An-Najah National University Faculty of Graduate Studies Effects of Salinity, Nutrients, Heavy Metals and Organic Matters on Growth, Yield and Uptake of Pea in Piped Hydroponics By Mohammad Ass'ad Saeed AL Jada Supervisor Prof. Dr. Marwan M. Haddad This Thesis is Submitted in Partial Fulfillment of the Requirement for the Degree of Master of Environmental Sciences, Faculty of Graduate Studies, An_Najah National University, Nablus, Palestine. 2014 III Dedication  My parents: Thank you for your unconditional support with my studies. I'm honored to have you as my parents. Thank you for giving me a chance to prove and improve myself through all my walks of life. Please do not ever change. I love you.  My family: Thank you for believing in me; for allowing me to further my studies. Please do not ever doubt my dedication and love for you.  My brothers: Hoping that with this research I have proven to you that there is no mountain higher as long as God is on our side, hoping that you will walk again and be able to fulfill your dreams. IV Acknowledgements I thank God for the completion of this work Thanked the people of thanking God I would like to express my deep gratitude to my advisor Prof. Dr. Marwan M. Haddad for supporting me My heartfelt thanks to my mother dear, that supported me by prayer and supplication to God And my gratitude to my wife and my children, for their patience and encouragement to me… My appreciation and respect to the staff and laboratories technicians: Zahran, Bilal and Rami. For all.… Thank you Mohammad VI List of Contents No. Subject Page Dedication III Acknowledgment IV Declaration V List of Contents VI List of Tables IX List of Figures XII List of Acronyms XIII Abstract VIX Chapter one: Introduction 1 1.1 Research background 2 1.2 Objectives 5 Chapter two: Literature review 6 2.1 General overview on Pea 7 2.2 Planting Date 7 2.3 Abiotic stresses 8 2.4 Salinity definition 9 2.5 Electrical conductivity of nutrient solution 10 2.6 Effect of excess salinity on water regime 12 2.7 Nutrient concentration versus crop performance 15 2.8 Salinity versus plant growth 16 2.9 Salinity versus plants mineral nutrition 18 2.10 Heavy metals uptake and impact 23 2.11 Accumulation of compatible osmolytes and osmotic stress 26 2.12 Palestinian statistics about peas planting 28 2.13 Summary 29 Chapter three: Methods and materials 32 3.1 Research plan 33 3.2 Experiment setup 35 3.2.1 Introduction 35 3.2.2 Growth chambers construction 35 3.2.3 Reservoir construction 37 3.3 Experiment program 38 3.3.1 Introduction 38 3.3.2 Seeds germination 39 3.3.3 Preparation of nutrient solutions 40 3.3.3.1 Introduction 40 VII 3.3.3.2 Preparation of nutrient solutions for all line 40 3.4 System Operation 42 3.4.1 Introduction 42 3.4.2 Transfer solutions to growth chambers 42 3.4.3 Monitoring and care of plants 42 3.5 Experiment management 42 3.5.1 Introduction 42 3.5.2 Physical measurements of plants 43 3.5.3 Chemical measurement 44 3.5.3.1 Analysis of plants 44 3.5.3.2 Analysis of nutrient solutions 45 3.6 Chemicals and reagents 45 3.7 Chemical analysis 46 3.8 Statistical analysis 46 Chapter four :Results and discussion 47 4.1 Results and discussion 48 4.1.1 Introduction 48 4.1.2 Nutrients solutions 48 4.1.2.1 Electrical conductivity(EC) 48 4.1.2.2 Nutrients concentrations of solutions 49 4.1.3 Characteristics of plants 55 4.1.3.1 Survival percentage 56 4.1.3.2 Pods characteristics 56 4.1.3.3 Leaves characteristics 61 4.1.3.4 Stems characteristics 66 4.1.3.5 Roots characteristics 71 4.1.4 The effect of salinity on whole plants 76 4.1.5 Nutrients in plants 78 4.1.5.1 Percentage of sodium and chloride in all parts of plants 78 4.1.5.2 Calcium in plant parts 80 4.1.5.3 Magnesium in plant parts 82 4.1.5.4 Potassium in plant parts 83 4.1.5.5 Phosphate in plant parts 85 4.1.5.6 Sulfate in plant parts 87 4.1.5.7 Nitrate in plant parts 89 4.1.5.8 Weight of micronutrients in plant parts 91 4.1.5.8.1 Copper in plant parts 92 4.1.5.8.2 Iron in plant parts 93 VIII 4.1.5.8.3 Manganese in plant parts 95 4.1.5.8.4 Zinc in plant parts 97 4.1.5.8.5 Molybdenum in plant parts 98 4.2 Summary 100 4.2.1 Electrical conductivity 100 4.2.2 Nutrients concentrations in solutions 100 4.2.3 Survival percentage 101 4.2.4 Pods characteristics 101 4.2.5 Leaves characteristics 102 4.2.6 Stems characteristics 102 4.2.7 Roots characteristics 103 4.2.8 Whole plants characteristics 104 4.2.9 Sodium and chloride in plant parts 104 4.2.10 Calcium in plant parts 105 4.2.11 Magnesium in plant parts 105 4.2.12 Potassium in plant parts 106 4.2.13 Phosphate in plant parts 106 4.2.14 Sulfate in plant parts 107 4.2.15 Nitrate in plant parts 107 4.2.16 Copper in plant parts 108 4.2.17 Iron in plant parts 109 4.2.18 Manganese in plant parts 109 4.2.19 Zinc in plant parts 110 4.2.20 Molybdenum in plant parts 110 Conclusion 111 Reference 115 ب الملخص IX List of Tables Table No. Title Page Table 1 Classification of water quality based on salt concentration 4 Table 2 Area harvested, yield and production of peas in Palestine in the period (2010-2013) 28 Table 3 The use of agricultural fertilizers in Palestine in the period (2004-2008) 29 Table 4 Nutrient solution components of six lines of the experiment 40 Table 5 Concentration of nutrients in Cooper solution 40 Table 6 Weight salts are required for preparing 153.6 liters of Cooper solution 41 Table 7 The date of obtaining samples of nutrient solutions 45 Table 8 The electric conductivity(ms/cm) at different times in nutrient solutions of various Lines 49 Table 9 Nutrients concentration(ppm) in Line1 solution at different times 50 Table 10 Nutrients concentration(ppm) in Line2 solution at different times 51 Table 11 Nutrients concentration(ppm) in Line3 solution at different dates 52 Table 12 Nutrients concentration(ppm) in Line4 solution at different times 53 Table 13 Nutrients concentration(ppm) in Line5 solution at different times 54 Table 14 Nutrients concentration (ppm) in line 6 solution at different times 55 Table 15 Relationship between survival percentage of pea plant and salinity levels(ms/cm): 56 Table 16 The average number, fresh and dry biomass weight of pods in different lines 57 Table 17 Average the area, fresh and dry biomass weight of leaves pea plants at different lines. 62 Table 18 average length, fresh and dry biomass of stems pea plants at different lines 66 Table 19 average length, fresh and dry biomass of roots pea plants at different lines 71 Table 20 Average fresh, dry biomass of whole plant of pea at different salinities(Electrical conductivity of various lines) 77 X Table 21 average percentage of chloride and sodium in all parts of pea plants at different lines 78 Table 22 The output of the ANOVA analysis and whether they have a statistically significant difference between percentage of sodium and chloride in plant parts 79 Table 23 average percentage (%) of calcium in all parts of pea plants at different lines 81 Table 24 The output of the ANOVA analysis and whether they have a statistically significant difference between percentage of calcium in plant parts 81 Table 25 average percentage(%) of magnesium in pea plants at different lines 82 Table 26 The output of the ANOVA analysis and whether they have a statistically significant difference between percentage of magnesium in plant parts 83 Table 27 Average percentage of potassium in all parts of plants at different lines 84 Table 28 The output of the ANOVA analysis and whether they have a statistically significant difference between percentage of potassium in plant parts 85 Table 29 Average percentage of phosphate in all parts of pea plants at different lines 86 Table 30 average percentage of sulfate in all parts of pea plants at different lines 88 Table 31 average percentage of nitrate in all parts of pea plants at different lines 90 Table 32 Average weight of copper(mg/Kg dry plant) in all parts of pea plants at different lines 92 Table 33 The output of the ANOVA analysis and whether they have a statistically significant difference between mean copper weight in plant parts 93 Table 34 Average weight of Iron(mg/kg dry plant) in all parts of pea plants at different lines 94 Table 35 The output of the ANOVA analysis and whether they have a statistically significant difference between mean iron weight in plant parts 94 Table 36 Average weight of manganese (mg/Kg dry plant) in all parts of pea plants at different lines 95 Table37 The output of the ANOVA analysis and whether they have a statistically significant difference 96 XI between mean manganese weight in plant parts Table38 Average weight of zinc (mg/kg dry plant) in all parts of pea plants at different lines 97 Table 39 The output of the ANOVA analysis and whether they have a statistically significant difference between mean zinc weight in plant parts 98 Table40 The output of the ANOVA analysis and whether they have a statistically significant difference between mean molybdenum weight in plant parts 99 Table 41 Average weight of molybdenum (mg/kg dry plant) in all parts of pea plants at different lines 100 XII List of Figures Page Title No. 35 Represent a true picture of one pipe in the system Figure 1 36 Represent general diagram of one pipe in PHS Figure 2 37 Represent the diagram of one line in PHS Figure 3 38 Represent complete Piped hydroponic system Figure 4 45 Samples are ready to measure the concentration of phosphate in plant nutrients or solutions Figure 5 60 Average of pods number of pea plants at different pipes in each line Figure 6 60 Average of fresh weight of pods of pea plants at different pipes in each line Figure 7 61 Average of dry biomass weight of pods of pea plants at different pipes in each line Figure 8 64 Average of area of leaves of pea plants at different pipes in each line Figure 9 65 Average of fresh weight of leaves of pea plants at different pipes in each line Figure 01 65 Average of dry weight of leaves of pea plants at different pipes in each line Figure 00 69 Average of length of stems of pea plants at different pipes in each line Figure 12 70 Average of fresh biomass weight of stems of pea plants at different pipes in each line Figure 13 70 Average of dry biomass weight of stems of pea plants at different pipes in each line Figure 14 75 Average of length of roots of pea plants at different pipes in each line Figure 15 75 Average of fresh biomass weight of roots of pea plants at different pipes in each line Figure 16 76 Average of dry biomass weight of roots of pea plants at different pipes in each line Figure 17 87 Average percentage of phosphate in whole pea plants at different lines Figure 18 89 Average percentage of sulfate in whole pea plants at different Lines Figure 19 91 Average percentage of nitrate in whole pea plants at different lines Figure 20 XIII List of Acronyms Cm Centimeter Cm 3 Cubic Centimeter DM Dry Matter EC Electrical Conductivity F FAO estimate FAO Food Agriculture Organization of The United Nation FAOSTAT FAO Statistical Databases FC Calculated data ICARDA ICARDA International Center for Agricultural Research in the Dry Areas. IM FAO data based on imputation methodology MS Millisiemens OD Official data OP Osmotic Pressure PCBS Palestinian Central Bureau of Statistics PHS Piped Hydroponic System PWA Palestinian Water Authority TDS Total Dissolved Salts WUE Water Use Efficiency XIV Effects of Salinity, Nutrients, Heavy Metals and Organic Matters on Growth, Yield and Uptake of Pea in Piped Hydroponics By Mohammad Ass'ad Saeed AL Jada Supervisor Prof. Dr. Marwan M. Haddad Abstract Population growth in Palestine and consequent increased water consumption lead to erosion of arable land and reduction in fresh water suitable for agriculture, so hydroponics may be the most appropriate alternative in these circumstances. The main objective