An-Najah National University Faculty of Graduate Studies Fate of Amoxicillin, Ibuprofen, and Caffeine in Soil and Ground Water Using Soil Columns By Halimeh Ahmad Sai , d Staiti Supervised Dr. Shehdeh Jodeh Prof. Marwan Haddad This Thesis is Submitted in Partial Fulfillment of the Requirement For The Degree of Master In Environmental Science, Faculty of Post Graduate Studies, An -Najah National University, Nablus, and Palestine. 2102 III Dedication To the most generous to all of us, the martyrs. To the wounded, prisoners and detainees. To give of himself in support of Palestine. To my parents who always encouraged me to science and progress and knowledge. To everyone who helped and supported me in my research with Love and Respect. IV Acknowledgments I would like to express my special thanks and gratitude to my supervisor Dr. Shehdi Joudi, in my first step in choosing the correct title and giving me the main lines for my research, and for his encouragement and support. I also would thank Dr. Marwan Haddad for helping and advising me with his experts’ knowledge. I would like to deeply thank Ms. Tamara Rinno for all the guidance, instruction, feedback and encouragement, her steadfast support. Actually this thesis could not be accomplished without the faithful help of technician’s team in the poison control and chemical biological center for their help and support during my study and analysis. Special thanks to my parents, for their help, encouragement and patience. Thanks and appreciation goes out to various people whose direct and indirect support has helped me to produce this thesis. V اإلقرار :أنا الموقع أدناه مقدم الرسالة التي تحمل العنوان Fate of Amoxicillin, Ibuprofen, and Caffeine in Soil and Ground Water Using Soil Columns أقر بأن ما اشتملت عليه هذه الرسالة إنما هي نتاج جهدي الخاص، باستثناء ما تمت اإلشارة إليه حيثما ورد، وأن هذه الرسالة ككل، أو أي جزء منها لم يقدم لنيل أية درجة أو لقب علمي أو بحثي .لدى أية مؤسسة تعليمية أو بحثية أخرى Declaration The work provided in this thesis, unless otherwise referenced, is the researcher's own work, and has not been submitted elsewhere for any other degree or qualification. Student's Name : : اسم الطالب Signature: : التوقيع :Date : التاريخ VI List of Contents No. Content Page Dedication Iii Acknowledgments Iv V اإلقرار List of Tables Ix List of Figures Xi Abstract Xiii CHAPTER ONE 1 1.1 General Introduction 1 1.2 Objective 3 1.3 Justification 4 CHAPTER TWO 5 2.1 Introduction 5 2.2 Amoxicillin 6 2.2.1 Definition &Uses 6 2.2.2 Mode of Action 7 2.2.3 Pharmacokinetics 7 2.2.4 Medical Uses 8 2.2.5 Veterinary Use 8 2.2.6 Adverse Effects 8 2.3 Ibuprofen 9 2.3.1 Definition 9 2.3.2 History of Ibuprofen 10 2.3.3 Mode of Action 10 2.3.4 Pharmacokinetics 11 2.3.5 Medical Use of Ibuprofen 11 2.3.6 Ibuprofen Interaction 11 2.3.7 Side effect of Ibuprofen 12 2.4 Caffeine 13 2.4.1 Definition & Use 13 2.4.2 History of Caffeine 14 2.4.3 Pharmacokinetics of Caffeine 14 2.4.4 Mechanism of Action of Caffeine 15 2.4.5 Medical Use of Caffeine 16 2.4.6 Caffeine Side Effect 16 2.5 Amoxicillin, Ibuprofen& Caffeine In Environment 17 2.6 Source Of Pharmaceutical In Environment 19 2.7 Definition of Soil 22 VII 2.8 Chemical Kinetics 24 2.8.1 Rate of Reaction 24 2.8.2 Order of Reaction 25 2.8.3 Zero-Order Reaction 25 2.8.2.2 first-Order Reaction 26 2.8.2.3 second-Order Reaction 27 2.9 Adsorption onto Soil 28 2.9.1 Adsorption Equilibrium Isotherms 30 2.10 Previous Studies 33 CHAPTER THREE 36 3 Methodology 36 3.1 Material & Methods 36 3.1.1 Chemicals & Reagents 37 3.1.2 Instrumentation 38 3.1.3 Soil Column Preparation 39 3.2 Soil Analysis 40 3.2.1 Soil Texture ( Hydrometer Test ) 40 3.2.2 Moisture 41 3.2.3 Specific Gravity 42 3.2.4 pH 43 3.2.5 Organic Carbon (Walkely ND Black 1934) 43 3.2.6 Wastewater Analysis 44 3.2.6.1 Determination of Chemical Oxygen Demand (COD) 45 3.2.6.2 Determination of Biological Oxygen Demand( BOD) 46 3.2.6.3 Determination of Nitrogen (Kjeldhal Test) 46 3.2.6.4 Determination of Phosphorus 48 3.2.6.5 Determination of Calcium & Magnesium 49 3.2.6.6 Determination of Heavy Metals 50 3.3 HPLC Scanning of Amoxicillin, Ibuprofen, &Caffeine 51 3.3.1 HPLC scanning of Amoxicillin 51 3.3.2 HPLC scanning of Ibuprofen 52 3.3.3 HPLC scanning of Caffeine 53 3.4 Calibration Curve 54 3.5 Isotherms 55 3.6 The Effect of Temperature On ( Amoxicillin , Ibuprofen Caffeine) -Soil Adsorption, 56 3.7 The Effect of pH On ( Amoxicillin, Ibuprofen, ,Caffeine) -Soil Adsorption 57 3.8 Amoxicillin ,Ibuprofen, & Caffeine Application to 58 VIII Soil-Column Experiment 3.9 Collecting and Storage of Leachate Water Samples 59 3.10 Distribution of Pharmaceutical in Soil Columns after Finishing the Leach ate Study 59 CHAPTER FOUR 60 4 Results &Discussions 60 4.1 Soil Tests 60 4.2 Waste water Test 61 4.2.1 pH, BOD ,COD 61 4.2.2 Calcium, Magnesium ,Phosphorus, Nitrogen 62 4.2.3 Heavy metals (Cadmium, Chrome, Zinc, Nickel). 63 4.2.4 HPLC Scanning of Amoxicillin, Ibuprofen, , &Caffeine Concentrations 64 4.3 The Effect of Temperature on The adsorption of (Amoxicillin, Ibuprofen, &Caffeine –Soil). 65 4.4 The Effect of pH on Drug-Soil Adsorption 73 4.5 Adsorption Isotherms 80 4.6 Polluted leach ate Water from Ibuprofen, Amoxicillin, &Caffeine Analysis 85 4.7 Mass Balance 100 Conclusion & Recommendation 104 References 109 ب الملخص IX List of Tables Table No. Subject Page )3.1( Wave length& retention time. 54 )4.1( Soil specific gravity, PH, texture, and moisture for soil before pollution. 60 )4.2( Wastewater pH, BOD &COD. 61 )4.3( wastewater Ca, Mg ,P,N. 62 )4.4( Heavy metals concentrations in the three pharmaceuticals companies’ wastewater. 63 )4.5( Retention time &peaks length of Ibuprofen, amoxicillin, &caffeine in pharmaceutical wastewater companies. 64 )4.6( Amoxicillin Standards at 230nm. 65 ) 4.7( Amoxicillin concentration in supernatant and soil- adsorption at 10, 30, 60,120 minutes. 67 )4.8( Ibuprofen Standards at 254nm 67 ) 4.9( Ibuprofen concentration in supernatant and soil- adsorption at 10, 30, 60,120 minutes. 68 )4.10( Caffeine Standards at 256 nm. 68 4.11( Caffeine concentration in supernatant and soil- adsorption at 10, 30, 60,120 minutes. 69 ) 4.12( Soil-adsorption of Amoxicillin, Ibuprofen, and Caffeine at temperature 15 cº. 70 )4.13( Soil-adsorption of Amoxicillin, Ibuprofen,caffeine,& soil adsorption concentration at 25 cº. 71 )4.14( Soil- adsorption of amoxicillin, ibuprofen, and caffeine at temperature 35 cº. 72 )4.15( Amoxicillin adsorption on soil at different pH, constant temperature 25 cº and one hour. 73 )4.16( Ibuprofen adsorption on soil at different pH and constant temperature 25 cº, and one hour. 75 )4.17( Caffeine adsorption on soil at different pH, constant temperature 25 cº and one hour. 76 )4.18( Concentration of Ibuprofen, Amoxicillin &Caffeine at different PH& constant temperature 25 cº. 78 X (4.19) The application of freundlich isotherm for the adsorption of ibuprofen on soil after 24 hours. 80 (4.20) The application of Langmuir isotherm for the adsorption of ibuprofen on soil after 24 hours. 81 (4. 21) The application of freundlich isotherm for the adsorption of amoxicillin on soil after 24 hours 82 (4.22) The application of freundlich isotherm for the adsorption of caffeine on soil after 24 hours. 83 (4.23) Freundlich isotherm constants (k & n) & the correlation coefficient R for ibuprofen, caffeine, &amoxicillin. 84 (4.24) Measured concentrations of polluted water flowed from amoxicillin versus time. 86 (4.25) Measured concentrations of polluted water flowed from Ibuprofen versus time. 88 ( 4.26) Measured concentrations of polluted water flowed from caffeine versus time. 90 (4.27) Represents concentrations of amoxicillin, in different soil depths. 93 (4.28) Represents concentrations of ibuprofen in different soil depths 95 (4.29) Represents concentrations of caffeine in different soil depths 97 (4.30) Mass balance for amoxicillin, ibuprofen and caffeine 101 XI List of Figures No. Figure Page (2.1) The chemical structure of amoxicillin 6 (2.2) The chemical structure of ibuprofen 10 (2.3) The chemical structure of caffeine 13 (2.4) Drug flow Pharmaceuticals and their metabolites enter environment from homes, health care facilities, and farms 20 (2.5) Soil structure 22 (2.6) The use of the characteristic kinetic plots 27 (3.1) Soil column apparatus 39 (4.1) Plot of amoxicillin calibration curve 66 (4.2) Plot of ibuprofen calibration curve 67 (4.3) Plot of caffeine calibration curve 69 (4.4) lot o concentrations o amoxicillin i upro en ca eine in soil at c 70 (4.5) Plot of concentrations of amoxicillin ,ibuprofen , ,&caffeine in soil at c 71 (4.6) lot o concentrations o amoxicillin i upro en ca eine in soil at c 72 (4.7) Plot of soil adsorption concentrations of amoxicillin in soil at different pH, temperature 25c º and one hour 74 (4.8) Plot of concentrations of amoxicillin in supernatant at different pH, temperature 25 c º, and one hour. 74 (4.9) Plot of soil adsorption concentrations of ibuprofen at different pH , temperature 25c º ,and one hour. 75 (4.10) Plot of concentrations of ibuprofen in supernatant at di erent p temperature c and one hour. 76 (4.11) Plot of soil adsorption concentrations of caffeine in soil at different pH, temperature 25c º, and one hour. 77 (4.12) Plot of concentrations of caffeine in supernatant at different pH, temperature 25c º, and one hour. 77 (4.13) Plot of soil adsorption concentrations of ibuprofen, amoxicillin and caffeine at different pH, constant temperature 25c º and one hour 78 (4.14) The application of freundlich isotherm for the adsorption of ibuprofen on soil after 24 hours. 81 (4.15) The application of langmuir isotherm for the 82 XII adsorption of ibuprofen on soil after 24 hours. (4.16) The application of freundlich isotherm for the adsorption of amoxicilline on soil after 24 hours 83 ( 4.17) The application of freundlich isotherm for the adsorption of caffeine on soil after 24 hours. 84 ( 4. 18) ln[A] versus time for polluted water flowed from Amoxicillin (one-year). 87 (4.19) ln[A] versus time for polluted water flowed from Amoxicillin (15-year). 87 (4.20) ln[A] versus time for polluted water flowed from Ibuprofen (one-year). 89 (4.21) ln[A] versus time for polluted water flowed from Ibuprofen (15-year). 89 (4.22) ln[A] versus time for polluted water flowed from caffeine (one-year). 91 (4.23) ln[A] versus time for polluted water flowed from caffeine (15-year). 