soil -0.51 -0.83*** -0.53 -0.79*** -0.72*** -0.76*** Cd soil -0.65* -0.80*** -0.8*** -0.96*** -0.79*** -0.83*** *, *** indicate significance at P<0.5, P<0.001 respectively Results showed significant (P<0.001) strong positive linear relationships among measured amaranths growth parameters (Table 4.15). Pearson correlation coefficient indicated a strong negative linear relationship between cadmium concentrations in soil and plants with measured amaranths growth parameters (Ratner, 2009). There was a little linear relationship between lead concentrations in plant tissues with measured growth parameters. However, there existed a strong negative linear relationship between measured growth parameters and soil lead concentrations (Ratner, 2009). Table 4. 13 Person correlations coefficient between measured amaranth’s growth parameters and lead and cadmium concentrations in soil and plant tissues Shoot length Root length Root dry Leaf dry Stem dry Leaf area Shoot length 1 Root length 0.84*** 1 Root dry 0.65** 0.86*** 1 Leaf dry 0.71*** 0.82*** 0.79*** 1 Stem dry 0.76*** 0.86*** 0.91*** 0.88*** 1 Leaf area 0.71*** 0.74*** 0.81*** 0.77*** 0.78*** 1 Stem Pb -0.08 -0.25 -0.23 0.0008 -0.04 -0.21 Root Pb -0.11 -0.21 -0.21 -0.06 -0.02 -0.22 Leaf Pb -0.07 -0.14 -0.15 -0.13 -0.04 -0.27 Stem Cd -0.82*** -0.80*** -0.65* -0.91*** -0.79*** -0.87*** Root Cd -0.82*** -0.80*** -0.66* -0.91*** -0.80*** -0.88*** Leaf Cd -0.80** -0.79** -0.68* -0.91*** -0.80*** -0.90*** Pb soil -0.59* -0.83*** -0.77** -0.89*** -0.80*** -0.77*** Cd soil -0.84*** -0.80** -0.58* -0.90*** -0.76*** -0.80*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001, respectively The coefficient of determination between root (X) and leaf/stem (Y), lead, and cadmium varied among the leafy vegetables. The lead coefficient of determination was kale stem (R2=0.9932) and also lowest in kale leaf (R2=0.9791). The cadmium coefficient of determination was highest in spinach leaf (R2=0.9997) and lowest in kale leaf(R2=0.9887). Results showed lead and cadmium concentrations in leaves and stem model of Equations IX, to XX. Spinach Stem Pb Y=0.851x R2=0.9925 IX Leaf Pb Y=0.653x R2=0.9830 X Kale Stem Pb Y=0.8854x R2=0.9932 XI Leaf Pb Y=0.6341x R2=0.9791 XII Amaranths Stem Pb Y=0.8125x R2=0.9848 XIII Leaf Pb Y=0.6095x R2=0.9920 XIV Spinach Stem Cd Y=0.8698x R2=0.9996 XV Leaf Cd Y=0.7424x R2=0.9997 XVI Kale Stem Cd Y=0.8539x R2=0.9984 XVII Leaf Cd Y=0.6962x R2=0.9887 XVIII Amaranths Stem Cd Y=0.8965x R2=0.9993 XIX Leaf Cd Y=0.7023x R2=0.9985 XX Whereby: Y= stem/leaf concentration of lead/cadmium X= Root concentration of lead/cadmium Pb= Lead, Cd= Cadmium Lead and cadmium had a negative correlation with measured growth parameters. The negative correlation was strong in cadmium than lead. Cadmium causes more toxicity in vegetables than lead. Cadmium's strong negative correlation corresponds with a similar study on root length, shoot length, leaf, stem, and root dry weights of spinach (Alia et al., 2015). The cadmium toxicity may have been higher in roots due to direct contact with soils and accumulation. Cadmium inhibited cell division at the root tips, reducing root elongation (Alia et al., 2015). The lower translocation index to shoots resulted in less inhibition of growth in shoot length than the root length. The reduced inhibition of shoot length in spinach corresponds with a similar study on tomatoes (Rehman et al., 2011). Lead negative association with root length and leaf area was also reported on tomatoes, radish, and soybeans (Rehman et al., 2011). Leafy vegetables have differential uptake and