of this research is to study the effect of three salinity levels(750, 1500 and 3750 ppm NaCl) , two levels of micro and macronutrients(Cooper, 1/4 Cooper solution) and of heavy metals(3.26 , 3.2 , and 2 ppm Zn, Cu and Fe, respectively ) on growth, yield, and uptake of pea plants grown in piped hydroponic. Six inches plastic (PVC) pipe have been used in closed hydroponics, that contain sites growth, injection tubes, sprayers, drainage tube, as well as 120 liter drums and pumps (1/2 Horse Power). The experiment was divided to six lines that depended on composition of nutrient solutions as mentioned above, each line included five pipes, each pipe contains a four seedlings, and all the pipes in various lines have been raised on a wooden stand about one meter. Then seeds are planted in organic soil for approximately 15 days, placed in a pot or a bin and installed by substrates and transferred to the pipelines of growth chambers that received tap water for one week and replaced with nutrient solutions that are previously prepared and pumps run three times for entirety 1.5 to 2 hours a day. XV Experience has shown survival percentage of six lines: 100%, 95%, 90%, 80%, 50% and 70%, respectively. The plants separated and divided into pods, leaves, stems and roots, then physical and chemical measurements conducted on them. It has been observed that the different salt proportion and nutrients and heavy metals affect significantly the quantities of some nutritional elements, and a negative effect on morphological characteristics of plants at high salinity of nutrient solution, that's where the plants under 3750 ppm did not produce pods. The best growth and yield and weight plants were in the Line 1, where fresh weight of pods, leaves, stems and roots were measured (6.53, 1.81, 1.58, 1.60 g), respectively . The least growth, yield and biomass plants were in the Line 5. Fresh biomass weight of pods, leaves, stems and roots (0.00, 0.23, 0.21 and 0.15g/plant), respectively. Stems and roots of plants that grew in Line 6, they had length 20.21, 21.07 cm that more than control . the performance of the plants have dropped under nutrient solution which has deficiency and decreased relative water content with increasing salinity (Ec) between 79.10% and 86.00%, where the relative water content relied directly on the salinity of the solutions, with the exception of the Line 6 was less than expected. Increasing the concentration of sodium chloride in the nutrient solutions that led generally to increase the concentration of sodium and chloride ions in plant parts, and particularly to increase chloride concentration in the roots( largest value 5.16% in Line 5 ) and sodium in the leaves and stems (largest values 4.90% and 6.11% in Line 5). Concentrations of nitrate and potassium in plants has decreased due to the impact and interact with chloride and sodium ions ,respectively. XVI Calcium percentage found in Line 2 is greater than Line 1, but in roots decreased (3.76% , 2.63% respectively), in roots of Line 6 more than Line3 (1.74% , 1.57% respectively), the greatest phosphate percentage in whole plants of Line 1 ( 0.64%). In general, high salinity led to decrease the elements in plant parts, but some elements increased in plant parts such as phosphorus in the roots, and other elements has increased in the leaves or stems, such as magnesium and calcium and phosphate in the pods peas, but nitrate was noted a slight increase in leaves of plants at 1500 ppm NaCl (Line 4) became (0.71%). The largest weight copper and zinc were found at the root of Line 6 (36.05 and 211.58 mg/kg dry plant, respectively). The effect of nutrients on plant peas have been positive, but increased nutrient value that needs the plant does not lead to growth, yield and production more than usual and decreased with increasing salinity, However there are complex interactions between the ions within the plants. The recommendations to the Palestinian community, it is desirable to use PHS because it does not need a large space and fertilizers as well as use water has electrical conductivity about 4 ms/cm, in addition it economically feasible, and recommendations fot researchers; study the effect of salinity on pea plants in hydroponics at different levels of salinity between the extent (1500 - 3750) ppm NaCl, in order to determine the maximum tolerable in peas without affecting the growth and performance of the plant and study the effect of a few types (pairs) of nutrients within different levels of salinity, due to the existence of relationships and complex interactions occur between the ions in nutrient solutions and within the plant tissue. 0 Chapter one Introduction 2 Introduction 1.1. Research background Pisum sativum, the common pea plant is an important legume grown as a garden or field crop throughout the temperate regions of the world; it is also grown as a cool season crop or hill country crop in the tropics. It ranks third in production among the grain legumes after soybean and beans, Pea is valued primarily for the nutritional quality of its seeds – pea protein is low in sulfur-containing amino acids, cysteine and methionine, but rich in lysine and other essential amino acids (Christou, 1994). World agriculture is facing a lot of challenges like producing 70% more food for an additional 2.3 billion people by 2050 while at the same time fighting with poverty and hunger, consuming scarce natural resources more efficiently and adapting to climate change (FAO, 2009 ) . However, the productivity of crops is not increasing in parallel with the food demand. The lower productivity in most of the cases is attributed to various abiotic stresses. Curtailing crop losses due to various environmental stressors is a major area of concern to cope with the increasing food requirements (Shanker and Venkateswarlu, 2011). The major abiotic stresses like drought, high salinity, cold, and heat negatively influence the survival, biomass production and yield of staple food crops up to 70% (Ahmad et al, 2012); thus, threaten food security in all parts of the world. 3 Salinity is one of the most brutal environmental factors limiting the productivity of crop plants because most of the crop plants are sensitive to salinity caused by high concentrations of salts in the soil. A considerable amount of land in the world is affected by salinity which is increasing day by day. More than 45 million hectares of irrigated land which account to 20% of total land have been damaged by salt worldwide and 1.5 M ha are taken out of production each year due to high salinity levels in the soil (Pitman and Lauchli, 2002 ; Munns and Tester, 2008 ). In most of the cases, the negative effects of salinity have been attributed to increase in Na + and Cl – ions in different plants hence these ions produce the critical conditions for plant survival by intercepting different plant mechanisms. Although both Na + and Cl – are the major ions which produce many physiological disorders in plants, Cl – is the most dangerous (Tavakkoli et al, 2010). Salinity at higher levels causes both hyperionic and hyperosmotic stress and can lead to plant demise. The outcome of these effects may cause membrane damage, nutrient imbalance, altered levels of growth regulators, enzymatic inhibition and metabolic dysfunction, including photosynthesis which ultimately leads to plant death (Mahajan and Tuteja, 2005; Hasanuzzaman et al, 2012). Palestinian population continues to increase; this requires more water consumption, and lead to a steady decline in the potable water, However, Israeli control over water sources, leading to a decrease in the quality and quantity of water used for agriculture. 4 Water quality is classified according to the total dissolved salts to several classes Table (1). Table (1) Classification of water quality based on salt concentration: Water designation (TDS) (ppm) EC(ds/m) Fresh water <500 <0.6 Slightly brackish 500 – 1000 0.6 – 1.5 Brackish 1000 – 2000 1.5 – 3.0 Moderately saline 2000 – 5000 3.0 – 8.0 Saline 5000 – 10000 8.0 – 15.0 Highly saline 10000 – 35000 15.0 – 45.0 Source: Pitman and Laüchli, 2002 ds/m: decisiemens per meter ppm: parts per million In general the response of various plants for different salinity levels vary from plant to another, but most of them involved in the low rate of growth, the number of leaves, fresh, dry weight of plants, productivity and plant height, that due to expended high energy for additional osmoregulation and ions uptake (Nawaz et al, 2010) . Study of abiotic stress is vital, and salt stress requires a modified environment where pea plants grown, and the best way to study the effect of salinity on pea plants were selected piped hydroponics, where it flows nutrient solution through the pipes and goes back to the reservoirs and pumps push solutions to the plants rotary and continuously, through hydroponics, it is possible to control the conditions of the environment that embraces pea plants to live. Where are controlled concentrations of sodium chloride salt and nutrients that are added to the nutrient solution, and control the environmental conditions of pea plants where different levels of salinity and nutrients, where translate these differences on the 5 characteristics of plants such as height, number of leaves, number of pods and root length in addition to the biomass of leaves, stems , roots and pods, not only that, but can identify the impact of salinity and nutrient concentration of elements in different parts of the pea plants .The importance of this study for Palestine, it may be exploited less land area and water quality, such as the utilization of saline water wells, and consumption of less quantity of fertilizers, control the conditions of the growth of plants to get more production of fruits and biomass and even outside of the planting season. 1.2. Objectives: The main aim of this research to identify the impact of increased salinity, nutrients and heavy metals on pea plants in piped hydroponics, and that by achieving the following objectives:  Comparison between the attributes of pea plants under different concentrations of salinity and nutrients in terms of the number of pods, leaves and the length of stems and roots.  Comparison between biomass weight of pea plants under different concentrations of salinity and nutrients.  Recognize the effects of salinity and concentrations of nutrients in pea plants.  Describe the impact ions of copper and zinc and iron on the natural properties of the pea plants and concentrations of different elements. 6 Chapter Two Literature review 7 Literature review 2.1. General overview on Pea: Peas(Pisum sativum) from the field crops of the family leguminous (Lazaro, 2006), which is characterized by the high proportion of protein and carbohydrates in seeds (Choudhury, 2007), is also characterized by its ability to fix nitrogen in atmosphere by bacteria root nodules of the genus (Rhizobium) (Rowland et al, 1994), which leads to raise the fertility of the medium cultivated and increase the proportion of nitrogen out. Moreover, dry pea seed is a rich source of protein (19–27%), and is relatively free of anti-nutritional substances (Petterson and Mackintosh, 1994). The pea plants are grown for use fresh (green pods) or dry seeds, as food for humans and sometimes as animal food. Pea is one of the most studied plants from genetics point of view and a source of immense variation (Marx, 1977; Choudhury, 2007). The wild pea's flowers have five sepals, five petals (two fused keel petals, two wing petals and a standard petal), ten another (nine fused into a filament tube and one partially free) and a single central carpel (Yaxley, 2001; Tucker, 1989; Ferrandiz et al., 1999). Further, pea is a self-pollinated, annual herb, with weak stem, alternate leaves, leaflets ovate or elliptic and terminal branched tendrils (Duke, 1981; Ghafoor, 2005). 