91 (4.24) Amoxicillin concentrations measured in soil layers for one-year. 94 ( 4.25) Amoxicillin concentrations measured in soil layers for 15 year. 94 (4.26) Ibuprofen concentrations measured in soil layers for one year. 96 ( 4.27) Ibuprofen concentrations measured in soil layers for 15 year. 96 (4.28) Caffeine concentrations measured in soil layers for one year. 98 (4.29) Caffeine concentrations measured in soil layers for 15- year. 98 (4.30) Ibuprofen, Amoxicillin& caffeine concentrations measured in soil layers for one year. 99 (4.31) Ibuprofen, amoxicillin and caffeine concentrations measured in soil layers for 15years. 99 XIII Fate of Amoxicillin, Ibuprofen, and Caffeine in Soil and Ground Water Using Soil Columns By Halimeh Ahmad Sai , d Staiti Supervised Dr. Shehdeh Jodeh Prof. Marwan Haddad Abstract The recent public interest regarding the presence of pharmaceutical pollutants in water has raised important concerns due to the unknown environmental impact especially on aquatic life, soil and underground water as emerging aquatic micro pollutants that have possibly been affecting the ecological system and lead research studies in recent years. In this study three pharmaceuticals were selected, ibuprofen, amoxicillin and caffeine as examples of pharmaceuticals that are released into the environment, all are marketed in the Palestinian market (pharmacies), private clinics, hospitals either for human veterinary use. In this research we have investigated the adsorption behavior of three pharmaceuticals on soil, the effect of temperature and pH on the adsorption process, in addition their effect on characteristics of underground water, all were studied using the UV-V is spectrophotometry. The results of experimental sorption data fitted very well the Freundlich isotherm model and first order kinetics model. XIV During the study of soil columns the study revealed that the concentration of caffeine in leachate higher than those of ibuprofen and amoxicillin, ecause ca eine has higher solu ility in water despite it didn’t reach 1ppm. While ibuprofen and amoxicillin were present in leachate with very small concentrations due to their degradation and decomposition into other substances that may be harmful, and would affect the natural properties of soil, groundwater and human health. Therefore, we recommend further studies of these drugs and find out how dangerous to human health. 1 Chapter One 1.1. General Introduction In the recent years, the occurrence and the fate of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging environmental issues that have possibly been affecting the ecological system, (Dietrich,D, D.R., et al.,2005); ( Kotchen, M., 2009). Pharmaceutical compounds may be described as any chemical used for diagnosis, treatment, alteration or prevention of diseases, (Thompson, A., 2005). Taking into account the use of thousands of pharmaceuticals types in human treatment and in agricultural sector (livestock), it’s not limited the size on the negative effects that these compounds will leave on the environment, as well as high cost to eliminate or mitigate these effects. It was discovered about more than 100,000 types of chemicals that are used in our everyday life either in households, industries or agriculture, (Kummerer, K., 2004). So pharmaceutical pollution is one of the most modern and chemical contaminants that pose to the environment. The pharmaceutical compounds used mainly by human and livestock are excreted only slightly transformed or even unchanged form which result from the bodies fluids (urine), or from disposing of the expired drugs, causing serious damage to the ecosystem, which is still in the eginning o the study in alestine that’s ecause our conventional 2 wastewater treatment is the primary mechanism by which pharmaceuticals introduced, (Karnjanapiboonwong, K., et al, 2001). According to a lot of recent researches variety of these compounds were detected in various water samples including hospitals wastewater, pharmaceutical industries, waste water treatment plant effluent, surface and ground water, this illustrated by a study entitled, (occurrence of pharmaceuticals and personal care products along the West Prong little Pigeon River in east Tennessee), by (Chane P.Yu and Kung H.Chu, in USA 2009), which pointed out the presence of concentrations of some drugs in ground water and soil. In addition to the quantities of expired medicines which are disposed o in unsa e ways it’s common to pour them down in the sink, flush them down in the toilet, or throwing them in the trash,without attention to their risks through landfills leahates that may eventually reach to ground water. Despite of the need for the drugs used for treatment of many diseases , it was found that they leave an adverse effects on non-target site , such as water, soil, air, health and others, therefore, they should be used and dealt in a scientific way to reduce as much as possible of their negative effects, by preventing drugs using randomly from general public, reducing distribution of physician free samples, separation of domestic waste, sewage recycling, improvement sewage infrastructure, public awareness, nutrition and health maintenance, drugs alternatives and research development. 3 Locally amoxicillin, ibuprofen, and caffeine are used in pharmaceutical manufacturing products for human and veterinary sector, whether used through physician prescription or by the person himself, this is clear from my reviews of many pharmacies and Palestinian ministry of health. This study will investigate the fate of amoxicillin, ibuprofen, and caffeine on the soil and ground water, the adsorption behavior of the three pharmaceuticals on soil, effect of pH and temperature on the adsorption process, and equilibrium isotherm. 1.2. Objectives: The study aims to achieve a set of goals and most important of which are the following: 1. To highlight the seriousness of the excessive use of drugs covered by the study. 2. To determine the adsorption of target ibuprofen, amoxicillin, and caffeine to soil. 3. To investigate the fate of ibuprofen, caffeine and amoxicillin on soil and ground water. 4. Create an alternative needs to minimize exposure to humane or to improve treatment process to protect human and environment. 4 1.3. Justification: The presence of a lot of factories that manufacture these drugs and others, in addition to a lot of governmental and specialized hospitals in Palestine. Although pharmaceuticals and personal care products (PPCPs) may exist in minute quantities, long-term release of them may result in significant environmental concentrations and consider being one of the most dangerous contaminant for environment. PPCPs enter the Palestinian land, via the hospitals and drugs manufacture factories, and disposal of residential wastewater which is the main source (85%) of influent to the plant. Since target PPCPs: amoxicillin, ibuprofen and caffeine may be found in septic tanks and consequently ground water due to incomplete human metabolism and excretion into the waste stream or by disposal of unused medication in the toilet or down to sink, and not be completely removed during the wastewater treatment process, these compounds may be discharged into streams and land application sites, and eventually contaminate aquatic environment or persist in surface water, groundwater, and soil. 5 Chapter Tow Literature Review 2.1. Introduction Every year large quantities of pharmaceuticals products are sold and consumed in Palestine and worldwide for diagnoses, treatment, alteration or prevention of human diseases. According to Palestinian ministry of health statistics , the rate of imported raw materials for local drug manufacturing was(17775) kg amoxicillin, (27780) kg ibuprofen, while imported rate of caffeine in 2007 was (425)kg, after that period it has not been imported for manufacturing or using in Palestine, (Ministry of Health, 2010). In addition to add about 10-15% on the purchased raw quantities as manufactured medicine that donated from foreign donors. The uses of these quantities leave a lot of adverse effects on the environment causing the so- called pharmaceutical pollution. This chapter will illustrate what has been written on the subject of previous study and research .Three pharmaceuticals were selected for study, these are amoxicillin , ibuprofen, and caffeine due to their large use, based on measured and predicted environmental concentration, risk to environment, potential to bioaccumulation and known removal in treatment processes. 6 2.2. Amoxicillin 2.2.1. Definition and Uses Formulations of AMOXIL contain amoxicillin, a semi synthetic antibiotic, an analog of ampicillin, with a broad spectrum of bactericidal activity against many gram-positive and gram-negative microorganisms, (Dimitrakopoulou, D. ,et al ., 2012.,) (Ball, P., 2007). Amoxicillin chemically is D- a-amino-p-hydroxybenzyl penicillin trihydrate. Figure 2.1: The chemical structure of amoxicillin (Gozlan I., et al., 2010). It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other beta-lactam antibiotics, (Bruhn, S. T. 2003). Amoxicillin is suscepti le to degradation y β-lactamase-producing bacteria, and so may be given with clavulanic acid to increase its susceptibility. Penicillin, which was first, discovered by Sir Alexander Fleming in 1928 when he noticed it being produced by the mould Penicillium notatum. http://www.bbc.co.uk/dna/h2g2/A354656 javascript:modelesswin('imageViewer?doc='+parent.myTitle+'&img=uspnf/pub/images/v28230/g-40.gif',500,500); 7 However, amoxicillin is an artificially altered (semi synthetic) variant of penicillin, and was first made in 1972, (Geddes, A.M, et al 2007). 2.2.2. Mode of Action Amoxicillin is a bactericidal antibiotic (kill the bacteria). It prevents bacterial cell wall mucopeptide synthesis by acylating the enzyme transpeptidase, thus making it unable to cross-link muramic acid containing peptidoglycan strands. This inhibition of the biosynthesis of dipeptidoglycan, a substance necessary for cell wall strength and rigidity, results in a defective cell wall. 2.2.3. Pharmacokinetics Amoxicillin is rapidly absorbed by the gastrointestinal tract after oral administration and is stable in the presence of gastric acid. Peak serum concentrations are usually attained within 1- 2 hours following oral administration. Amoxicillin diffuses readily into most body tissue and fluids, with the exception of the cerebrospinal fluid, although higher concentrations of the drug may be attained in patients with inflamed meningitis. Its elimination half-life ranges from 0.7 to 1.4 hours in patients with normal renal function. Amoxicillin is partially metabolized to microbiologically inactive metabolites and both are then rapidly excreted in urine, small amounts of the compounds are excreted in feces and bile. (Ball, P., 2007), (Paintaud, G., et al., 1992). 