translocation index of lead and cadmium. Plants accumulate heavy metals more in roots and translocate poorly to the shoots (Cannata et al., 2013). Cadmium has higher mobility than lead in plants, hence a strong positive Pearson correlation coefficient (Sekara et al., 2005). The amount of cadmium in roots is proportional to the stems and leaves concentrations due to passive translocation (Sekara et al., 2005). Higher cadmium translocation and the transfer were also due to weak adsorption by soil colloids and its easier accumulation by plants (Zaprjanova et al., 2010). The translocation was also affected by transpiration rate and phytoavailable cadmium influenced by soil concentrations, soil pH, clay mineral, and organic matter (Grant et al., 1998; Tariq Rafiq et al., 2014). The cadmium coefficients were within the range of those reported on sugar beets (Sekara et al., 2005), and in spinach (Alia et al., 2015). Lead ions form stable chelates as root deposits easily than cadmium ions, hence lower mobility (Cannata et al., 2013). The lead was translocated to the leaves and stems, however, it was commonly accumulated in roots (Usman et al., 2019). The low translocation was also due to apoplastic barriers and precipitation of lead insoluble salts in roots (Usman et al., 2019). Cadmium concentrations' strong positive relationship between roots with stems and leaves were also reported on maize (Grant et al., 1998) and Phaseolus vulgaris L.(Hardiman et al., 1984). stronger cadmium correlations than lead correspond with a similar study on red beets (Sekara et al., 2005), Myriophyllum spicatum L. (Yabanli et al., 2014), and Tetraenaqataranse(Usman et al., 2019). CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Lead and cadmium reduced leaf, stem, and root dry weight, shoot and root length, and leaf area of leafy vegetables. The growth inhibition was higher in roots than on aboveground biomass. Cadmium inhibited growth more than lead on leafy vegetables. The vegetables had differential tolerance to lead and cadmium. Spinach had the highest tolerance, followed by amaranths and kale in that order. Results showed strong negative correlations between cadmium concentrations with the growth of vegetables. Silicon application on contaminated soils enhanced leafy vegetables lead and cadmium tolerance. Lead and cadmium had significant transfers from the soil to tissues of leafy vegetables. Cadmium transfer to tissues was higher than lead transfer. The transfer factor was highest to roots, followed by stem and leaves in that order. Leafy vegetables had differential accumulation and transfer of lead and cadmium. Amaranths had the highest lead transfer, while spinach had the highest cadmium transfer. There was a strong positive relationship between root cadmium concentrations with stem and leaf concentrations. Silicon application reduced uptake of lead and cadmium by the leafy vegetables. Silicon treatment reduced lead and cadmium mobility in vegetables hence higher accumulation on roots. Silicon significantly reduced cadmium mobility highest in kale, amaranths, and spinach in that order. It reduced lead mobility in spinach, followed by kale and amaranths in that order. 5.2 Recommendations The study recommends spinach production on soils contaminated with heavy metals due to its high growth tolerance, especially for phytoremediation. Silicon fertilization on lead and cadmium polluted soils, for enhanced leafy vegetable tolerance and increased productivity. The study recommends non-production of leafy vegetables on soils contaminated with lead and cadmium for human utilization. The leafy vegetables had the potential to accumulate metal ions beyond the allowable limits. The concentrations were high in roots and stems, where human consumption is intended, the plant tissues should be discarded due to high accumulations. 