2.2. Planting Date : Pea crop is one of the winter crops that fit with moderate cold weather, and grow from October to mid-November (Tar'an, 2005), and the temperature 8 range suitable for growth ( between 10-23 ◦ C), with an optimum daily temperature of 17 ◦ C, a minimum of 10 ◦ C and a maximum of 23°C, where they can get a bumper crop and high quality, the normal growing period is 65 to 100 days for fresh pea, with an additional 20 days for dry peas, even though the growing period is extended under cool conditions. Under irrigation, pea yields 2 to 3 ton/ha shelled fresh pea (70% to 80% moisture), 0.6 and 0.8 ton/ha dry pea (12% moisture) (Tzitzikas, 2005). The major component of pea seed is a starch, which accounts for up to 50% of the seed dry matter (DM) (Borowska et al. 1996; Wang et al. 1998). Protein and total dietary fiber account for about 24% and 20% DM, respectively, whereas lipids are present in lower amounts (2.5% DM) (Black et al. 1998). However high variation in starch and protein contents were observed frequently, whereas the variations in the other components are usually lower (Borowska et al, 1996). 2.3. Abiotic stresses: Abiotic stresses remain the greatest constraint to crop production worldwide. It has been projected that more than 50% of yield reduction is the direct result of abiotic stresses (Rodriguez et al, 2005; Acquaah, 2007. The major abiotic stresses like drought, high salinity, cold, and heat negatively influence the survival, biomass production and yield of staple food crops up to 70% (Mantri et al, 2012; Ahmad et al, 2012); hence, threaten the food security worldwide. 9 2.4. Salinity definition: The term salinity refers to the amount of dissolved salts that are present in water. Sodium and chloride are the predominant ions in seawater, and the concentrations of magnesium, calcium, and sulfate ions are also substantial. Naturally occurring waters vary in salinity from the almost pure water, devoid of salts, in snowmelt to the saturated solutions in salt lakes. The quantity and kind of salts present in the groundwater is probably the most important single parameter for evaluating the suitability of water for irrigation. Salinity of irrigation water is usually determined by measuring its electrical conductivity and is the most important parameter in determining the suitability of water for irrigation. The electrical conductivity is expressed as mmho/cm or decisiemens per meter (dS/m). Salinity is expressed also into (TDS) which is the concentration of soluble salts in the water sample in mg/l (FAO, 1985). There are many studies and researches on the effect of salinity on cultivated plants hydraulically, as different plants vary in their response to salinity. The effect of salinity on plant reality is very complicated, depending on the type of plant and the duration of exposure to the salinity and type of nutrients (elements and ions) and the stage of growth( Gunes et al, 2000). The influence of the complex environmental stress resulting from salinity doesn't affect the property osmotic plant only, but it includes toxic effects and unrest in nutrients within the plants. Also disturbing the absorption of 01 ions by the plant and making the proportion of the nutritional elements unstable (Yorgancilar and Gül Yeğin, 2012; Munsuz et al, 2001). The plants grown in cultural solution aren't absorbing the ions as per their present proportions in the solution; some ions are absorbed more than the others. These ions selection depends on the plant species (Black, C. A. 1970). The accumulation of toxic ions take a long time, and the effects may be the emergence of a slower pace. Damage grade depends on the time of exposure to toxic substances, and the concentration of toxic ions, the sensitivity of the plant, and finally in the Evapotranspiration from plant(Yurtseven, 2004). Crop performance may be adversely affected by salinity-induced nutritional disorders. These disorders may result from the effect of salinity on nutrient availability, competitive uptake, transport or partitioning within the plant. For example, salinity reduces phosphate uptake and accumulation in crops grown in soils primarily by reducing phosphate availability but in solution cultures ion imbalances may primarily result from competitive interactions (Grattan and Grieve, 1999). 2.5. Electrical conductivity of nutrient solution: The total ionic concentration of a nutrient solution determines the growth, development and production of plants (Steiner, 1961). The total amount of ions of dissolved salts in the nutrient solution exerts a force called osmotic pressure (OP), which is a colligative property of the nutrient solutions and 00 it clearly depends on the amount of dissolved solutes (Landowne, 2006). Also, the terms solute potential or osmotic potential are widely used in nutrient solution, which represent the effect of dissolved solutes on water potential; solutes reduce the free energy of water by diluting the water (Taiz and Zeiger, 1998). Thus, the terms osmotic pressure and osmotic potential can be used interchangeably, still important considering the units that are used, commonly atm, bar and MPa (Sandoval et al, 2007). An indirect way to estimate the osmotic pressure of the nutrient solution is the electrical conductivity (EC), an index of salt concentration that defines the total amount of salts in a solution. Hence, EC of the nutrient solution is a good indicator of the amount of available ions to the plants in the root zone (Nemali and van Iersel, 2004). Estimation of the osmotic pressure of a nutrient solution from EC can be done by using the following empirical relations (Sandoval et al, 2007): OP (atm) = 0.36 X EC (in dS m -1 at 25 o C) OP ( ar) 0.36 X EC (in dS m -1 at 25 o C) OP (MPa) = OP (bars) X 0.1 The ions associated with EC are Ca 2+ , Mg 2+ , K + , Na + , H + , NO3 - , SO4 2 , Cl - , HCO3 - , OH - (United States Department of Agriculture [USDA], 2001). The supply of micronutrients, namely Fe, Cu, Zn, Mn, B, Mo, and Ni, are very small in ratio to the others elements (macronutrients), so it has no significant effect on EC (Sonneveld and Voogt, 2009). The ideal EC is specific for each crop and depends on environmental conditions (Sonneveld and Voogt, 2009); however, the EC values for hydroponic 02 systems range from 1.5 to 2.5 ds m -1 or 4 dS m -1 (Ayers and Westcot, 1994). Higher EC hinders nutrient uptake by increasing osmotic pressure, whereas lower EC may severely affect plant health and yield (Samarakoon et al, 2006). The decrease in water uptake is strongly and linearly correlated to EC. 2.6. Effect of excess salinity on water regime: The main cause of reducing plant growth in the presence of salt can be impairment of water regime. Increasing the salt concentration in the nutrient solution that increases the osmotic pressure of the nutrient solution and plants cannot uptake the water as easily as in the case of relatively non saline solution. Therefore, as the concentration of salt i.e. solution EC increases, water becomes less accessible to plants, even if water is available(AL-Jobori and AL-Hadithy, 2014); (Ayers & Westcot, 1994). Osmotic pressure depends on the number of particles contained in the solution and the temperature, physiological stress symptoms appear it due to lack of water flowing to the plants. In saline solutions, despite the fact that water can exist physically, it becomes inaccessible to plants and the phenomenon is known as physiological drought (Ayers and Westcott, 1994). The first effects of solution salinity, especially when the plants classified as sensitive and moderate sensitive, can be attributed to the increase of osmotic value of the nutrient solution (Munns and Termaat, 1986). With the increasing salinity of nutrient solution, uptake of water through the root 03 system becomes more difficult which leads to decreased evapotranspiration and yield. There are several reasons why evapotranspiration decreases with increase in the salinity of nutrient solution. Due to decreased accessibility of water to the root system, root growth is reduced which leads to a reduction in the total absorption area for water uptake. At the same time, total leaf area e.g. transpiration surface is reduced. As one of the mechanisms by which plants protect their cells from harmful effect of high concentration of salts is dilution, then increasing of water retention in the tissues of the plant further reduces transpiration. These factors reduce the efficiency of water usage and ultimately result in reduction of vegetable growth and yield. The period of growth and vegetation is shortened, water regime of plants is disrupted and the uptake and distribution of essential elements in both semi-controlled and field conditions is altered (Maksimović et al, 2008, Maksimović et al, 2010). At very low water potential, the uptake of water and maintenance of turgor pressure in the tissues becomes very difficult. Water potential of leaves of plants well provided with water ranges from - 0.2 to about -0.6 MPa, but the leaves of plants in arid regions can have significantly lower values, from -0.2 to 5 MPa even in extreme conditions (Taiz and Zeiger, 2006). Since the uptake of water is spontaneous process, the water potential of root cells must be more negative than potential of nutrient solution. If, due to increased salt concentration, the difference between water potential of nutrient solution and of root cells differs very slightly, plants may adapt osmotically by accumulation of so-called 04 compatible osmolites in their cells. In that way, water potential of plant cells is kept more negative in relation to the nutrient solution( water) potential, thus permitting continuous uptake of water (Guerrier, 2006; Ghoulam et al, 2002). Increasing the salinity of a medium in which is the root leads to a reduction in the osmotic potential of leaves (Sohan et al, 1999, Romero-Aranda et al, 2001). Reduced osmotic potential of leaves is reflected in many processes in plants. Several authors have reported that water and osmotic potential of plants become more negative with increase in nutrient solution salinity, while turgor pressure concomitantly increases (Meloni et al, 2001, Romero- Aranda et al, 2001). Ashraf, (2001) found that leaf water potential and evapotranspiration significantly decreased with increasing salt concentration in six species of the genus Brassica. At 200 mM NaCl B. campestris and B. carinata held a significantly higher water potential of leaves than other species in their experiment and therefore can be considered more tolerant to stress caused by salts. According to (Sohan et al, 1999), the decrease in water potential can be explained by: 1) The influence of high concentrations of salts due to which plants accumulate more NaCl in the leaves than usual, and 2) By the reduced flow of water from root to aboveground organs due to the reduction of water conductivity, causing water stress in the tissues of leaves. After (Katerji et al 1997), a decrease in RWC indicates loss of turgor which occurs due to disturbances in the increase in the area of individual leaves, in other words in leaf expansion. The connection between the impact of salt on gas 05 exchange in leaves and growth isn't completely understood. Many experimental results indicate that gas exchange in leaves of plants remains unchanged under the influence of soil water potential, until it reaches a certain threshold value (Ritchie, 1981). Results of (Shalhevet, 1994) suggest that the expansion of leaves is the most affected by osmotic stress and that there was a linear relationship between transpiration and the synthesis of organic matter in different agro ecological conditions. The slope of this function represents the efficiency of water utilization by plants (water use efficiency, WUE). More recently, stomatal traits have been proven to critically affect WUE. In absence of stress, it has been demonstrated that low stomatal density reduces transpiration water fluxes (Zhang et al, 2001) and improves water use efficiency (Masle et al, 2005). 