8 2.2.4. Medical uses Amoxicillin is used to treat certain infections caused by bacteria, such as pneumonia, bronchitis, gonorrhea, and infections of the ears, nose, throat, urinary tract, and skin. It is also used in combination with other medications to eliminate H. pylori bacteria that cause ulcers. Amoxicillin is in a class of medications called penicillin-like antibiotics, and it works by stopping the growth of bacteria, (Ball, P., 2007). 2.2.5. Veterinary Use The amoxicillin/clavulanic acid combination has trade names include Clavaseptin, Clavamox and Synulox. that used in veterinary medicine for treatment of urinary tract, skin, enteritis, respiratory tract infections, soft tissue infections, metritis and mastitis. Amoxicillin/clavulanic acid is banned from use in domestic-food animal (cattle, swine, etc.) in both the US and Europe, (Georgios, A.D., 2010). 2.2. 6. Adverse Effects Side-effects of amoxicillin include nausea, vomiting, rashes, and antibiotic-associated colitis, diarrhea also may occur. Rarer, but patient- reported, side-effects include mental changes, lightheadedness, insomnia, confusion, and anxiety, sensitivity to lights and sounds, and unclear thinking. Use of the amoxicillin/clavulanic acid combination for more than one week has caused mild hepatitis in some patients. Young children http://en.wikipedia.org/wiki/Urinary_tract_infections http://en.wikipedia.org/wiki/Enteritis http://en.wikipedia.org/wiki/Respiratory_tract_infections http://en.wikipedia.org/wiki/Metritis http://en.wikipedia.org/wiki/Mastitis http://en.wikipedia.org/wiki/Nausea http://en.wikipedia.org/wiki/Colitis http://en.wikipedia.org/wiki/Hepatitis 9 having ingested acute overdoses of amoxicillin manifested lethargy, vomiting and renal dysfunction, (Denes, A., et al., 2009). 2.3. Ibuprofen 2.3.1. Definition Ibuprofen is one of the most prescribed and consumable global non- steroidal anti-inflammatory drugs (NSAIDs), with analgesic and antipyretic properties, which is a prop ionic acid derivative, (Jacobs, L.E., et al., 2011). Ibuprofen is one of the most consumed medications corresponds to the classification of the non-Steroidal anti-inflammatory drugs (NSAIDs), and more than 70 million annual prescriptions in the world, (Me´ndez-Arriaga, F., et al., 2010). Ibuprofen is a core medicine in the world health 0rganization, have an anti- platelet effect, though it is relatively mild and short-lived when compared with aspirin or other better-known anti platelet drugs. Originally marketed as Brufen, and is available under a variety of popular trademarks, including Motrin, Nurofen, Advil, and Nuprin. Its half life is 1.9-2.2 hours, with molecular weight is 206.281 g/mol, (Jarrr, A.A., 2003), (Zheng, J.P., 2007) and have the structural formula: http://en.wikipedia.org/wiki/Lethargy http://en.wikipedia.org/wiki/Renal_failure http://en.wikipedia.org/wiki/World_Health_Organization http://en.wikipedia.org/wiki/Antiplatelet http://en.wikipedia.org/wiki/Aspirin http://en.wikipedia.org/wiki/Trademark http://en.wikipedia.org/wiki/Nurofen http://en.wikipedia.org/wiki/Advil 10 Figure 2.2: The chemical structure of Ibuprofen, (Siskou, I.C., et al., 2007). 2.3.2. History of Ibuprofen Ibuprofen was discovered as a form of propionic acid in 1961, how its name came is unknown. It was made and discovered at the Boots Company in the 1960s in the United Kingdom. It was discovered by Andrew RM Dunlop, with colleagues Stewart Adams, John Nicholson, Vonleigh Simmons, Jeff Wilson and Colin Burrows, and was patented in 1961. Ibuprofen was made available under prescription in 1969, and in the United States in 1974, (Rang, H.P, et al, 1995). 2.3.3. Mode of Action The exact mechanisms of action of Ibuprofen is unknown, its anti inflammatory effects are believed to be due to inhibition of both cylooxygenase-1 (COX-1) and cylooxygenase-2 (COX-2) enzymes which leads to the inhibition of prostaglandin synthesis, and this is the cause for the analgesic and anti-inflammatory action of the drug. (Rang, H.P, et al, 1995). http://en.wikipedia.org/wiki/Patent http://upload.wikimedia.org/wikipedia/commons/6/69/Ibuprofen2.svg 11 2.3.4. Pharmacokinetics: Ibuprofen is well absorbed from the gastrointestinal tract. It is highly bound (90-99%) to plasma proteins and is extensively metabolized to inactive compounds in the liver, mainly by glucuronidation. Both the inactive metabolites and a small amount of unchanged ibuprofen are excreted rapidly and completely by the kidney, with 95% of the administered dose eliminated in the urine within four hours of ingestion, (Rang, H.P, et al, 1995). 2.3.5. Medical Uses of Ibuprofen: Ibuprofen is a non steroidal anti-inflammatory drug (NSAID), used in both human and veterinary medicine for treatment of a wide range of conditions. It is used to treat headaches, muscle aches, backaches, dental pain, menstrual cramps, arthritis, or athletic injuries. It is also used to reduce fever and to relieve minor aches and pain due to the common cold or flu, by the enzyme in the body that makes prostaglandins, and decreasing prostaglandins helps to reduce pain, swelling, and fever, (Jarrr, A.A., 2003). 2.3.6. Ibuprofen Interactions Ibuprofen can interact with an anticoagulants, including warfarin- ibuprofen may increase risk of severe bleeding and sometimes fatal hemorrhage, especially from the gastrointestinal tract. Ibuprofen should 12 only be used in patients taking warfarin if absolutely necessary and they must be closely monitored. It may reduce the anti-hypertensive effect of beta-blockers and diuretics and may cause hyperkalemia in patients under these treatments. Ibuprofen may prolong bleeding time in patients treated with zidovudine, and may also interact with probenecid, antidiabetic medicines and phenytoin, (Hersh, E.V., et al., 2007) 2.3.7. Side Effects of Ibuprofen: Adverse effects with non-prescription or short-term use ibuprofen are rare and may include:  gastrointestinal–dyspepsia, heartburn, nausea, loss of appetite, stomach pain, and diarrhea.  Central nervous system (CNS)–dizziness, fatigue, headache, and nervousness.  Hypersensitivity reactions-skin rashes and itching. Rarely dermatitis and epidermal necrolysis have been reported with ibuprofen.  Rare cases of photosensitivity.  Cardiovascular-fluid retention and in some cases oedema, these effects are rare at non-prescription doses.  Allergic reactions such as skin rash, itching, swelling of the face or breathing difficulties may also occur, (Al-Nasser, A., 2000). http://www.pubmedhealth.30/9/2011.(Al-Nasser,I.A) 13 2.4. Caffeine 2.4.1. Definition and Use Caffeine is white crystalline xanthine alkaloid, very common substance that acts as a central nervous system stimulant drug , which is found in leaves, seeds and/or fruits of at least 63 plant species worldwide, and is part of a group of compounds known asmethylxanthines, have molecular weight 194.19g/mole and structural Formula: C8H10N4O2 as shown: Figure (2.3): The structural formula of caffeine The most commonly known sources of caffeine are coffee, cocoa beans, tea, chocolate, soft drinks, and can also be purchased as capsules, tablets, or powder, (Jacobs,L.E., et al., 2012). Other sources of caffeine include over-the-counter pain relievers and cold medications. Caffeine is an adjuvant to increase the rate at which the medication is absorbed into the body; it can be present in these products ranging from 16-200 mg. http://en.wikipedia.org/wiki/Crystalline http://en.wikipedia.org/wiki/Xanthine http://en.wikipedia.org/wiki/Alkaloid http://www.aginternetwork.net/whalecomwww.sciencedirect.com/whalecom0/science/article/pii/B0123694000001708 14 2.4.2. History of Caffeine Ca eine is one o the world’s most widely used drugs that were first extracted from coffee in 1821. Coffee originated in Ethiopia and later introduced to Arabia and the rest of the east. Ethiopian nomads discovered coffee through their animals, the animals would eat the fruits from the trees they would have an energy boost; the nomads tried eating the seeds and had an increase in energy. In 1573 coffee was introduced to the Europeans, tea was introduced later in 1657 and became very popular to the people of Europe. Near the end of the 19th century cola products started to appear around the world and became one of the larger drank caffeine drinks, (Smith, A., (2002). Caffeine was first isolated from coffee in 1820 by the German chemist Friedlieb Ferdinand Runge, and then independently by French chemists Pierre Robiquet, Pierre Pelletier, and Joseph Caventou, (Waston, J., 2003). 2.4.3. Pharmacokinetics of Caffeine Caffeine is administered orally and intravenously, and is well absorbed from the gastrointestinal tract, following oral administration, peak plasma concentrations in adults are reached within 50-75 minutes. Therapeutic caffeine concentrations are reported to be 5-25 mg/L in adults. http://en.wikipedia.org/wiki/Coffee http://en.wikipedia.org/wiki/Friedlieb_Ferdinand_Runge http://en.wikipedia.org/wiki/Pierre_Jean_Robiquet http://en.wikipedia.org/wiki/Pierre_Joseph_Pelletier http://en.wikipedia.org/wiki/Joseph_Bienaim%C3%A9_Caventou 15 Caffeine is distributed rapidly to all body tissues and readily crosses the blood-brain and placental barriers and is distributed into breast milk, it is roughly 36% bound to plasma proteins. In adults, caffeine is partially metabolized in the liver via demethylation reactions, dependent on the Cytochrome P450 isoenzymes; major metabolites include paraxanthine (80%), theobromine (10%), and theophylline (4%). The plasma half-life of caffeine is 3-7 hours in adults. Caffeine metabolism in neonates is limited due to their immature hepatic enzyme systems, therefore unchanged caffeine and its metabolites are excreted in the urine. Plasma half-life for neonates may vary widely, from 65-100 hours, and the fraction of caffeine excreted unchanged in the urine is roughly 86% within 6 days. Young infants have a plasma half-life of caffeine of 3-4 days, (Abel, P; Joju George, A.D., 2008). 2.4.4. Mechanism of Action of Caffeine Caffeine is a mild, direct stimulant at all levels of the CNS and also stimulates the heart and cardiovascular system. The related xanthine, theophylline, shares these properties and is widely used in the treatment of pulmonary disease. Caffeine also stimulates the medullar respiratory center and relaxes bronchial smooth muscle, also it stimulates voluntary muscle and gastric acid secretion, increases renal blood flow, and it have a mild diuretic effect, (Rang H.P., et al., 1995). 