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APPENDICES Appendix 1: Person correlations coefficient between measured Spinach growth parameters and lead and cadmium concentrations in soil and plant tissues in Field Experiment Short rains, 2019 Shoot length Root length Root dry Leaf dry Stem dry leaf area Shoot length 1 Root length 0.66* 1 Root dry 0.74** 0.81*** 1 Leaf dry 0.69** 0.63* 0.94*** 1 Stem dry 0.61* 0.62* 0.88*** 0.95*** 1 leaf area 0.68** 0.78*** 0.97*** 0.94*** 0.94*** 1 Stem Pb -0.68* -0.74** -0.94*** -0.93*** -0.96*** 0.99*** Root Pb -0.67* -0.78** -0.97*** -0.93*** -0.93*** -0.99*** Leaf Pb -0.7** -0.79** -0.97*** -0.92*** -0.93*** -0.99*** Stem Cd -0.68** -0.78** -0.98*** -0.94*** -0.94*** -0.99*** Root Cd -0.68** -0.79** -0.97*** -0.95*** -0.94*** -0.99*** Leaf Cd -0.69** -0.78** -0.96*** -0.94*** -0.93*** -0.99*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 2: Person correlations coefficient between measured Kale growth parameters and lead and cadmium concentrations in soil and plant tissues in Field Experiment Short rains, 2019 Shoot length Root length Root dry Leaf dry Stem dry leaf area Shoot length 1 Root length 0.73** 1 Root dry 0.78** 0.54 1 Leaf dry 0.84*** 0.76** 0.57* 1 Stem dry 0.56* 0.57** 0.5 0.83*** 1 leaf area 0.84*** 0.85*** 0.58* 0.98*** 0.85*** 1 Stem Pb -0.84*** -0.86*** -0.64* -0.97*** -0.86*** -0.99** Root Pb -0.8*** -0.88*** -0.55 -0.96*** -0.84*** -0.99*** Leaf Pb -0.82*** -0.83*** -0.59* -0.97*** -0.88*** -0.99*** Stem Cd -0.84*** -0.85*** -0.57* -0.98*** -0.86*** -0.99*** Root Cd -0.83*** -0.85*** -0.58* -0.98*** -0.86*** -0.99*** Leaf Cd -0.83*** -0.82*** 0-.57* -0.97*** -0.85*** -0.99*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 3: Person correlations coefficient between measured Amaranth’s growth parameters and lead and cadmium concentrations in soil and plant tissues in field experiment Short rains, 2019 Shoot length Root length Root dry Leaf dry Stem dry leaf area Shoot length 1 Root length 0.84*** 1 Root dry 0.78** 0.86*** 1 Leaf dry 0.86*** 0.85*** 0.70** 1 Stem dry 0.88*** 0.84*** 0.84*** 0.98*** 1 leaf area 0.85*** 0.81*** 0.76** 0.99*** 0.98*** 1 Stem Pb -0.85*** -0.82*** -0.75** -0.99*** -0.99*** -0.99*** Root Pb -0.86*** -0.83*** -0.79** -0.99*** -0.99*** -0.99*** Leaf Pb -0.92*** -0.86*** -0.83*** -0.98*** -0.99*** -0.98*** Stem Cd -0.84*** -0.81*** -0.78** -0.99*** -0.99*** -0.99*** Root Cd -0.85*** -0.83*** -0.79** -0.99*** -0.99*** -0.99*** Leaf Cd -0.83*** -0.82*** -0.78** -0.99*** -0.99*** -0.99*** **, *** indicate significance at P<0.01, P<0.001 respectively Appendix 4: Person correlations coefficient between measured Spinach growth parameters and lead and cadmium concentrations in soil and plant tissues in field experiment long rains, 2020 Shoot length Root length Root dry Leaf dry Stem dry Leaf area Shoot length 1 Root length 0.63* 1 Root dry 0.83*** 0.87*** 1 Leaf dry 0.87*** 0.78*** 0.93*** 1 Stem dry 0.96*** 0.67** 0.89*** 0.93*** 1 Leaf area 0.88*** 0.82*** 0.97*** 0.94*** 0.94*** 1 Stem Pb -0.91*** -0.76** -0.93*** -0.92*** -0.97** -0.97*** Root Pb -0.89*** -0.82*** -0.97*** -0.93*** -0.92*** -0.99*** Leaf Pb -0.86*** -0.84*** -0.97*** -0.92*** -0.94*** -0.99*** Stem Cd -0.89*** -0.82*** -0.98*** -0.94*** -0.95*** -0.99*** Root Cd -0.85*** -0.82*** -0.95*** -0.90*** -0.91*** -0.99*** Leaf