2.7. Nutrient concentration versus crop performance In the absence of salinity, plant growth in relation to the concentration of an essential nutrient element in the root media is often described by the "generalized dose response curve'' (Berry and Wallace, 1981). There is a nutrient-concentration window where plant growth is optimal. Concentrations below this optimal range are considered sub-optimal and growth is reduced. When the concentration or activity of the essential nutrient element exceeds this optimal range, growth may be inhibited due to either toxicity or to a nutrient-induced deficiency. It is important to mention that these dose response curves can apply not only to vegetative and reproductive organs of a particular crop in a quantitative sense but can 15 06 be modified to include a qualitative aspect as well. For example, excessive NO3 - accumulation in spinach leaves may not affect yield but may pose a health risk to the consumer. Therefore this window of nutrient adequacy would be narrowed and could be re-labelled "nutrient acceptability''. (Marschner, 1995). The plant may not exhibit the same response function under saline conditions as it does under non saline conditions. In some cases the optimal range may be widened, narrowed, or it may shift in one direction or the other depending upon the plant species or cultivar, the particular nutrient, the salinity level, or environmental conditions (Grattan and Grieve, 1994). 2.8. Salinity versus plant growth Response of field crops to the presence of increased amounts of salts is primarily stunted growth (Romero-Aranda et al, 2001). The ultimate impact of excess salts is of course very dependent on the other environmental factors such as humidity, temperature, light and air pollution (Shannon et al, 1994). The accumulation of salts in the leaves cause premature aging, reduces the supply of plant parts with nutrients and products of carbon assimilation of the fastest-growing plant parts and thus impair the growth of the entire plant. In more sensitive genotypes salts accumulate more rapidly and because cells aren't able to isolate the salt ions in vacuoles to the same extent as more tolerant genotypes, the leaves of more sensitive genotypes usually die faster (Munns, 2002). Neumann, 07 (1997) suggests that growth inhibition due to excessive salt concentration in the leaves reduces the volume of new leaf tissue in which excess salts can accumulate and therefore, in combination with the continuous accumulation of salts, it can lead to an increase in salt concentration in the tissue. It is often difficult to determine the relative influence of osmotic effect and the effect of the toxicity of specific ions on vegetable yield. In any case, yield losses due to osmotic stress can be very significant even before symptoms of toxicity on leaves become noticeable. Under the influence of salt stress growth of many species of vegetables is reduced, such as tomato (Romero-Aranda et al, 2001, Maggio et al, 2004), pepper, celery (De Pascale et al, 2003a, ) and peas (Maksimović et al, 2008, Maksimović et al, 2010). There are significant differences in salt tolerance between plant species and genotypes and similar goes for the ability to tolerate water deficiency (Munns, 2002; Luković et al, 2009). Salinity causes anatomical changes in leaves of many plant species. For example, the epidermis and mesophyll leaves of beans, cotton and Attriplex become thick, length of palisade mesophyll cells and diameter of spongy mesophyll cells increase and thickness of palisade and spongy layers and increasing as well (Longstreth and Nobel, 1979). In some other plant species, there were recorded adverse effects. In spinach leaves the presence of salt reduces the intercellular spaces (Delfine et al, 1998) and stomatal density in tomato (Romero- 08 Aranda et al, 2001), ut it increases stomatal density in pea (Maksimović et al, 2010). 2.9. Salinity versus plants mineral nutrition: Increased salt concentration in the vicinity of the root system can interfere with mineral nutrition of plants and limit field crop yield due to salinity or osmotic value of the nutrient solution. Salinity affects nutrient availability to plants in many ways. It modifies binding, retention and transformation of nutrients in the solution and affects the uptake and/or absorption of nutrients by the root system due to antagonism of ions and reduced root growth. It disrupts the metabolism of nutrients in the plant, primarily through water stress, thus reducing the efficiency of utilization of nutrients. In the presence of increasing concentrations of salts some species-specific symptoms may be present, such as necrosis and burns of leaf edges because of the accumulation of Na + and Cl - ions (Wahome, 2001). The high concentration of ions can disrupt the structure and function of cell membranes. Mineral nutrition of plants depends on the activity of membrane transporters which participate in the transfer of ions from the nutrient solution into the plant and regulate their distribution within and between cells (Marschner, 1995; Epstein and Bloom, 2005). Changes in membranes may finally lead to disturbances in chemical composition of cells and can therefore be displayed as symptoms of deficiency of some essential elements, similarly as it happens in the absence of salts (Grattan and Grieve, 1999). High concentrations of NaCl act antagonistically to the 09 uptake of the other nutrients, such as K + , Ca 2+ , N, P (Cramer et al, 1991, Grattan and Grieve, 1999). Increased concentrations of NaCl increase concentrations of Na + and Cl - and reduce concentrations of Ca 2+ , K + and Mg 2+ in many plant species (Bayuelo-Jimenez et al, 2003). In the presence of NaCl, the concentration of K + , Ca 2+ and P in vegetative parts decreased and in pods and grains increased. The deleterious effects of salinity on tomato biomass production can be ameliorated by an enhanced supply of calcium. Similarly to the effect on the uptake of macroelements, salt stress can exert stimulatory and inhibitory influence on the uptake of some trace elements (Grattan and Grieve, 1999). Under the conditions of salt stress, the uptake of nitrogen is often disrupted and numerous studies have shown that excess salts can reduce the accumulation of nitrogen in plants (Pardossi et al, 1999, Silveira et al, 2001, Wahid et al, 2004). Increase in uptake and accumulation of Cl - is accompanied by a reduction in the concentration of NO3 - in eggplant (Savvas and Lenz, 2000) and NO3 - reduction in pea plants (Shahid et al, 2012). There are authors who have attributed this reduction to the antagonism between Cl - and NO3 - (Bar et al, 1997) and those who explain it by reduced water uptake (Lea-Cox & Syvertsen, 1993). The rate of nitrate uptake or interactions between NO3 - and Cl - is associated with tolerance of examined plant species to salts; In addition, rate of nitrification of ammonia is often significantly reduced due to the large direct toxic effects of Cl - and the total amount of salt on the activity of nitrifying bacteria (Stark and Firestone, 1995). Level of salinity doesn't 19 21 affect necessarily the overall uptake of nitrogen by plants which may continue to accumulate nitrogen in the presence of excess salts despite a reduction in yield of dry matter. With the increase in nutrient solution salinity, total removal of nitrogen through the yield often decreases. Reduction in nitrogen fertilizer use efficiency is primarily a result of reduction of plant growth rate rather than the reduction of nitrogen uptake rate. Excess water and poor aeration that lead to anaerobic conditions can reduce the accessibility and absorption of nitrogen through the root system. In anaerobic conditions, the intensity of reduction of NO3 - to NO2 - is higher. Graham and Parker ( 1964) found that the highest EC that can tolerate Rhizobium strain compatible with pea is of 4.5 dS m -1 . On the basis of tolerance to salt concentration, (Elsiddig and Elsheikh, 1998) proposed the division of strains of bacteria from the genera Rhizobium and Bradyrhizobium in four groups: sensitive strains, 0-200 mM; moderately sensitive, 200-500 mM; tolerant, 500-800 mM; and highly tolerant, more than 800 mM. The classification should be considered with precaution, as a great influence on the overall tolerance to salinity has pH value of the nutrient solution, temperature and carbon source that bacteria use. Water stress, that is the result of high osmotic pressure of the nutrient solution, leads to the disturbance of nitrogen metabolism in plant tissues. In the presence of excess salts the synthesis of proteins is disturbed as well. (Nightingale and Farnham, 1936) found that with increase in osmotic pressure the amount of soluble organic nitrogen and proteins in sweet peas decreased, while the nitrate form of nitrogen accumulated. Naeem (2008) 20 found that Nodulation was completely inhi ited at Hoagland’s nutrient concentration of half and one fourth strength whereas better nodulation was observed at one sixth and one-eighth nutrient concentrations after 14 days of inoculation. Frechilla et al (2001) found salinity affects the uptake of several nutrients in different ways, depending on the nitrogen source. Thus, chloride accumulated mainly in nitrate- fed plants, displacing nitrate, whereas sodium accumulated mainly in ammonium-fed plants, especially in roots, displacing other cations suck as ammonium and potassium. It is concluded that the nitrogen source (ammonium or nitrate) is a major factor affecting pea responses to saline stress, plants being more sensitive when ammonium is the source used. The different sensitivity is discussed in term of a competition for energy between nitrogen assimilation and sodium exclusion processes, (Yorgancilar and Gül Yeğin, 2012) found that the effects of different salt proportions in the nutrient solution (0, 25, 50, 100 mM NaCl) on pea plant (Pisum sativum L. cv. Jofs) macro and micro elements involved in its growth. Statistically the different salt proportion affects significantly, the quantities of some nutritional elements. The essential elements proportions in roots (P, Mg, S, B, Cu, Mn and Zn) and in stem (K, Ca, Mg, Na, B, Fe, Mn, and Zn) are different. Common essential elements in roots and stem are Mg, B, Mn and Zn. The response of nine pea (Pisum sativum) genotypes, under salt stress, root/shoot sodium (Na + ) was increased with increasing salinity levels, which enhanced the Na + : K + ratio and seemed to affect the bioenergetics processes of photosynthesis. Whereas, root and shoot of tested genotypes 22 exhibited a considerable reduction in phosphorus (P) and potassium (K) contents, the tested genotypes were categorized into salt tolerant and salt sensitive categories. Tolerant genotypes were successful in maintaining the maximum dry matter, low Na + , while high P and K + under saline conditions( Shahid et al, 2012). Nenova ( 2008) found both nutrient supply and nutrient balance are important factors for plant growth and development. Nutrient interactions consisting of mutual influence on absorption, distribution and functioning exist and proven by numerous data in literature, most interactions are complex i.e. a nutrient interacts simultaneously with more than one nutrient. Besides the drop of Fe concentration in shoots and roots, Fe deficiency caused a decrease in shoot N, an increase in Mn, Cu, Zn, P and Na in shoots and roots; and an increase only in shoot K and Mg. Excess Fe decreased the shoot concentration of Mn, Zn and Na, and the root concentration of Mn, Cu and Mg. Besides the great increase in Na, salinity was associated with an increase in root P, Cu and Zn, with a decrease in K, Ca, Mg, Fe, Mn in both parts, and a decrease in shoot Cu and P. The interaction between nutrients can occur at the root surface or within the plant and might be due to: i) formation of precipitates and complexes between ions with different chemical properties, and ii) competition between ions with similar properties (Robson and Pitman, 1983; Fageria, 2001). Interactions between Fe and P fall in the first category, whereas interactions between (Fe and Zn, Mn and Cu) and between (Na and K, Ca 23 and Mg) fall in the second category. More detailed information about the mechanisms of nutrient interactions might be found in (Foy et al, 1978; Grattan and Grieve, 1999; Fageria, 2001; Rabhi et al, 2007). (Abdul Jabbar and Saud, 2012) found the rhizobia inoculation with phosphorus application for soybean crops caused increasing in both yield and its components (increasing nitrogen fixation). 