16 2.4.5. Medical Use of Caffeine: Caffeine is used as a central nervous system (CNS) stimulant, anorexiant, diuretic, and in a number of analgesic and cold medication compounds. It is also used in the treatment of spinal headaches and has been used as a respiratory stimulant in preterm infants. Some people use caffeine for asthma, gallbladder disease, attention deficit-hyperactivity disorder (ADHD), shortness of breath in newborns, and low blood pressure. It is also used for weight loss and for type two diabetes, and is one of the most commonly used stimulants among athletes. Caffeine creams are applied to the skin to reduce redness and itching in dermatitis. Healthcare providers sometimes give caffeine intravenously (by IV) for headache after epidural anesthesia, breathing problems in newborns, and to increase urine flow. In foods, caffeine is used as an ingredient in soft drinks, energy drinks, and other beverages. People with voice disorders, singers, and other voice professionals are often advised against using caffeine, (Waston, 2003). 2.4.6. Caffeine Side Effect Whether caffeine is consumed in food or as a medicine, it changes the way your brain and body work and changes how you behave and feel, it is a central nervous system stimulant, (Smith A., 2002). Ca eine’s main http://www.webmd.com/asthma/default.htm http://www.webmd.com/digestive-disorders/picture-of-the-gallbladder http://www.webmd.com/add-adhd/default.htm http://www.webmd.com/heart/understanding-low-blood-pressure-basics http://www.webmd.com/diet/default.htm http://diabetes.webmd.com/guide/type-2-diabetes http://diabetes.webmd.com/guide/type-2-diabetes http://www.webmd.com/skin-problems-and-treatments/picture-of-the-skin http://www.webmd.com/skin-problems-and-treatments/guide/default.htm http://www.webmd.com/skin-problems-and-treatments/understanding-dermatitis-basics http://www.webmd.com/lung/breathing-problems-causes-tests-treatments 17 effect on body is make feel more awake and alert for a while, but it can also cause problems according to several studies, most important of them the study that conducted by the Organization of Teratology Information Specialists that caffeine can make shaky, hard to fall asleep, increase heart beat speed, cause an uneven heart rhythm, raise blood pressure cause headaches, nervousness, and/or dizziness, addiction, (make you dependent on it so you need to take more of it), (Organization of Teratology Information Specialists, 2011); (Waston J., 2003). 2.5. Amoxicillin, Ibuprofen and Caffeine in Environment Each year large quantities of these drugs are sold and consumed in the worldwide and USA for diagnosis, treatment, and prevention of human diseases, the reasons are that these amount keep increasing because of improving health care system and long life expectations of people, (Xia, K., et al.,2005).,( Skadsen J.M., et al .,2004). People all over the world consume millions of doses of medication through physician prescription and over-the-counter, as well as millions of other veterinary either orally or by injection. As mentioned earlier, and according to the statistics of Palestinian ministry of health, tons of raw materials are consumed every year, the average of their consumption in both humanitarian and veterinary sector in the year of 2010 reached 17,775 kg of amoxicillin, 27780 kg of ibuprofen, and 425 kg of caffeine just for the 2007, for local manufacturing, adding 10-15 % on the 18 purchased raw materials as a donated medicine from foreign donors, (Palestinian Ministry Of Health, 2010). In addition to the quantities of expired medicines which are disposed o in unsa e ways it’s common to pour them down in the sink or flush them down in the toilet, also manufacturing waste, waste from human or animal excretion. Once the pharmaceuticals in the environment, it may metabolite from the parent compound and distributed between major environment compartments, the concentration in these compartment depend on numerous factors, including how the parent compounds is released in the environment, how fast it degrades, the half life of metabolite, soil and sediment and subsequent movement to air and water, (University of Aveiro, 2008). Environmental monitoring has identified a number of pharmaceuticals including ibuprofen, mood stabilizers (caffeine), antibiotics, present in some environments at level high to harm aquatic organism. (Chane, P.Yu, 2009). Through a review of several references is clear that the drugs found in the environment, where it was clear in a study conducted in the United States in 1999-2000 by a geological survey, that the most water ways contain at least some antibiotics synthetic hormones, steroids, or other common drugs. The survey sampled 139 streams in 30state, found that about 80% contained antibiotics, analgesics, steroids, caffeine, hormones, and other contaminants, half of the stream contained seven or more chemical compounds, one-third of streams contained 10 or more 19 compounds and one sample contained 38 chemical,( Morse, A., Jackson, A., 2003) . 2.6. Source of Pharmaceutical in Environment In spite of all benefits of these drugs in the treatment of many diseases for human and animals, but the added to the environment in large quantities, through several sources that include:  Direct disposal at manufacturing.  Excretion with urine and feces (sewage water).  Drugs in animal manure. The amount of pharmaceutical that can be discharged by the producer to the sewage can be 1-5% of total production, this is low compared to other type of industry, this type of industry produce several kinds of chemicals from drugs, catalyst, solvent and other per unit of actual product that goes to municipal sewage treatment plants (STPs). Industrial waste water may be possible source for contamination of surface water. The following picture shows the routes of the pharmaceuticals entering to the environment: 20 Figure (2.4): Drug flow Pharmaceuticals and their metabolites enter environment from homes, health care facilities, and farms, (Bethany Halford, 2008). The pharmaceutical administrated (in its medical term or other words consumed) by human after required action in the body get excreted with urine and feces as (original compound), and usually as a number of metabolites. The important issue about the pollution that occurs with medication that result from household disposal of unused or expired medications is that some of these products can cause environmental damage if disposed into the sewer system ,because these materials are not adequately destroyed by sewer treatment plants, (Dietrich D.R., et al ., 2005), toilet wastewater is mixed with other wastewater stream forming finally a sewage that enter the municipal sewer, human pharmaceuticals have to pass through “ST s ”prior to entering rivers or streams. In sewage treatment plants (STP) effluents many pharmaceutical compounds do not get removed to a sufficient degree, this because (STP) are not efficient enough to remove these micro pollutants, consequently they enter the 21 surface water where they may pose effect onto aquatic life, (Katarzyna, K-R., et al., 2007) In animal husbandry, pharmaceuticals are used worldwide not only for therapeutic purpose but also for animals growth promotion, large proportion of antibiotics is excreted through manure, some of manure used as fertilizer, the remaining antibiotics or other pharmaceuticals and their metabolites enter soil, where they might persist or reach ground water, (Binh C.T. Th, et al., 2007). The resulting pharmaceuticals residues especially antibiotics can affect the natural soil microbial community and soil function and may even harm animals and human via the food chain,(Institute of soil science, 2003, and Environmental Toxicology and Pharmacology , 2008). Once PPCPs are released to the environment, their behavior will be determined by their physical and chemical properties such as water solubility, lipophilicity, vapor pressure, and sorption coefficients. These properties may help determine whether they are likely to partition between water, air, or soil environments. Photolysis, biodegradation, and sorption of PPCPs can contribute to their elimination and mobility in the environment, (Todd, A. A., 2010), (Xu .J, et al 2009). One of the aims of this study was to determine the presence of all concentrations of amoxicillin and ibuprofen and caffeine in the soil, from 22 here should be identified on soil properties and mechanical process of absorption and adsorption of these drugs on soil. 2.7. Definition of Soil Soil is the unconsolidated mineral or organic material on the immediate surface of the earth and serves as a natural medium for the growth of land plants. The soil is composed of particles of broken rocks that have been changed by environmental and chemical processes that include weathering and erosion. It’s a mixture o minerals and organics components that are in gaseous, solid, and aqueous state. Most soils have a density between 1-2 gram\cm 3 . Soil particles are loosely packed, forming a soil structure that filled with pore and spaces, these pores contain air (gas) and solution (liquid), as shown in following figure: Figure 2.5: Soil structure, (Daniel H, (1998) 23 Most soil consists of combination of three types:  Sandy soil : particles size 0.5-2 mm, texture is gritty, compaction is low), porous, and usually holds less water for plants than other soil types, and it have the property of dryness, its particles tend to be weakly bonded together , so sandy soil easily affected by erosion .  Silt soil: particles size 0.02-0.5 mm, texture is smooth and slippery, compaction is medium, have small particles and weaker than those formed by clay particles.  Clay: a particle size less than 0.02 mm, texture is sticky, compaction is high, and have small particles that tend to bonded together strongly, can tend to hold water. The important physical properties of soil is the pH, which controlled by the concentration of hydrogen ion in the soil. The availability of hydrogen ions in soil matrix is caused by chemical weathering reaction and by dissociation of water by action of roots. Soil with large concentration of hydrogen ion tend to be acidic, and many of nutrients become soluble and readily leached from the soil to the ground water, but soil with low concentration of hydrogen ions tend to be alkaline so nutrients become insoluble and plant cannot extract them. The chemicals movement(transport) processes through soil are diffusion, convection, and hydrodynamic dispersion, and chemical 24 properties of soil like solubility, sorption, and density influence the ability of substances to be transported within soil. More soluble chemical tend to move more easily within water than that are less soluble water, which will tend to attach to clay particles and organic matter near the soil surface, ( Naser.O.Z. M, 2011). 