Cd -0.83*** -0.77** -0.94*** -0.90*** -0.86*** -0.94*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 5: Person correlations coefficient between measured Kale growth parameters and lead and cadmium concentrations in soil and plant tissues in Field Experiment Long rains, 2020 Shoot length Root length Root dry Leaf dry Stem dry Leaf area Shoot length 1 Root length 0.63* 1 Root dry 0.83*** 0.87*** 1 Leaf dry 0.87*** 0.78*** 0.93*** 1 Stem dry 0.96*** 0.67*** 0.89*** 0.93*** 1 Leaf area 0.88*** 0.82*** 0.97*** 0.94*** 0.94*** 1 Stem Pb -0.91*** -0.76** -0.93*** -0.92*** -0.97*** -0.97*** Root Pb -0.89*** -0.82*** -0.97*** -0.93*** -0.92*** -0.99*** Leaf Pb -0.86*** -0.84*** -0.97*** -0.92*** -0.91*** -0.99*** Stem Cd -0.89*** -0.82*** -0.98*** -0.94*** -0.95*** -0.99*** Root Cd -0.85*** -0.82*** -0.95*** -0.9*** -0.94*** -0.99*** Leaf Cd -0.83*** -0.77** -0.94*** -0.9*** -0.86*** -0.94*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 6: Person correlations coefficient between measured Amaranth’s growth parameters and lead and cadmium concentrations in soil and plant tissues in Field Experiment Long rains, 2020 Shoot length Root length Root dry Leaf dry Stem dry Leaf area Shoot length 1 Root length 0.82*** 1 Root dry 0.83*** 0.59* 1 Leaf dry 0.97*** 0.87*** 0.78*** 1 Stem dry 0.95*** 0.83*** 0.82*** 0.98*** 1 Leaf area 0.95*** 0.89*** 0.75** 0.99*** 0.97*** 1 Stem Pb -0.96*** -0.87*** -0.77** -0.99*** -0.97*** -0.98*** Root Pb -0.96*** -0.86*** -0.82*** -0.99*** -0.99*** -0.98*** Leaf Pb -0.97*** -0.86*** -0.77** -0.98*** -0.95*** -0.98*** Stem Cd -0.94*** -0.85*** -0.79** -0.98*** -0.95*** -0.96*** Root Cd -0.92*** -0.83*** -0.73** -0.95*** -0.96*** -0.97*** Leaf Cd -0.93*** -0.87*** -0.76** -0.97*** -0.99*** -0.97*** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 7: Effects of silicon on lead shoot uptake by spinach, kale and amaranths in greenhouse experiment Vegetable Mean Median Minimum Maximum Lower quartile Upper quartile Spinach Si 0.00023c 0.0002 0.0002 0.0002 0.0002 0.0002 Control 0.00021c 0.0002 0.0002 0.0002 0.0002 0.0002 Pb+Si 0.00207b 0.0020 0.0019 0.0024 0.0019 0.0023 Pb 0.00272a 0.0028 0.0024 0.0029 0.0025 0.0029 P *** Kale Si 0.00009b 0.0001 0.0001 0.0001 0.0001 0.0001 Control 0.00002b 0.0000 0.0000 0.0000 0.0000 0.0000 Pb+Si 0.00085a 0.0008 0.0007 0.0010 0.0008 0.0009 Pb 0.00107a 0.0010 0.0009 0.0012 0.0010 0.0012 P *** Amaranths Si 0.00026b 0.0003 0.0002 0.0003 0.0002 0.0003 Control 0.00006b 0.0001 0.0001 0.0001 0.0001 0.0001 Pb+Si 0.00184a 0.0019 0.0015 0.0021 0.0016 0.0020 Pb 0.00186a 0.0019 0.0016 0.0021 0.0017 0.0020 P *** *, **, *** indicate significance at P<0.5, P<0.01, P<0.001 respectively Appendix 8: Effects of silicon on lead shoot uptake by spinach, kale and amaranths in greenhouse experiment Vegetable Mean Median Minimum Maximum Lower quartile Upper quartile Spinach Si 0.0066c 0.0067 0.0062 0.0069 0.0063 0.0068 Control 0.0112c 0.0111 0.0102 0.0122 0.0104 0.0120 Cd+Si 1.0840a 1.0800 1.0720 1.1000 1.0740 1.0950 Cd 1.5900a 1.5440 1.4540 1.7720 1.4770 1.7150 P *** Kale Si 0.0018b 0.0017 0.0017 0.0019 0.0017 0.0018 Control 0.0025b 0.0023 0.0022 0.0029 0.0022 0.0027 Cd+Si 0.3280a 0.3220 0.3210 0.3410 0.3210 0.3360 Cd 0.3480a 0.3690 0.2960 0.3790 0.3150 0.3770 P *** Amaranths Si 0.0045b 0.0044 0.0039 0.0054 0.0040 0.0051 Control 0.0076b 0.0074 0.0074 0.0081 0.0074 0.0079 Cd+Si 1.1110a 1.1860 0.9240 1.2240 0.9890 1.2140 Cd 1.1180a 1.1320 0.9990 1.2230 1.0320 1.2000 P *** *** indicate significance at P<0.001 respectively Appendix 9: National Commission 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