2.10. Heavy metals uptake and impact: Some of heavy metals (Fe, Cu and Zn) are essential for plants, plants need of these metals (140, 4.15, (8-100) mg/kg dry wt. respectively) (Nagajyoti P. et.al, 2010), the essential heavy metals (Cu, Zn, Fe, Mn and Mo) play biochemical and physiological functions in plants, two major functions of essential heavy metals are participation in redox reaction, and direct participation, being an integral part of several enzymes. Plants are often sensitive both to the deficiency and to the excess availability of some heavy metal ions as essential micronutrients, at higher concentrations, the plants are exposed to poisoning. Researches have been conducted throughout the world to determine the effects of toxic heavy metals on plants (Fernandes and Henriques, 1991; Reeves and Baker, 2000). Zinc (Zn) is an essential micronutrient that affects several metabolic processes of plants (Cakmak and Marshner, 1993). The phytotoxicity of Zn and Cd is indicated by decrease in growth and development, metabolism and an induction of oxidative damage in various plant species such as 24 Phaseolus vulgaris (Cakmak and Marshner, 1993) and Brassica juncea (Prasad et al, 1999). Cd and Zn have reported to cause alternation in catalytic efficiency of enzymes in Phaseolus vulgaris (Van Assche et al, 1988; Somasekharaiah et al, 1992) and pea plants (Romero-Puertas et al, 2004). Zinc toxicity in plants limited the growth of both root and shoot (Choi et al, 1996; Ebbs and Kochian, 1997, Fontes and Cox, 1998). Zinc toxicity also causes chlorosis in the younger leaves, which can extend to older leaves after prolonged exposure to high nutrient medium Zn levels (Ebbs and Kochian, 1997). The chlorosis may arise partly from an induced iron (Fe) deficiency as hydrated Zn +2 and Fe +2 ions have similar radii (Marschner, 1986). Excess Zn can also give rise to manganese (Mn) and copper (Cu) deficiencies in plant shoots. Such deficiencies have been ascribed to a hindered transfer of these micronutrients from root to shoot. This hindrance is based on the fact that the Fe and Mn concentrations in plants grown in Zn-rich media are greater in the root than in the shoot (Ebbs and Kochian, 1997). Another typical effect of Zn toxicity is the appearance of a purplish-red color in leaves, which is ascribed to phosphorus (P) deficiency (Lee et al, 1996), transport chain (Demirevska- kepova et al. 2004). Excess of Cu in nutrient medium plays a cytotoxic role, induces stress and causes injury to plants. This leads to plant growth retardation and leaf chlorosis (Lewis et al, 2001). Exposure of pea plants to excess Cu generates oxidative stress and ROS (Malecka, 2014; Stadtman and Oliver, 1991). Oxidative stress causes disturbance of metabolic pathways and damage to macromolecules (Hegedus et al, 2001). 25 Copper (Cu) is considered as a micronutrient for plants(Thomas et al. 1998) and plays important role in CO2 assimilation and ATP synthesis. Cu is also an essential component of various proteins like plastocyanin of photosynthetic system and cytochrome oxidase of respiratory electron Iron (Fe) as an essential element for all plants has many important biological roles in the processes as diverse as photosynthesis(Nenova, 2006), chloroplast development and chlorophyll biosynthesis. Iron is a major constituent of the cell redox systems such as heme proteins including cytochromes, catalase, peroxidase and leghemoglobin and iron sulfur proteins including ferredoxin, acontiase and superoxide disumutase (SOD) (Marschner, 1995). The appearance of iron toxicity in plants is related to high Fe +2 uptakes by roots and its transportation to leaves and via transpiration stream. The Fe +2 excess causes free radical production that impairs cellular structure irreversibly and damages membranes, DNA and proteins (Arora et al, 2002; de Dorlodot et al, 2005). The presence of three metals iron, zinc and copper, happens interactions among them (Nenova, 2008). (Luo and Rimmer, 1995) found that Cu-Zn interactions in a soil were synergistic compared with an antagonistic effect in solution culture. Iron readily forms insoluble Fe-oxides and Fe-phosphates in solution, with these being unavailable to plants (Halvorsan and Lindsay, 1972; Cline et al, 1982). To keep Fe available and prevent deficiency, Fe is often added to nutrient solutions in chelated form. Many researchers have shown that chelates reduce the plant uptake of metals in nutrient solutions (Bachman and Miller, 1995). However, Cu and Zn uptake at low to medium solution 26 concentrations has been shown to be increased by the presence of EDTA (Checkai et al, 1987). An increase in Zn supply resulted in a decrease in the concentrations of Ca, Mg, P in the roots and an increase of Ca and N levels in the stems and leaves. The amount of Zn in roots, stems and leaves increased with greater Zn rates (Stoyanova and Doncheva, 2002). Although only extremely small amounts of Mo and Cu are required for normal plant growth, reduced supply with Mo and Cu to the growth medium decreased activities of the enzymes (nitrate reductase and glutamine synthetase)( Hristozkova et al, 2006). 2.11. Accumulation of compatible osmolytes and osmotic stress: One of the ways plants can adapt to conditions of osmotic stress is the accumulation of salt ions, if these salts are isolated in individual cell compartments by which their involvement in metabolism is prevented. The ability to regulate the concentration of salts through compartimentation is an important aspect of tolerance to increased salt concentrations (Romero- Aranda et al, 2001). In the presence of salts plants often accumulate low molecular weight substances which are called compatible osmolites. These substances don't interfere with normal biochemical reactions in cells (Hasegawa et al, 2000, Ashraf and Foolad, 2007). Compatible osmolites are low molecular weight molecules such as proline and glycine betaine (Ghoulam et al, 2002; Ashraf and Foolad, 2007). It is believed that under conditions of stress, proline has a role in osmotic adjustment of cells, enzymes and membrane protection and also as a source of nitrogen for a moment when conditions of stress are over (Ashraf and Foolad, 2007). The role of glycine betaine is also in maintaining pH of the cells, cell 27 detoxification and binding of free radicals. Conditions of salt stress also lead to the accumulation of the other nitrogen compounds such as amino acids, amides, proteins and polyamines, which is often correlated with tolerance to salt (Mansour, 2000). Another group of compatible osmolytes are carbohydrates, both simple sugars (glucose, fructose, sucrose, fruktani), and starch. Their most important roles, beside in osmotic adjustment, is carbon storage and neutralization of free radicals (Parida et al, 2002). A similar role is attributed to the polyols that may accumulate under conditions of salt stress as well (Bohnert et al, 1995). Ionic status of plants is highly correlated with tolerance to salts so that it can serve as a selection criterion in breeding to help create genotypes more tolerant to excess salt (Ashraf and Khanum, 1997). The beneficial effect of salt acclimation was also evident in the prevention of K + leakage and Na + accumulation, primary in roots, suggesting that here the physiological processes play the major role. 2.5% (polyethylene glycol) PEG 6000 wasn't as efficient as salt in enhancing salt tolerance and acclimation appears to be more related to ion-specific rather than osmotic component of stress. We also recorded an increase of the xylem K/Na in the salt acclimated plants. Therefore, the present study reveals that short- term exposure of the glycophyte P. sativum species activates a set of physiological adjustments enabling the plants to withstand severe saline conditions, and while acclimation takes place primary in the root tissues, control of xylem ion loading and efficient Na + sequestration in mesophyll cells are also important components of this process (Pandolfi et al, 2012). 29 28 2.12. Palestinian statistics about peas planting: Field crops are an important crop in Palestine, which are considered as food for humans and animals, in 2010 were planted about 245,414 acres (PCBS, 2013). Peas were planted in 2007 on an area of approximately 4807 acres (PCBS, 2009), and the production of peas was 2218 tons (PCBS, 2013), the percentage of agricultural and arable land in a steady decline in the period (2000-2011) (FAOSTAT, 2014), (Table 2) shows area harvested, yield and production of peas in Palestine in the period (2010-2013). Table (2) Area harvested, yield and production of peas in Palestine in the period (2010-2013): Item Year Area harvested (Ha) Yield (Hg/Ha) Production (Tones) P ea s, d ry 2010 31(OD) 27741.94(FC) 86(IM) 2011 40(F) 26250.00(FC) 105(F) 2012 45(F) 24444.44(FC) 110(F) 2013 46(F) 26086.96(FC) 120(F) P ea s, g re en 2010 305(OD) 74622.95(FC) 2276(IM) 2011 250(F) 80000.00(FC) 2000(F) 2012 240(F) 104166.67(FC) 2500(F) 2013 ---- ---- ---- Source :( FAOSTAT, 2013) F: FAO estimate IM: FAO data based on imputation methodology FC: Calculated data OD: Official data It is noted that yield and production of green and dry peas in Palestine increase with time, and shows the importance of food for people and animals, (Table 3) shows the value of fertilizer consumed in Palestine in the period (2004-2008), as it shows an increase in fertilizer use over the years, and the resulting contamination of groundwater. 29 Table (3) The use of agricultural fertilizers in Palestine in the period (2004-2008): Years Value in thousands of US dollars 2004 34,446 2005 35,246 2006 36,595 2007 39,590 2008 47,290 Source: (PCBS, 2014) Salinity of the water varies according to scattered areas in the Palestinian governorates, which is the least in Qalqilya and Tulkarm, and most in Jericho and the Gaza Strip, and the average salinity in all the provinces about 750 ppm (PWA,2013). 