2.8. Chemical Kinetics 2.8.1 Rate of Reaction The rate is de ined as change in concentration (Δc) with time (Δt). The rate can be positive or negative: a positive rate means that the concentration is increasing with time e.g. a product; a negative rate means that the concentration is falling with time e.g. a reactant. A rate law is a mathematical equation that describes the progress of the reaction. In general, rate laws must be determined experimentally. There are two forms of a rate law for chemical kinetics: • Di erential rate law. • Integrated rate law. The differential rate law describes how the rate of reaction varies with the concentrations of various species, usually reactants, in the system. 25 Each rate law contains a constant” k” called the rate constant. The units or the rate constant depend upon the rate law, and the concentration always has units of mole\L. The rate is the derivative of concentration with respect to time. r = − d [A] / dt…………………………………………………...…… ( . ). Integrated rate law relates the concentration to time. r = k [A] n …………………………………………………..……….... (2.2). 2.8.2 Order of Reaction The power to which its concentration term in the rate equation is raised. For example, given a chemical reaction 2A + B → C with a rate equation. r = k [A] 2 [B] …………….……..…………………………...……….. (2.3). The reaction order with respect to A would be 2 and with respect to B would be 1; the total reaction order would be 2 + 1 = 3. 2.8.2.1 Zero-Order Reaction The rate of reaction is a constant when the limiting reactant is completely consumed. Differential Rate Law: 26 r = k……………………………………………………….……......... (2.4). The rate constant, k, has units of mole/ L / sec. 2.8.2.2 First-Order Reaction For a first-order reaction, the rate of reaction is directly proportional to the concentration of one of the reactants. Differential Rate Law: r = k [A] ………………………………...…………………….............. (2.5) – [dt] / [A] = k*A…………………………………………….……….. (2.6) [A] = [A] 0 e - k t …………………….………………….….................. (2.7) [A] / [A] 0 = 1/2 = e –kt 1/2 ….………………………….….………… (2.8) Taking the ln of both sides ln (1/2) = - ln 2 = - k t 1/2…..………….……………….………........... (2.9) Or t 1/ = (ln ) / k………………..………………………..……….. (2.10) For any order other than first order, the half-life of the reaction is dependent on the concentration. The rate constant “k” has units o sec-1. 27 2.8.2.3 Second-Order Reaction For a second-order reaction, the rate of reaction is directly proportional to the square of the concentration of one of the reactants. Differential Rate Law: r = k [A] 2 ..................................................…………………..…...… (2.11). -d [A] / dt = k*[A] 2 .……………….…………………...…………... ( . ). [A] / 1+ kt [A] 0 = [A] …………..………………………….…....... (2.13). t1/ 2 = 1/ k [A] 0 ………………..……………………………...….... (2.14). The rate constant, k, has units of L mole -1 sec -1 . Zero-order first-order second-order Figure. 2.6: The use of the characteristic kinetic plots (Attaallah, M.A., 2011). 28 2.9. Adsorption onto Soil Adsorption phenomenon in solution systems plays an important role in many areas of practical environmental technology. In water and wastewater treatment, for example, the adsorption technique has found wide applicability because of several advantages such as high efficiency, simple operation, and easy recovery/reuse of adsorbent. It is of general interest to understand pharmaceutical adsorption from an aqueous environment to a solid surface. The characteristics of the adsorption behavior are generally understood in terms of both equilibrium, and adsorption kinetics. Adsorption: is the phenomenon of attraction or adhesion of solute molecules to the surface of soil particles at an interface between two phases which can be solid –liquid. The driving force for adsorption result from specific affinity of solute to the soil where the atoms at the solid surface subjected to unbalanced force of attraction , so adsorption is essentially a surface phenomenon, (LeVan, M.D., 2008). Absorption: is the uniform distribution of the substance throughout the bulk, adsorption essentially happens at the surface of the substance. When both adsorption and absorption processes take place simultaneously, the process is called sorption. Most soils absorb water and chemicals, although in amount much less than those adsorbed. 29 Adsorption process involves two components adsorbent and a dsorbate. Adsorbent is the substance on the surface of which adsorption takes place. A dsorbate is the substance which is being adsorbed on the surface of adsorbent. The adsorption process includes electrical attraction of the solute to adsorbant, vander-waal attraction or chemical nature, (Aly, O.M and Faust, S.D., 1964). Adsorption can be classified into two types: Physical Adsorption or Chemical Adsorption:  Physical Adsorption or Physisorption: occur when the force of attraction existing between a dsorbate and adsorbent are weak Vanderwaal forces of attraction, Physical Adsorption takes place with formation of multilayer of a dsorbate on adsorbent. It takes place at low temperature, and its fully reversible reaction where adsorption occurs at the same temperature and the process may be slow because of diffusion effects. Molecules in the physical adsorption are free to undergo translation movement within interface.  Chemical Adsorption or Chemisorption : occur when the force of attraction existing between a dsorbate and adsorbent are by chemical bond, includes the transferring of electrons, as a result there is chemical bond and chemical adsorption is irreversible it takes place with formation of unilayer of a dsorbate on adsorbent. Molecules in chemical adsorption are not 30 considered to be free to move on the surface where they attached to active center. Chemical interaction between adsorbent and a dsorbate is favored by high temperature, (Ruthven, M.D., (2004). 2.9.1. Adsorption Equilibrium Isotherms Adsorption equilibrium is the physic-chemical aspect which determine the adsorption capacity, as the adsorption process continue, the solute that is adsorbed tend to desorbs so equal amount of solutes are adsorbed and desorbed at the same time, where no change can be observed in the solute concentration and the system reach equilibrium. The equilibrium position is characteristic of entire system, the solute, adsorbent, solvent, temperature, pH, and so on, (Aly, O.M., and Faust, S.D., 1964); (Ruthven, M.D., 2004). Adsorption Isotherm: is the amount of solute adsorbed per unit of adsorbent as a function of equilibrium concentration of adsorbent in the bulk under a set of experimental conditions. Adsorption isotherm can be generated based on theoretical models where Langmuir and Freundlich models are the most commonly used ones, (Helby, W.A., 1952). The Langmuir isotherm, originally derived for the adsorption of gas molecules on solid surfaces, was modified to fit the adsorption isotherm of solutes onto solid surfaces in solution systems, (Sohn, S., and Kim, D., 2005); (Pei, J., and Zhang, 2012). 31 When solute molecules adsorbed on the surface of the soil, these adsorption may appear in two forms: single layer adsorption or multi molecular layer adsorption. Several type of isotherm may occur Langmuir and Freundlich isotherms are valid for single layer, where for multilayer Brunauar Emmett and Teller (BET) isotherm are valid. The Langmuir equation is expressed as: X / m = Xm bCe / 1 + bCe (equation 2.15) Where: X : amount of solute adsorbed in moles (mg, mole). m: weight of adsorbent (mg, g). b: a constant related to the heat of adsorption, 1/ unit weight. Xm: amount of solute adsorbed per unit weight of adsorbent required for monolayer. Ce: equilibrium concentration of the solute. For linearization of equation (2.15), it can be written in the form: Ce* m / x = 1/ bxm + Ce / xm (equation 2.16) 32 Or m / x = 1/ Xm + Ce / bxm * 1/ Ce (equation 2.17) Any of these equations may be used to evaluate “b “ and “Xm” from experimental data using graphic or linear least squares analysis, (Rubin, A.J., and Mercer, D.L., 1984). Freundlich adsorption equation is perhaps the most widely used mathematical description of adsorption in aqueous systems. The Freundlich equation is an empirical equation based on adsorption on a heterogeneous surface. The equation is commonly represented as: log Cs = log K + log Ci (equation 2.18) Where: Cs: is the concentration of pharmaceutical adsorbed on red soil (mg/g). Ci : is the concentration of pharmaceutical remained in solution (mg/L). K & n: are the Freundlich constants characteristics of the system, indicating the adsorption capacity and adsorption intensity respectively, (Soil Science and Plant Nutrition, 2001); ( Aly, O.M and Faust, S.D, 1964). 33 2.10. Previous Studies: Adsorption is integral to a broad spectrum of physical, biological, and chemical processes and operations in the environmental field. Both adsorption and desorption of the different materials in the environment can be studied kinetically by using different equations like Freundlich and Langmuir isotherms and by knowing especial conditions like PH, time, temperature , concentration and other different condition . Many researchers in the world and in Palestine studied the kinetics behavior and fate of may pharmaceuticals in the environment like:  Adcharee Karnjanapiboonwong, et al, (2011): Occurrence of PPCs Wastewater Treatment Plant and in Soil and Groundwater at a Land Application Site, in USA. Adcharee Karnjanapiboonwong and his team studied the presence of eight types of drugs (estrone 7β-estradiol estriol 7α- ethynylestradiol, triclosan, caffeine, ibuprofen, and ciprofloxacin) in wastewater, sewage sludge, soil, and groundwater. The results indicated that concentrations of PPCPs in wastewater influent, effluent, sludge solid phase, and sludge liquid phase were in the range of non-detected (ND)- 8 μg/L concentrations in soil and groundwater samples were in the range of ND-319 ng/g in soil and ND- 74 μg/L in ground water respectively Overall data indicate that PPCPs in the wastewater effluent from the waste water http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/?Author=Adcharee+Karnjanapiboonwong http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/y2975343k4l25123/ http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/y2975343k4l25123/ http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/y2975343k4l25123/ http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/?Author=Adcharee+Karnjanapiboonwong 34 treatment plant WWTP transport both vertically and horizontally in the soil, and eventually reach groundwater following land application of the effluent, (Karnjanapiboonwong, A., et al. 2011).  