2.13. Summary: The effect of salinity on plant reality is very complicated, depending on the duration of exposure to the salinity and type of nutrients (elements and ions) and the stage of growth( Gunes et al, 2000). The influence of the complex environmental stress resulting from salinity doesn't affect the property osmotic plant only, but includes toxic effects and unrest in nutrients within the plants (Yorgancilar and Gül Yeğin, 2012); (Munsuz et al, 2001). Also disturbing the absorption of ions by the plant and making the proportion of the nutritional elements unstable. The plants grown in cultural soil aren't absorbing the ions as per their present proportions in the solution; some ions are absorbed more than the others(Black, 1970). The accumulation of toxic ions take a long time, and the effects may be the emergence of a slower pace. Damage grade depends on the time of 31 exposure to toxic substances, and the concentration of toxic ions, the sensitivity of the plant, and finally in the evapotranspiration from plant (Yurtseven, 2004). Crop performance may be adversely affected by salinity induced nutritional disorders. These disorders may result from the effect of salinity on nutrient availability, competitive uptake, transport or partitioning within the plant (Grattan and Grieve, 1999). Higher EC hinders nutrient uptake by increasing osmotic pressure, whereas lower EC may severely affect plant health and yield, Duzdemir et al (2009) stated pea plants is very sensitive to salinity. Since salinity causes high yield losses on pea, and Shahid et al (2012) said that there is variation in the ability of pea genotypes to tolerate various levels of salinity as Climax and Samarina zard, (Shahid et al, 2012) explained pea genotypes acclimated to salinity that is highly associated with concentration of osmolytes and antioxidant enzymes and (Nenova, 2008) stated that the impact of salt stress on the plant peas be greater if combined with the stress of iron deficiency and (Yorgancilar and Gül Yeðin, 2012) stated that the effects of salinity levels in the irrigation water on pea plant’s macro and micro elements involved in its growth. It affects significantly on the quantities of some nutritional elements. The essential elements proportions in roots (P, Mg, S, B, Cu, Mn and Zn) and in stem (K, Ca, Mg, Na, B, Fe, Mn, and Zn) are different. Common essential elements in roots and stem are Mg, B, Mn and Zn. The main cause of reducing plant growth in the presence of salt can be impairment of water regime, the period of growth and vegetation is shortened, water regime of plants is disrupted and the uptake and distribution of essential elements in both semi-controlled and field conditions is altered. The salinity leads to dramatic changes in root 30 anatomy as stated (Kukavica et al, 2013). Plants are often sensitive both to the deficiency and to the excess availability of some heavy metal ions as essential micronutrient. The demand for plant peas continues to increase in Palestine, despite the decline in the planting peas land, and to increase production; farmers used an increasing amount of fertilizer. This study is important because studies and researches related to hydroponics in Palestine are still rare, and this study differs from previous studies in many things: First, the concentration of NaCl was between 750 ppm to 3750 ppm, whereas previous studies have relied on different concentrations , secondly, the conditions of the experiment were part of the circumstances, weather factors and natural disparate momentarily terms of temperature, humidity and wind speed, while the previous studies, the conditions and weather factors have been controlled and fixed during the planting season, thirdly, the nutrient solution, which used Cooper solution full concentration in one of the section of the experiment and quarter Cooper solution in another section, while other types of solutions have been used in previous studies such as solution Hoagland and Arnon solution and Steiner solution, and Fourthly, solution of 3.26 ppm zinc and 3.2 ppm Cu and 2 ppm iron have been used in one part of the experiment, the type and concentration of these elements have been used unique compared with other studies. 32 Chapter three Materials and Methods 33 Materials and Methods 3.1. Research plan: As already mentioned that the aim of the research is to study the effect of salinity on plant peas in hydroponics in terms of the nature of the plant (morphology) and nutrients uptake and concentration of chloride and sodium ions in different plant parts. Therefore, measurement of biomass of fresh and dry plant, plant height, number of leaves, root length and the yield are the key things in this research as well as analysis of ions and salts in the nutrient solution and the various parts of the plant is also important to recognize the impact of the salinity of the nutrient solution to these ions in the plant. The experiment was divided to six lines that were depended on composition of nutrient solutions, the schema below describes the different lines in experiment, and they had various salinity and concentration of nutrients and traces heavy metals. The experiment was under normal weather conditions, where the temperature was 13-25 ° C, relative humidity 53-73% and the rate for the number of hours of solar radiation brightness 8-9 hour \ Day (PCBS, 2015) 34 The third line was considered the control line, which contains a concentration of salinity 750 ppm NaCl, this resembles the salinity rate of the water in the various regions and governorates of the West Bank and Gaza Strip in Palestine (Jebreen, 2014). Tools and materials required to conduct the experiment are as follows: 15 Plastic tubes 6" length 3 meters , Six pumps (1/2HP) Italy , plastic agro- tube 1", plastic agro-tube 1/2" , 120 sprayers nozzle ,30 plastic drain fittings 1", six plastic Water valves 1" inlet, 1" outlet ,60 plastic tube caps 6" and six tanks (120 Liter), As for the materials described in the section 3.3.2 page 38, and details of materials and procedures are described in below sections. 35 3.2. Experiment setup: 3.2.1. Introduction: System which was used in the study, was used closed hydroponic system and constructed by using 6 inches plastic pipes and length 1.5 meters, where pipes was placed on the wooden stand at a height of almost 1 meter, and the system has provided a nutrient by pumps that pumped nutrients solution from drums to growth chambers (pipes), So that, nutrient solutions injected in the form of spray on the roots of seedlings of pea plants for distributing of nutrients to all plants equally. 3.2.2. Growth chambers construction: During the process of seed germination (see section 3.3.1.) was built growth chambers, Piped Hydroponics System that will be used for the cultivation of the plant peas can be installed as follows, see (Figure 1 and 2) bellow. Figure (1) Represent a true picture of one pipe in the system. 36 Figure (2) Represent general diagram of one pipe in (PHS). The pipes had diameter of 6 inches and length 1.5m, then growth sites cut for seedlings (diameter 27/8" inches), drainage holes 1", and holes injectors4". (Figure 2) early explains this. Internal spray Lines (injector tubes) were fitted in the growth chambers by using half inch inside diameter flexible polyethylene. The spray Lines were attached inside of the chambers with the plastic zip ties by small holes. Nozzle sprayers were placed near the growth bin (growth site) exact around the roots of pea plants. Every line of hydroponics consists of five pipes, so that each pipe accommodates four seedlings (Figure 2), and injector tubes were connected with pumps, the pumps were received the nutrient solution from tanks (Figure 3), and the nutrient solution were pumped to sprayers inside the chambers through injector tubes to increase the solubility of gases (particularly oxygen) in the nutrient solution to prevent root rot and distribution of nutrients to the seedlings evenly. 37 Each pipe was full of nutrients solution, so it was filled with more than half of the tube slightly, Nutrient solutions in excess of this level back to the tanks (reservoirs) through the drainage holes. Figure (3) Represent the diagram of one line in Piped Hydroponic system. 3.2.3. Reservoir construction: Tanks (reservoirs) which were contained nutrients solutions for each Line in the experiment, the size of about 120 liters, and the tanks were received solution returning from the pipe through the drain holes, which is connected to a pump that was moved solutions strongly to the injection pipes and sprinklers in the chamber. The pump(1/2 HP) is connected to the tank(1 inch inlet) as described in the diagram as above (Figure 3) and a discharge from growth chamber(1 inch outlet). 38 Pipes in the experiment were raised from the ground, about one meter to allow for solutions to return to the tanks, depending on gravity, so the system was needed to support the pipeline, which was built by wood panels as in (Figure 4). After building the support system, the pipes had been placed on the system, that's where all five pipelines' within the single-Line, and the injection tubes were inserted in the growth chambers, and sprinklers were installed in the pipeline injection versus growth holes that seedlings had been put there. Figure (4) Represent complete Piped hydroponic system. 3.3. Experiment program: 3.3.1. Introduction: Experiment starts actually, after the process of planting seeds in organic soil, through that, the nutrient solutions are prepared. When the seedlings 39 become limited length, and then they are transferred to the growth chambers containing tap water, and after a week of planting is replaced tap water with nutrient solutions, and through growth process follow-up seedlings and an interest to making sure the arrival of the nutrient solution to plants. 3.3.2. Seeds germination: Pea seeds were chosen from agricultural shops in the Palestinian market, farmers use these seeds in agriculture land. These seeds were reserved and sterilized in special containers from exporting company, then seeds were planted in organic potting soil (ECO TERRA) for approximately 15 days (starting from the mid of January until the end of month of the year 2014), and the seedlings at that time became about 5-7 cm, then Seedlings were placed in a pots or a bins, they were installed by small stones(substrates) , they were transferred to the pipelines of growth chambers that contained tap water for a period of one week and so, they were adapted to a new situation, during that period, the pumps were operated to move the water through the pipes that prevented root rot and growth of algae and the roots of plants got oxygen required for normal growth. 41 3.3.3. Preparation of nutrient solutions: 3.3.3.1. Introduction: The plants in different lines were put in growth chambers, they are wanted nutrient solutions that are divided into six solutions (S1, S2, S3, S4, S5 and S6), and the solutions have been prepared as (Table 4). 3.3.3.2. Preparation of nutrient solutions for all lines: Solutions nutrients necessary for plant growth has been prepared based on (Table 4) Table (4) Nutrient solution components of six lines of the experiment: Solution No. Composition Solution1 Cooper solution prepared as (Table 2 and 3) and 115.2g Sodium Chloride dissolved in 153.6 liters Solution2 1/4 Cooper solution(1/4 amounts of Table 2 and 3) and 115.2g Sodium Chloride dissolved in 153.6 liters Solution3 115.2 g Sodium Chloride dissolved in 153.6 liters Solution4 230.4 g Sodium Chloride dissolved in 153.6 liters Solution5 576 g Sodium Chloride dissolved in 153.6 liters Solution6 2.2 g Zinc Sulfate, 1.93 g Copper Sulfate, 2.1g Iron- EDTA and 115.2 g Sodium Chloride dissolved in 153.6 liters Cooper solution was prepared according to the concentrations shown in (Table 5 and 6) Table (5) Concentration of nutrients in Cooper solution: Nutrients N P K Ca Mg Fe Mn Cu Zn B Mo S Concentration (ppm) 200 60 300 170 50 12 2 0.1 0.1 0.3 0.2 69 Source: Trejo-Téllez et al. 2007 40 Table (6) Weight salts are required for preparing 153.6 liters of Cooper solution: Salt Chemical formula Molecular weight(g/mol) Required weight(g) Calcium nitrate Ca(NO3)2.4H2O 236 154.06 Potassium nitrate KNO3 101 89.55 Monopotassium phosphate KH2PO4 136 40.40 Magnesium sulphate MgSO4.7H2O 246.5 78.80 Iron -EDTA Fe-EDTA 367 12.13 Manganese sulphate MnSO4.H2O 169 0.94 Boric acid H3BO3 62 0.26 Copper sulphate CuSO4.5H2O 249.7 0.06 Ammonium molybdate (NH4)6Mo7O24.4H2O 1236 0.06 Zinc sulphate ZnSO4.7H2O 287.6 0.07 Previous salts had been divided into two solutions: Solution I: calcium nitrate was dissolved well in 10 liters tap water and then salt iron- EDTA added to the calcium nitrate solution which was dissolved also well. Solution II: Another salt were dissolved one by one in 10 liters tap water, with good stirring until dissolved completely. After Preparation of the two solutions, they were mixed with tap water until the final solution became a total volume 153.6 liters, two solutions were separated so as not to precipitate salts. 42 3.4. System Operation: 3.4.1. Introduction: The pumps are running, which paid the nutrient solutions to the growth chambers, which include plants, and the movement of the nutrient solutions constantly. This is important to prevent root rot, and when stems become long and begins to bend. Stems are connected with Tightropes and install, even stems grow vertically to the top. 3.4.2. Transfer solutions to growth chambers: After the seedlings were planted in pipes which contained tap water, the pipes have been discharged from the tap water, and prepared nutrient solutions (S1, S2, S3, S4, S5 and S6) see (Table 4) were transferred to growth chambers (Line 1, Line 2, Line 3, Line 4, Line 5 and Line 6) respectively, and pumps were run three times for entirety 1.5 to 2 hours a day. 3.4.3. Monitoring and caring of plants Pea plants have been installed with yarns and ropes, so that seedlings grow in a vertical and observe plants on a daily basis and continuously. 3.5. Experiment management: 3.5.1. Introduction: To identify the effect of different levels of salinity, nutrients and heavy metals on plants peas through measurements of the physical properties and morphological characteristics and chemical analyzes of nutrients in the nutrient solution and parts of various plants. 43 3.5.2. Physical measurements of plants: Length of seedlings were measured centimeter unit once a week, and metal ruler was used in the measurement, and leaves were counted, which were divided into three kinds of: large, middle and small, as well as the pods and flowers of peas were counted once a week, and that work had started from the beginning of February until the end of March of the year 2014. At the end of the season (end of March), all seedlings were harvested, and they were cut into leaves, stems, pods and roots, where those parts had been dealt with individually, the length of the stems had been measured, and the leaves had been counted which were calculated the total area, the pods were counted, length of roots were measured centimeter unit and metal ruler was used in the measurement. Fresh stems, fresh leaves, fresh roots and fresh pods were weighed by a sensitive balance, then weighted parts plants were placed in a furnace under 80 o C for two days in order to dry well. After plants parts had dried, weighed and the relative water content (RWC) was calculated for the different parts of plants. RWC = ((fresh weight – dry weight)/fresh weight) X 100 Leaves each plant area was measured by counting squares on graph paper, painted with horizontal and vertical lines, so that the distance between each two adjacent was 0.5 cm, leaves are arranged on graph paper, and then calculate the area per plant which is equal to the length of leaves position multiplied by the width. 44 3.5.3. Chemical measurement: Chemical analysis were conducted on nutrient solutions and parts of various plants in the six sections of the experiment. 3.5.3.1. Analysis of plants: Preparation methods of samples of plants and solutions had been adopted by (ICARDA) called (Dry Ashing), plant samples were Weighed 0.5 – 1.0 g dry matter of plants (pods , leaves , stems or roots) and plant material were put in a 30 – 50 mL porcelain crucibles . porcelain crucibles were placed into a cool muffle furnace, and temperature were increased gradually to 550 °C for 7 hours after attained 550 °C, the muffle furnace were Shut off and opened the door cautiously for rapid cooling, when had cool, took out the porcelain crucibles carefully, cold ash were dissolved in 10 mL portions 2 N HCl and mix with a plastic rod. After 15 – 20 minutes, brought to the volume 250-mL used distilled water, mixed thoroughly, allowed standing for about 30 minutes, and used the supernatant. the aliquots were Analyzed for P by Colorimetry(ascorbic acid, ammonium molybdate, sulfuric acid 5N, potassium antimonyl tartrate) (Figure 5) , for Na by Flame Photometry, for Cl by titration with silver nitrate, and for other by ICP-MS, for S by Colorimetry ( by Hydrochloric acid and Barium chloride) . 45 Figure (5) Samples are ready to measure the concentration of phosphate in plant nutrients or solutions: 3.5.3.2. Analysis of nutrient solutions: Samples from nutrient solutions were taken four times (Table 7), In order to compare nutrient concentrations before and after planting seedlings. Table (7) The date of obtaining samples of nutrient solutions. First Second Third Fourth 26/1/2014 16/2/2014 6/3/214 26/3/2014 Nutrient solutions had been mitigated, even devices could be measured within acceptable concentration, and nutrients have been measured in a manner similar to measure nutrients in plants. 3.6. Chemicals and reagents: Hydrochloric acid 2N is used to prepare solutions from ash of plant. 46 Nitrate reagent: HI93728-0, for measuring the concentration of nitrate in nutrient solution and plants. Phosphate reagent :( ascorbic acid, ammonium moly date, sulfuric acid 5N, potassium antimonyl tartrate) had used to measuring the concentration of phosphate in nutrient solution and plants. Sulfate reagent :( 25% BaCl2, 1N HCl) had used to measuring the concentration of sulfates in nutrient solution and plants. Silver nitrate (0.0141M) and potassium dichromate (indicator): for measuring the concentration of chloride in nutrient solution and plants. 3.7. Chemical analysis: The ICP-MS analysis is equipped with an Agilent 7500s ICPMS for the determination of trace elements at the ppb and sub-ppb concentration levels. Nitrate meter: HANNA Instrument, HI 93728-0. Spetronic 21D spectrophotometer has used to determining of phosphate and sulfate (Colorimetry). 3.8. Statistical analysis: Treatments in the experiment were arranged in a Completely Randomized Design (CRD), with five pipes, four replicate in each pipe. Collected data were subjected to the analysis of variance (one way ANOVA) using SPSS program version 21. Means separated will be used to Tukey HSD at 0.05 level of probability. Microsoft excel vs 2010 is used to make some tables and diagrams in results, and Pearson correlation is used to find the relation between the concentrations of nutrients and dates sampling . 47 Chapter four Results and Discussion 48 4.1. Results and Discussion 4.1.1. Introduction: The results are classified into two main parts, first, results competent in the impact of salinity and nutrient concentration on chemical properties of solutions and nutrients in different of pea plant parts include the value of electrical conductivity and concentrations of nutrients, and second, results competent in the morphological and physical characteristics include the number of pods and leaves and stems length and roots and biomass weight and water content of different pea plant parts. 4.1.2. Nutrients solutions: Electrical conductivity and the concentration of nutrients must be measured in an experiment to determine what effect of the nutrient solution on plants. 4.1.2.1. Electrical conductivity (EC): The electrical conductivity of the various nutrient solutions had decreased with time, indicating that the plants had absorbed the proportion of ions nutrient solution with growth stages. A Pearson product-moment correlation was run to determine the relationship between date sampling and the salinity at different lines. The data showed no violation of normality, linearity or homoscedasticity. There are a strong, negative correlation between date sampling and the salinity at Lines 3 and 6, which are statistically significant, (Table 8). 49 (Table 8) shows the electrical conductivity(ms/cm) at different times in nutrient solutions of various lines, although it was noted that the electrical conductivity has increased slightly at a specific time, and the reason of increasing in the electrical conductivity might result from plants to get rid of some of the ions e.g. Potassium and nitrate affected by the increase of salinity in plants (Pandolfi et al. 2012). Table (8) The electrical conductivity (ms/cm) at different times in nutrient solutions of various lines: Date Line 1 Line 2 Line 3 Line 4 Line 5 Line 6 25th Jan. 2014 4.43 3.19 2.83 4.34 8.56 2.84 16th Feb. 2014 4.60 3.34 2.70 4.14 8.27 2.70 06th Mar. 2014 4.35 3.18 2.60 4.22 8.29 2.45 26th Mar. 2014 3.81 2.83 2.31 3.56 6.93 2.34 Average EC 4.30 3.14 2.60 4.07 8.10 2.58 Pearson correlation -0.798 -0.741 -0.969 * -0.842 -0.857 -0.989 * Sig.(2-tailed) 0.202 0.259 0.031 0.158 0.143 0.011 *Correlation is significant at the 0.05 level (2-tailed). ms/cm:millisiemens/centimeter 4.1.2.2. Nutrients concentrations of solutions: A Pearson product-moment correlation was run to determine the relationship between date sampling and nutrients concentrations at Line 1. The data showed no violation of normality, linearity or homoscedasticity. There are strong, negative correlations between date sampling and nutrient concentrations as phosphate, sulfate, chloride, magnesium, iron and calcium, which are statistically significant, (Table 9). The concentration of nutrients in all lines solutions decreased with over time, due to be absorbed by pea plants in growth chambers , but potassium 51 and nitrate often had increased with over time , due to interact between the sodium and chloride with other nutrients. Potassium ions have been replaced by sodium, while nitrates have been replaced by chloride ions (Gomez et al. 1996; Silveira et al. 1999), and nitrate concentrations had increased due to nitrogen fixation and the pea plants are considered of leguminous plants(Fatima et al. 2008), see (Tables 9-14). Table (9) Nutrient concentrations (ppm) in solution of line 1 at different times in 2014: *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). A Pearson product-moment correlation was run to determine the relationship between date sampling and Nutrient concentrations at Line 2. The data showed no violation of normality, linearity or homoscedasticity. There was a strong, negative correlation between date sampling and Nutrients 25th Jan. 16th Feb. 6th Mar. 26th Mar. Pearson correlation Sig. 2-tailed Phosphate 29.00 16.00 8.96 5.46 -0.964* 0.036 Sulfate 60.00 46.75 23.75 13.50 -0.990* .010 Nitrate 354.28 487.14 752.86 647.22 +0.842 0.158 Chloride 749.83 699.84 649.85 624.86 -0.990* 0.010 Sodium 290.20 263.00 255.50 254.50 -0.886 0.114 Magnesium 67.61 65.11 63.46 60.72 -0.996** 0.004 Iron 10.66 6.39 2.91 2.62 -0.949 0.051 Manganese 2.11 0.43 0.04 0.04 -0.836 0.164 Copper 0.17 0.05 0.01 0.01 -0.889 0.111 Zinc 0.21 0.05 0.06 0.05 -0.771 0.229 Potassium 336.54 1413.36 1413.36 1413.36 -0.775 0.225 Calcium 93.50 81.17 76.92 69.23 -0.980* 0.020 Molybdenum 0.20 0.01 0.01 0.01 -0775 0.225 50 nutrient concentrations as phosphate, sulfate, chloride and magnesium, which are statistically significant, see (Table 10). Table (10) Nutrients concentration (ppm) in Line 2 solution at different times in 2014: Nutrients 25th Jan. 16th Feb. 6th Mar. 26th Mar. Pearson correlation Sig. 2- tailed Phosphate 16.18 10.733 5.022 3.078 -0.981* 0.019 Sulfate 37.50 28.125 13.75 12.125 -0.964* 0.036 Nitrate 296.71 243.57 177.14 388.89 +0.303 0.697 Chloride 732.68 674.84 649.85 624.86 -0.974* 0.026 Sodium 291.96 264.25 256.25 251.50 -0.923 0.077 Magnesium 38.84 38.177 38.0743 37.24 -0.964* 0.036 Iron 1.85 0.6727 0.41 0.304 -0.890 0.110 Manganese 1.17 0.1232 0.004 0.003 -0.826 0.174 Copper 0.10 0.058 0.093 0.064 -0.407 0.593 Zinc 0.12 0.0594 0.0639 0.0564 -0,797 0.203 Potassium 109.02 94.3667 103.5207 98.045 -0.479 0.521 Calcium 54.09 46.6226 48.5385 46.48 -0.758 0.242 Molybdenum 0.0349 0.0044 0.0046 0.0043 -0.776 0.224 *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). A Pearson product-moment correlation was run to determine the relationship between date sampling and Nutrient concentrations at Line 3. The data showed no violation of normality, linearity or homoscedasticity. There are strong, negative correlations between date sampling and nutrient concentrations as chloride, sodium and copper, which are statistically significant, see (Table 11). 