Awartani Lama, (2010): Fate of Oxytetracycline and Doxycycline in Soil and Underground Water in Palestine. Awartani studied the adsorption behavior of two antibacterials, oxtetracycline and doxycyclin that are released into soil, also studied the effect of organic matter and magnesium chloride hepta hydrate addition on the adsorption of the two antibacterials. The results showed that increasing organic matter increases the adsorption of tetracycline more than doxycycline, also showed that the composition of oxytetracycline complex with magnesium ion was more stable than doxycycline complex with magnesium. The study also revealed a higher concentration of doxycycline in leachate water from the soil than those of oxtetracycline due to its higher solubility in water, in addition to decrease of the concentrations for both substances over time in leachate over time in leach ate water due to degradation, (Awartani, L., 2010).  Hattab Areej, (2010), Palestine: Adsorption of some fluoroquinolones on selected adsorption. Hattab Areej studied the adsorption of some fluoroquinolones (antibiotic) such as Gemifloxacin Mesylate and Levofloxacin Hemihydrate by using selected adsorbents such as charcoal, kaolin, silica gel and alumina. For http://www.aginternetwork.net/whalecomwww.springerlink.com/whalecom0/content/?Author=Adcharee+Karnjanapiboonwong 35 equilibrium studies, two adsorption isotherms were used Freindlich and Langmuir. The study showed that maximum adsorption capacity for fluoroquinolones occurred into adsorbent charcoal. The amount adsorbed at equilibrium decreases as adsorbate s concentration increased and the weight of adsorbent increases the amount of drug adsorbed increases, (Hattab, 2010). 36 Chapter Three Methodology The research experimental work basically depends on determining the concentration of residues of amoxicillin, ibuprofen, and caffeine versus time in soil, and leachate (underground water) based on times–terms concentration which is considered by three terms, Zero-term, short-term (one year), and long –term concentration (fifteen years). Samples of soil, wastewater, and leachate were analyzed by UV spectrophotometer and HPLC. The room temperature recorded ranged between 18c ە -25cە. All glassware used were cleaned and dried before measurement and each measurement of this study was the average of three readings to ensure that consist values were obtained. Standard readings were obtained for amoxicillin, ibuprofen, and caffeine and were plotted against absorbance readings in order to calculate the concentrations of these compounds. 3.1. Material & Methods This chapter of study addressed on each of the tools that used to implement the experiment, method of work, and materials used in the analysis in addition to the three main drugs which are the subject of study and the mathematical equations needed for the experimental procedures. This chapter will explain the procedure and chemicals for all experiments. 37 3.1.1 .Chemicals and Reagents All pharmaceuticals and chemicals were purchased from the Sun Company in Palestine and all chemicals were HPLC grade. The other pharmaceutical materials (samples for studies) were manufactured in Palestine, (Beir zait ,Dar ashefa, Jerusalem pharmaceutical companies in Ramallah ). The following list is for all chemicals used in this study:  Pharmaceuticals compound (amoxicillin, ibuprofen, and caffeine).  Chemicals for HPLC and spectrophotometer. - Acetonitril. - Monobasic potassium phosphate. - Sodium hydroxide. - Trichloroacetic acid. - Ammonium hydroxide. - Glacial acetic acid. - Sodium acetate. - Distilled water.  Reagents 38 - Potassium dichromate. - Sulfuric acid. - - Ammonium Sulfate. - Potassium hydroxide. - Copper Sulfate. - Boric acid. - Potassium thiosulfate. - Phenolphthalein. - Ethylene diamine tetra acetic acid EDTA. 3.1.2 .Instrumentation Absorbance readings of ibuprofen, amoxicillin, and caffeine were detected using UV-VIS SHIMADZU, Model No: UV-1601, double beam spectrophotometer wave length range 190-1100 nm, accuracy ± 0.004. The detecting wavelengths for the pharmaceutical compound were confirmed using high performance liquid chromatography (HPLC), (SHIADZU CORPOROATION), with Lichoro CART®, C18 column(250 mm×4mm μm) Detector DIODE ARRAY. The wave lengths were 254nm, 230nm, and 256nm for ibuprofen, amoxicillin, and caffeine respectively. 39 3.1.3. Soil Column Preparation In this study seven soil PVC plastic columns were prepared, with 2meter long and 6 inchs diameter (15.25cm), )Cordy,G.E ., et al, 2004). Sample of soil was collected from 1000m 2 area located on the north of Jenin district (Marj Iben Amer), far away from any expected source of contamination with any pharmaceuticals type. A 1.0 Kg of soil sample was collected from different sites randomly, mixed, sieved, and filled inside the columns and was analyzed before any treatments. The first column was used as blank using tap water, which is the same water that has been used for preparation of medicines, and added to the soil as in the other columns, as shown in the following figure. Figure (3.1): Soil column apparatus 40 3.2. Soil Analysis One kilogram of soil sample was weighted accurately, sieved in 2.0 mm sieve, and dried at 105 cە. Before any treatment with pharmaceuticals several tests were conducted on soil as the following: 3.2.1. Soil Texture (Hydrometer Test) The determination of the size distribution of soil particles is known as mechanical or particle size analysis. The particle size distribution of soil expresses the proportions o various size classes (clay ‹0.00 mm silt 0.002-.0.02mm, and sand 0.02-2.0 mm particle size), represented by weight percentages of total soil, using hydrometer method, (Bouyoucos,G.J., 1962) .based on the stoke Law,( the law that the force that retards a sphere moving through a viscous fluid is directly proportional to the velocity of the sphere, the radius of the sphere, and the viscosity of the fluid) ,and used to predict the settling times for various sized particles and it states that the rate which particles fall in suspension is directly proportional to their size and the force due to gravity. The soil was sieved using 2 mm sieve, and dried at 105 cە for 24 hours by Elle oven, and the soil texture was determined by ASTM 152-H hydrometer device. 41 3.2.2. Moisture The results of soil analysis are to be calculated on the basis of oven dried sample weight. Therefore, the moisture analysis is executed before any other analysis. The result on basis of air-dry weight was multiplied by a moisture correction factor (mcf), which is a ratio of the dry weight to the wet weight. A crucible was placed in Ari J.Levy oven at a temperature of 105 cOfor 2 hours, then cooled down to room temperature in a desiccators; the weigh the empty crucible was recorded. A 10g of soil sample were weighted and placed at least 12 hours in the oven at 105 cO, then cooled it down to room temperature in a desiccator and reweighed again. The moisture content for the soil content was calculated using the following equation: M (moisture content) % = (B-C)/ (C - A ) * 100% Mcf (moisture correction factor) = 100+M% / 011 Where: A: weight of empty crucible B: Sample weight + crucible weight before drying. C: Sample weight + crucible weight after drying, (Buurman, P., et al., 1996), (Dhyan.S., et al., 1999). 42 3.2.3.Specific Gravity Specific gravity is the ratio of the mass of unit volume of soil at a stated temperature to the mass of the same volume of gas-free distilled water at a stated temperature. Take 500 mL of distilled water and determined their weight (W1), then weigh out about 100 g of dried soil, and placed in volumetric flask with distilled water. entrapped air by boiling gently for 10 minutes, occasionally rolling volumetric flask to assist in removal of air, cooled the contents at room temperature. The volumetric flask weight (soil plus bottle plus water) (W2). Using a plastic squeezing bottle, wash the inside of the flask and make sure that no soil is left inside then determine the weight of the dry soil in the evaporating dish(W3). The specific gravity of soil sample can be calculated by the following equation: Specific Gravity (Gs) = W3/(W1+W3) W2 Where: W1: Weight of distilled water with flask. W2: weight of (soil plus bottle plus water) W3: weight of the dry soil in the evaporating dish. 43 Specific gravity is generally reported on unit weight of water at temperature T=20cº. The specific gravity at (20 cO) = Gs (at T1C)*A with value of A at (20 cº) =1 3.2.4. pH Add 10 g of air dried of 2 mm sieved sample into 100 ml distilled water and shake for one hour (Dhyan, S., et al., 1999). The results of moisture, pH, soil texture, specific gravity will be shown in chapter four. 3.2.5. Organic Carbon (Walkely ND Black 1934) The organic carbon in the sample is oxidized with potassium dichromate and sulphuric acid. The excess potassium dichromate is titrated against ferrous ammonium sulfate. Weigh accurately 1gram of soil to contain into a dry 500 mL conical flask 10 mL 1 N K2Cr2O7 and 20 mL concentrated H2SO4 were added. Immediately swirl the flask until the soil and the reagent are mixed. Insert a 200 cº thermometer and heat while swirling the flask and the contents on a hot plate or over a gas burner and gauze until the temperature reaches 135 cە (approximately ½ minute). Then 1ml of diphenylamine indicator 44 was added and the resulted suspension was titrated against 0.5 N ferrous ammonium sulfate solution until green color started appearing indicating the end point. The carbon content was calculated using the following equation. Organic Carbon (%) = 0.003 g x N x 10 mL x (1 T/S) x100 / ODW = 3(1− T/S)/ W Where: N = Normality of K2Cr2O7 solution. T = Volume of FeSO4 used in sample titration (mL). S = Volume of FeSO4 used in blank titration (mL). ODW = Oven-dry sample weight (g). % of organic matter = 1.72 X % of organic carbon, (Walkely, A.J., and Black. 1934; Jackson, M.L., 1962). 3.2.6. Wastewater Analysis Three pharmaceutical factories in the West Bank discharge their wastewater into the public sewer system with and without prior treatment. All wastewater samples were taken around 9:00 a.m, and collected at the discharge point from these factories in Ramallah. 