52 Table (11) Nutrients concentration (ppm) in Line 3 solution at different dates in 2014. Nutrients 25th Jan. 16th Feb. 6th Mar.2014 26th Mar Pearson correlation Sig. 2-tailed Phosphate 0 0 0 0 ----- ----- Sulfate 13.900 13.750 13.250 12.250 -0.944 0.056 Nitrate 31.200 44.268 43.400 175.000 +0.818 0.182 Chloride 669.492 654.8591 649.853 624.859 -0.965* 0.035 Sodium 292.001 264.000 255.250 242.500 -0.967* 0.033 Magnesium 31.343 28.6728 31.046 26.0309 -0.709 0.291 Iron 0.205 0.1646 0.1525 0.1136 -0.982* 0.018 Manganese 0.0757 0.009 0.0017 0.0009 -0.829 0.171 Copper 0.0265 0.0218 0.0215 0.0155 -0.954* 0.045 Zinc 0.0268 0.0177 0.0153 0.0134 -0.927 0.073 Potassium 33.1753 34.1747 38.8519 33.6002 +0.292 0.708 Calcium 40.9501 19.9921 22.4434 18.9259 -0.793 0.207 Molybdenum 0.0088 0.0066 0.0051 0.0054 -0.899 0.101 *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). A Pearson product-moment correlation was run to determine the relationship between date sampling and Nutrient concentrations at Line 4. The data showed no violation of normality, linearity or homoscedasticity. There was a strong, negative correlation between date sampling and Nutrient concentrations as sulfate, chloride, iron and copper, which are statistically significant, see (Table 12). 53 Table (12) Nutrients concentration (ppm) in Line 4 solution at different dates in 2014. Nutrients 25th Jan 16th Feb 6th Mar 26th Mar. Pearson correlation Sig. 2-tailed Phosphate 0 0 0 0 ---- ---- Sulfate 13.95 13.25 12.5 12 -0.997** 0.003 Nitrate 32.111 75.2871 55.8 155.56 +0.846 0.154 Chloride 1298.65 1284.74 1279.71 1249.72 -0.951* 0.049 Sodium 584.8733 363.75 350.75 345 -0.815 0.185 Magnesium 31.769 30.8971 31.948 27.23 -0.737 0.263 Iron 0.199 0.1404 0.133 0.0772 -0.966* 0.034 Manganese 0.0675 0.0033 0.0035 0.0012 -0.791 0.209 Copper 0.0265 0.0235 0.0206 0.0119 -0.958* 0.042 Zinc 0.0288 0.0157 0.0273 0.0173 -0.439 0.561 Potassium 32.7695 37.0972 39.3635 34.15 +0.280 0.720 Calcium 38.8224 19.6159 20.3788 17.2856 -0.828 0.172 Molybdenum 0.0081 0.0055 0.0091 0.0071 +0.050 0.950 *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). A Pearson product-moment correlation was run to determine the relationship between date sampling and Nutrient concentrations at Line 5. The data showed no violation of normality, linearity or homoscedasticity. There was a strong, negative correlation between date sampling and Nutrient concentrations as molybdenum, which is statistically significant, see (Table 13). 54 Table (13) Nutrients concentration (ppm) in Line 5 solution at different times in 2014. Nutrients 25th Jan 16th Feb 6th Mar 26th Mar. Pearson correlation Sig. 2-tailed Phosphate 0 0 0 0 ---- ---- Sulfate 13.5 12.5 12 12 -0.913 0.087 Nitrate 32.4 66.8714 60.6714 166.67 +0.873 0.127 Chloride 4097.29 3824.1 2399.46 2349.47 -0.933 0.067 Sodium 1479.359 801.5 778.5 761.5 -0.803 0.197 Magnesium 32.00 31.72 32.18 31.78 -0.092 0.908 Iron 0.21 0.196 0.21 0.20 -0.105 0.895 Manganese 0.0712 0.0104 0.007 0.0023 -0.835 0.165 Copper 0.03 0.0213 0.0569 0.0344 +0.415 0.585 Zinc 0.0293 0.0118 0.0126 0.011 -0.796 0.204 Potassium 33.4588 40.3779 44.5178 40.1451 +0.683 0.317 Calcium 39.2342 23.871 25.165 2.593 -0.928 0.072 Molybdenum 0.0086 0.0072 0.0062 0.0027 -0.959* 0.041 *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). A Pearson product-moment correlation was run to determine the relationship between date sampling and Nutrient concentrations at Line 6. The data showed no violation of normality, linearity or homoscedasticity. There was a strong, negative correlation between date sampling and Nutrient concentrations as chloride and sodium, which are statistically significant, see (Table 14). 55 Table (14) Nutrients concentration (ppm) in Line 6 solution at different times in 2014. Nutrients 25th Jan 16th Feb 6th Mar 26th Mar Pearson correlation Sig. 2-tailed Phosphate 0 0 0 0 ---- ---- Sulfate 15.45 13.5 12 12 -0.935 0.065 Nitrate 31.00 79.71 60.99 166.67 +0.860 0.140 Chloride 669.49 654.86 649.853 624.859 -0.965* 0.035 Sodium 290.92 254.75 228.75 161.25 -0.979* 0.021 Magnesium 31.99 31.079 31.61 31.60 -0.218 0.782 Iron 1.91 0.234 0.148 0.192 -0.787 0.213 Manganese 0.0716 0.0043 0.0049 0.0095 -0.732 0.268 Copper 2.78 1.26 1.09 1.042 -0.837 0.163 Zinc 3.01 1.67 1.2723 1.054 -0.922 0.078 Potassium 33.12 37.94 37.78 36.85 +0.632 0.368 Calcium 38.88 21.24 21.71 22.86 -0.723 0.277 Molybdenum 0.0086 0.0084 0.004 0.0057 -0.762 0.238 *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). Finally the relationship between the date of sampling and the concentration of nutrients in nutrient solutions mostly, isn't linear because of the complex interactions among themselves and the availability of nutrients for plants and the effect of salinity on nutrient uptake by plants. 4.1.3. Characteristics of plants: Morphological characteristics of the pods, leaves, stems and roots should be known to determine the impact of the above factors on plants. 56 4.1.3.1. Survival percentage: The percentage of survival in pea plants was depended on the concentration of nutrients and salinity levels, and (Table 15) shows the relationship between the salinity levels and survival percentage of pea plants. Table (15) Relationship between survival percentage of pea plant and salinity levels (ms/cm): Line number Average Salinity(ms/cm) Survival percentage % Line 1 4.3 100 Line 2 3.14 95 Line 3 2.61 90 Line 4 4.07 80 Line 5 8.10 50 Line 6 2.58 70 A Pearson product-moment correlation was run to determine the relationship between salinity levels and survival percentage. The data showed no violation of normality, linearity or homoscedasticity. There is a strong, negative correlation between salinity levels and survival percentage, which is statistically significant (r = - 0.999, p < 0.05), while there isn't a strong, positive correlation between nutrients levels and survival percentage, which is statistically significant (r = 0.984, p > 0.05). 4.1.3.2. Pods characteristics: The number and fresh and dry biomass weight of the pods of pea plants in different lines had differed depending on the quality and quantity of nutrients and salinity in the nutrient solutions, according to analysis of variance (ANOVA) the (Table 16) shows the average number and fresh 57 and dry biomass weight of the pods in different lines, the average of measurements were significantly differed at (α ≤ 0.05). Table (16) The average number, fresh and dry biomass weight of pods in different treatments. Treatment Average number of pods Fresh pods weight (gram/plant) Dry pods weight (gram/plant) Line 1 2.55a 6.53a 1.175a Line 2 1.58b 3.70b 0.687b Line 3 1.17b 2.16c 0.3983c Line 4 1.20b 1.10cd 0.202d Line 5 ---------- ---------- ---------- Line 6 0.35c 0.022de 0.014de  Averages in columns with the same small letters are not significantly different at P≤0.05 level, according to Tukey HSD. It is found that number, fresh and dry weight increased significantly in the solution which owned more of the nutrients , as Line 1 more than Line 2, and the number of pods and weight decreased significantly when the increase of salinity in the nutrient solution (Line 5 less than Line 4 and Line 4 less than Line 3), but number of pea pods has increased in Line 4 plants(contained 1500 ppm NaCl) compared in Line 3(contained 750 ppm NaCl), it may be due to a period of premature aging and to maintain the life cycle of a physiologically, and this result supports the findings of (Duzdemir, 2009; Farooq et el, 2009) and in plants of Line 6 (contained heavy metals) was owned pod, fresh and dry biomass pods weight less than Line 3 (Control Line) , While plants Line 5 (containing 3750 ppm NaCl) hadn't produce pods, it can be seen clearly in (Table 16). 58 (Table 16) shows the average of number and fresh and dry biomass weight of pods in plants of Line 1 more than Line 2, because the plants of Line 1 had absorbed the adequacy of nutrients, whereas the plants of Line 2 didn't get adequacy of essential nutrients that are required for metabolic processes and performance in pea plants, But increasing the salinity of the nutrient solution lead to stress the plants and then lead to low and poor metabolic processes and the performance of plants, the number and fresh and dry biomass of pods pea plant were increased significantly in Line 3 more than Line 4, Line 4 more than Line 5 , that consistent with (Nenova V., 2008 and Yorgancilar M. et.al., 2012). Line 6 was contained 2 ppm Fe, 3.2 ppm Cu and 3.26 ppm Zn, Copper is an essential heavy metal for higher plants particularly for photosynthesis, Cu is a constituent of primary electron donor in photosystem of plants. Because Cu can readily gain and lose an electron, it is a cofactor of oxidase, mono- and di-oxygenase (e.g., amine oxidases, ammonia monoxidase, ceruloplasmin, and lysyl oxidase) and of enzymes involved in the elimination of superoxide radicals (e.g. Superoxide dismutase and ascorbate oxidase). Several enzymes contain Zn, such as carbonic anhydrase, alcohol dehydrogenase, superoxide dismutase and RNA polymerase, Zinc is required to maintain the integrity of ribosome, and it takes part in the formation of carbohydrates and catalyzes the oxidation processes in plants. Zinc also provides a structural role in many transcription factors and is a 59 cofactor of RNA polymerase; Iron Fe is an essential element in many metabolic processes. ( Nagajyoti P. et.al, 2010). The number, fresh and dry biomass weight of pods of pea plants in Line 6 less than Line 3, because of the presence of nutrients, iron, copper and zinc in the nutrient solution which have a significant role in biological processes in pea plants, but the concentrations of copper and zinc in a solution of Line 6 have been high, so the plants have been exposed to a case of toxicity which reduces the production of pods. Average of number, fresh and dry biomass of pod in Line 1 significantly differ than other lines but they don’t significantly among Lines 2, 3 and 4, and the average of Line 6 significantly differ than Line 3 (Control Line), according to tukey HSD test. ( Figures 6, 7 and 8) show the averages of number and fresh and dry biomass weight of pods at different pipes in each line, each line had five pipes and four replications in each pipe, the average of number and fresh and dry biomass of pods of pea plant among pipes in each line were not differ depending on Tukey HSD ,(P> 0.05), With the exception of the fifth pipe of each line, where it is often value more than the other pipes in all measurements on the pods, and the reason for that, it is expected, the fifth pipe in each line was exposed to the sun more than other pipes. 61 Figure (6) Average of pods number of pea plants at different pipes in each line; each value is the average of four replications in each pipe, Tukey HSD, P ≥ 0.05 Figur