45 Wastewater samples were collected in glass containers previously cleaned by washing with non-ionic detergent, rinsed with tap water and later rinsed with distilled water prior to usage. The samples were labeled and transported to the laboratory, stored in the refrigerator at about 4 c o prior to analysis as the following: 3.2.6.1. Determination of Chemical Oxygen Demand (COD): The Chemical Oxygen Demand (COD) test measures the oxygen equivalent consumed by organic matter in a sample during strong chemical oxidation. 2.5 ml of sample was taken into a refluxing flask; 3.5 ml of sulfuric acid H2SO4 with 1.5 potassium dichromate (K2Cr2O7) were added to the solution. Digest the tubes for two hours, then cooled at room temperature, and titrated the samples using ferrous ammonium sulfate (FeNH4SO4) and indicator. The milliliter equivalent of O2 is equal the milliliter equivalent of ferrous ammonium sulfate (FeNH4SO4) FAS. Therefore COD of wastewater samples can be calculated by the following equation: COD as mg of O2/L = (A –B) * M* 8000/) sample volume) 46 Where: A: ml FAS used in blank B: ml FAS used in sample M: morality of FAS 8000: ml equivalent of O2*1000L/ml, (Andrew, E., et al., 1995). 3.2.6. 2. Determination of Biological Oxygen Demand( BOD) Biological oxygen demand (BOD) measure the amount of oxygen requires by bacteria for breaking down to simpler substances the decomposable organic matter present in wastewater or treated effluent. It is also taken as a measure of the concentration of organic matter present in any water. The greater the decomposable matter present, the greater the oxygen demand. Fill three dissolved oxygen bottles with sample wastewater, place the sample bottle in the dark and incubate for five days at 20c . After five days, the level of dissolved oxygen (in mg/L) of this sample was determined, (Andrew, E., et al., 1995), both results of BOD, COD, will be shown in chapter four. 3.2.6.3.Determination of Nitrogen (Kjeldhal Test) Nitrogen in wastewater is mostly present in an organic form with small quantities of ammonium and nitrate. The samples is first digested in 47 a catalyst mixture which convert all nitrogen into ammonium sulfate, then the distillation processes of ammonia (librated after sodium hydroxide is add to ammonium sulfate), over boric acid and finally the amount of the ammonia that has been trapped is determined by titration with a standard solution, and a calculation made. 143 ml of concentrated H2SO4 to 25ml of wastewater, the sample was allowed to cool, and then a 50 mL of reagent (a mixture of 134 g of potassium sulfate (K2SO4), 7.3 grams copper sulfate CusO4 catalysts solution) were added. Cool and dilute to reach one litter, store at room temperature and do not allow salt to precipitate, if precipitation does occur, put reagent bottle in warm water bath for about 30 minutes. Stir on stirrer plate until precipitation is no longer evident. Add 25ml sample of boric acid (The boric acid captures the ammonia gas) into the mixture, forming an ammonium-borate complex (H2BO-3). As the ammonia collects, the color of the receiving solutions changes. The flask was placed under the condenser of distillation apparatus to separate ammonia–nitrogen from the digestate; this is accomplished by raising the pH with sodium hydroxide 50% (NaOH). This converted the ammonium (NH4 + ) ion to ammonia (NH3). Add an indicator to the distilled solution, and titrated with hydrochloric acid Cl “0. M“to the violet end point. The percent of nitrogen was calculated using the following equation: 48 %Nitrogen = V (HCl) xM (HCl) Mwt (N) / (V of sample *1000)*100% N Concentration ppm = %N*10000, (Andrew, E., et al., 1995) 3.2.6.4. Determination of Phosphorus Phosphorus occurs in natural waters and in wastewaters almost solely as phosphates. Add 0.5 mL of sulfuric acid to a 100 mL wastewater sample with 15 ml potassium thiosulfate K2SO4 in a 125 mL Erlenmeyer flask. Boil gently on a pre-heated hot plate for approximately 30-40 minutes. Add few drops of phenolphthalein indicator to the sample solution. Acid samples must be neutralized with 1 N sodium hydroxide (40 g NaOH/L), and get pink color, then add distilled water to volume 100ml mark and known (V1). Take 1 ml of the mixture was added to 4 ml to phosphorus reagent that consist of 0.132 g ascorbic acid were dissolved in 25ml distilled water), then continue the volume to 25 ml (V2). If color is blue this indicates for the presence of phosphorus. A blank test shall be carried out in parallel by the same procedure, using the same quantities of reagent (4ml) and (25ml) distilled water sample. By using spectrophotometer at 880 nm wave length to measure the concentration of phosphorus. The concentration of phosphorus can be calculated by using the following equation: 49 Conc. of phosphorus in ppm = (C*V 0 *V2)/ (V1*1000ml) *100 Where: C: Conc. that measured by UV. V1:100ml V2:25ml, (Bowman, G.T., and Delfino, J.D., 1982). 3.2.6.5. Determination of Calcium and Magnesium The titration method using the chelating agent, EDTA (ethylenediamine tetraacetic acid), used to determine both calcium and magnesium. The titration is conducted under conditions where both ions react with the EDTA, and the end point is detected after both are titrated. To deter mine magnesium, take 25ml of the wastewater sample in an Erlenmeyer flask then add at least one ml of pH 10 buffer solution (ammonium hydroxide) to the sample. The pH should be 10. To check pH, use standardize pH meter, a few drops Eriochrome Black indicator were added to the Erlenmeyer flask. Fill the burette with standardized EDTA; begin to titrate the sample two drops at a time. Be careful to titrate slowly near the endpoint, as the color will take about 5 seconds to develop, the endpoint color is blue. 50 To determine calcium take 25-ml of the water sample into an Erlenmeyer flask, add at least one ml of pH 12 buffer solution (Sodium hydroxide) to the sample. The pH should be 12, to check pH, use standardize pH meter. Add a few drops hydroxynaphthol blue indicator to the Erlenmeyer flask. Fill the burette with standardized EDTA; begin to titrate the sample two drops at a time. Be careful to titrate slowly near the endpoint, as the color will take about 5 seconds to develop, the endpoint color is blue,(Andrew, E., et al., 1995). The concentration of both calcium and magnesium can be calculated by using the following equations: Conc. of calcium (ppm) = Volume of EDTA*Mwt(Ca)*M (EDTA)*100 Volume Sample of *1000 Conc. of Magnesium (ppm) = (Volume of EDTA*Mwt (Mg)*M (EDTA)*100) Volume Sample of *1000 Determination of Heavy Metals The analysis of wastewater for trace heavy metal contamination is an important step in ensuring human and environmental health. Spectrophotometric method is the most important for determining heavy metals in wastewater. In comparison with atomic emission spectroscopy, 51 atomic absorption spectroscopy and similar techniques, it offers the advantage of having calibration graphs that are linear over a wider range. The heavy metals analyzed in this study included cadmium, chromium, nickel, and zinc using atomic absorption flame emission spectrophotometer instrument. This served as the stock solution equivalent to 100 ppm. Subsequently lower concentrations of 1 ppm, 2 ppm, 4 ppm and 6 ppm, were prepared from the stock by serial dilution. The same method was adopted for Cr, Ni, Cu and Zn, (Andrew, E., et al., 1995). 3. 3. HPLC Scanning of Amoxicillin, Ibuprofen, and Caffeine A rapid analytical procedure for routine identification and quantification of amoxicillin, Ibuprofen, and caffeine in wastewater by high performance liquid chromatography (HPLC), the processes for detection of these compounds involve three solution preparations (mobile phase, standard, and sample solutions). 3.3.1 HPLC Scanning of Amoxicillin For detection of amoxicillin use UV at 230 nm, chemicals and reagents (Acetonitril solution, monobasic potassium phosphate, Sodium hydroxide, distilled water, and amoxicillin raw material). In this experiment three solutions will prepared: 52 Mobile phase solution: solution prepared from diluents and acetonitrile (96:4), the diluents was a mixture of (6.9 g of monobasic potassium phosphate with 1000 ml distilled water), adjusted with 45%NaOH solution to pH of 5.0 +0.1. The standard solution: it’s prepared y dissolving an accurately weighed quantity of amoxicillin raw material in diluents to obtain solution having a known concentration of about 1.2 mg\ml, degassed by ultrasonication before use. The mobile phase was prepared daily (use the solution within 6 hours). Waste water samples: that were collected from drug manufacturers must be filtered before making injection. Procedure: separately inject equal volume (10 μ liters) of standard solution into HPLC to take retention time, and then inject equal volume of three sample solutions into same device, with cleaning by mobile phase after each sample. 3.3.1. HPLC Scanning Of Ibuprofen : For detection ibuprofen use UV at 254 nm, (chemicals and reagents are Trichloroacetic acid, distilled water, ammonium hydroxide, ibuprofen active ingredients material). Three solutions will prepare: Mobile phase solution: its prepared from 4 grams of trichloroacetic acid with 400 ml distilled water, adjusted with ammonium hydroxide solution 53 to pH of 3, then 600 ml from acetonitril solution was added, finally filtration for all solution made and degassed by ultrasonication before use . Standard solution: it’s prepared rom 0. grams of ibuprofen active ingredients with 10 ml volumetric from mobile phase solution and degassed well. In addition to the wastewater samples that collected from drug manufacturers must be filtered before make injection. Procedure: separately inject equal volume (10 μ liter) of standard solution into HPLC to take retention time, and then inject equal volume of the three samples solution into same device, with cleaning by mobile phase after each sample. 3.3.2. HPLC Scanning of Caffeine For detection of caffeine use UV at 256 nm, chemicals and reagents are (glacial acetic acid CH3COOH, distilled water, sodium acetate, caffeine raw materials). As in previous scanning, three solutions prepared which are: Mobile phase solution: 20% CH3COOH (volume\volume), with 100 ml distilled water buffered to pH 5.08 with saturated sodium acetate solution. Standard solution: prepare 0.05mg\ml (5ppm) of caffeine raw material. In addition to the waste water samples that were collected from drug manufacturers must be filtered before making injection. 54 Procedure : separately inject equal volume (10 μ liter) of standard solution into HPLC to take retention time, then inject equal volume of the three samples solution into same device, with cleaning by mobile phase after each sample, (United States Pharmacopeia Convention, Inc. 1995), The following table showed retention time and wavelength for each drug. Table (3.1): wave length and retention time: Drug ’ s Name Wave Length(nm) Retention Time(minute) Amoxicillin 230 9.16 Ibuprofen 254 2.0 Caffeine 256 20.7 3.4. Calibration Curve A standard calibration curves for amoxicillin, ibuprofen, and caffeine were performed by preparing diluted solutions from stock solution containing 500mg\L, 50 mg of ibuprofen ,amoxicillin and caffeine reference standards were accurately weighed each of which alone, transferred into 100 ml volumetric flasks, mobile phase solution was added to volume and stirred until completely dissolved. Several dilutions were made of each of them by taking 1ml(10ppm), 3ml(30ppm), 4ml(40ppm), 55 6ml(60ppm) and 8ml(80ppm) from stock solution and transferred into 50 ml volumetric flasks, distilled water was added to volume. Absorbance readings were recorded at 254nm for ibuprofen, 230 nm for amoxicillin, and at 256 nm for caffeine. 3.5. Isotherms The most widely used equation to fit empirical data from solute – solvent adsorbent system is the Freundlich equation. Due to its simplicity and versatility in fitting data from systems, Freundlich relationship will be used in this study to describe the quantitative adsorption of ibuprofen, amoxicillin, and caffeine onto soil. Four different concentrations 50ppm, 40ppm, 30ppm, 20 ppm and of each drug solutions were prepared, each in 125 ml Erlenmeyer flask, 5 grams of oven-dried sieved sample were added to each flask, 50 ml of each concentration for each substance were added to each flask, all samples were covered with teflon screw caps and mounted on Comfort Hetro Master Shaker for 24 hrs. Soil was left to settle, and centrifuged at 3000 rpm for 10 minuts. Absorbance readings were recorded at 254nm for ibuprofen, 230nm for amoxicillin and at 256 nm for caffeine sample solutions using UV-1601 PC, SHIMADZU spectrophotometer. 56 3.6. The Effect of Temperature on (Amoxicillin, Ibuprofen, and Caffeine) -Soil Adsorption The purpose of this task is to determine the effect of temperature on the process of hydrolysis and adsorption of amoxicillin, ibuprofen, and caffeine onto soil. Stock solutions of amoxicillin, ibuprofen, and caffeine (500mg\L) were used as a dsorbate and solution of 50 ppm (50mg\L) concentration were obtained by diluting the stock solution with distilled water. In the kinetic experiments for Ibuprofen, 50ppm was prepared from stock solution (1ml of ibuprofen solution/10ml distilled water) was mixed with 10 gram of oven dried sieved soil in 100ml volumetric flask. The adsorbent and a dsorbate suspension were separated by shaking and by centrifugation at 3000 rpm for 10 minutes using Hermel Z200A Centrifuge. Thermodynamic study of adsorption experiments were performed following the same procedure at 15 c ە , 25 c ە , and 35c .ە Then the supernatants were filtered through filter paper and the three drugs concentrations were analyzed using UV-1601 PC, SHIMADZU spectrophotometer at different time (10, 30, 60, 120,and 180 minutes) to show the effect of contact time of their uptake by the adsorbent (soil) . To determine the concentrations of thesis pharmaceuticals that adsorbed on soil by subtracted the concentration in the supernatant solution from the origin one (50ppm) then plotted graphs of the pharmaceutical 57 concentration versus time intervals. Finally plot the curve as Time on x- axis and the concentration Y-axis, (Sruchi M. K., 2011). 3.7. The Effect of pH on (Amoxicillin, Ibuprofen, and Caffeine) -Soil Adsorption. The purpose of this experiment is to determine the extent to which adsorption was influenced by varying pharmaceutical ionization state and aquifer material surface properties with varying pH, and the properties of the pharmaceuticals and surfactants studied. So we have to do the following: Take the 50 ppm sample of each drug and mix it with 10 g of the soil sample and use 0.1 M HCL to acidify the solution be sure the final PH is a out “ . ”. Do the same thing ut change the p to 4 then you already have it at 7 then use NaOH 0.1M) to have it 12. Keep in mind the concentration stays 50 ppm. Then shake it for 60 min and keep the temperature at 25c . Centri uging the samples iltering them using Whatman ilter papers “no. 4 ” and measuring the concentration of the drugs using spectrophotometer, use the calibration curve that was made in (Kinetic and Isotherms of amoxicillin, ibuprofen, caffeine) experiment. Finally plot the curve as pH on x-axis and the concentration Y-axi,( Hari, A.C., et al., 2005), the results will shown in chapter four. 58 3.8. Amoxicillin, Ibuprofen, and Caffeine Application to Soil Column Experiment Seven columns were prepared for the pollution process, two columns for each drug was labeled according to the pollutant type, quantity and time. The first column was considered as blank, i.e. hasn’t any pharmaceuticals, but tap water was added to it. The second one was polluted with Ibuprofen; a solution containing (0.2034) gram of Ibuprofen/4L) contained 50mg/L was prepared and added to the column, therefore it was labeled (ibuprofen one-year), the third column contained (3.054) gm of Ibuprofen powder 4\L contained 50mg/L of ibuprofen, a product of the Palestinian Companies for Pharmaceuticals, Ramallah). The third and four columns were polluted using amoxicillin contained of 50 mg\L, solution containing 0.2034 g for one year and 3.054 for 15-years. The fifth and sixth columns were polluted using caffeine 50mg\L solution containing 0.2034 g for one year, and 3.054 for 15-years. After the addition of pharmaceuticals to the columns, Soil columns were left for 24 hrs to ensure a complete adsorption process on soil. 59 3.9. Collecting and Storage of Leachate Water Samples Water samples were collected and kept in well closed HDPE plastic bottles, and stored in a refrigerator (at 7c°). HDPE plastics are known for their low adsorption properties, low moisture absorption, and high tensile strength. HDPE is also non-toxic and non-staining and meets FDA and USDA certification, (http://www.boedeker.com/polye_p.htm;Boedeker Plastics, Inc. Polyethylene Specifications Texas / USA). Leach ate water was centrifuged to remove solid particles and then the supernatant was iltered using Whatman ilter papers no “4 “ e ore analysis. 3.10. Distribution of Pharmaceutical in Soil Columns after Finishing the Leachate Study: At the end of the experiment, each soil column was cut into eight parts (0-25cm, 25-50cm, 50-75cm, 75-100cm, 100-125cm, 125-150cm, 150-175cm, 175-200cm . The soil samples were prepared as follows: 20 gm of polluted soil were weighed and transferred in to 250 ml conical flask, 100 ml of distilled water were added and stirred for 30 minutes using Freed Electric magnetic stirrer, the suspension was filtered through Whatman ilter papers “no. 4 ” and measured the concentration using spectrophotometer. 60 Chapter Four 4. Results & Discussion The results of this work are represented in tabular and graphical form. Results were devoted to understand the behavior and the fate of amoxicillin, ibuprofen, and caffeine in soil and underground water for the next 15 years, including their adsorption in soil. Discussion of the results follows each part of experimental work. 4.1. Soil Tests Samples of red soil were analyzed in order to evaluate the soil texture, moisture p value and speci ic gravity. Ta le” 4. ” shows the results obtained from these tests. Table (4.1): Soil specific gravity, pH, texture, and moisture for soil before pollution. Soil Properties Result Specific Gravity 2.34 pH value 7.14 Clay (%) 41% Silt (%) 59% Moisture content 9.5% Organic Carbon % 2.34% Organic Matter % 4.36% 61 From the table above it was noticed that the silt percentage is larger than that in the clay , organic carbon and organic matter are less than the percentage that increase the adsorption (3%, 5%) respectively ,which indicates that low percentage of adsorption will occur. 4.2. Waste water Test Samples of wastewater from three pharmaceuticals industrial companies were collected and analyzed in order to evaluate the following: 4.2.1. pH, BOD, COD Table (4.2): wastewater PH, BOD and COD Company Name pH BOD mg\L COD mg\L Alquds 6.62 189.00 5440.00 Dar Alshefa 5.74 210.00 11200.00 Beir –zait 7.44 91.00 64.00 From the table above it was noted that pH in both Dar Alshefa, Alquds companies is acidic, which indicates that these companies used acidic materials in the process of preparation of medicines, and low bacterial level in their wastewater, a while the pH in Beir –Zait company is around neutral . 62 The rate of COD in Dar Alshefa and Alquds companies is higher than BOD in comparison with Beir-Zait Company, and this shows that thesis companies use chemical treatment for waste water, and they had manufactured a batch of antibiotics in the same day of sample collection. It was also known that wastewater containing antibiotics characterized by the high proportion of COD and low BOD, which is difficult to make biological treatment, (Elmolla, E., and Chaudhuri, M., 2009), this is consistent with the information that was known about the mechanism of the companies wastewater treatment when I visited them. 4.2.2.Calcium, Magnesium ,Phosphorus, Nitrogen: Table (4.3): wastewater Ca, Mg, P, N. Company Name Ca ppm Mg ppm P ppm Nitrogen ppm Alquds 973.00 51.66 ND 85.00 Dar Alshefa 158.00 88.50 ND 105.00 Beir –zait 121.68 3.69 ND 89.80 It is noted from the table above that there is arising in the concentration of both calcium and magnesium in wastewater collected from the pharmaceutical manufacturing companies, this may be due to the use of some materials in drug filling phase which contains calcium and 63 magnesium, or manufacturing other types of drugs that also contain calcium and magnesium especially drugs used to treat osteoporosis. For nitrogen, it is normal to note increase in concentration and this is due to the presence of bacteria whether dead or alive in the wastewater. 4.2.3. Heavy metals (Cadmium, Chrome, Zinc, Nickel). Table (4.4): Heavy metals concentrations in the three pharmaceuticals companies’ wastewater: WHO Standards Company name (Cr) conc. ppm (Cd) conc. ppm (Zn) conc. Ppm (Ni) conc. ppm Cr ppm 0.050 Cd ppm 0.003 Alquds 0.050 0.017 0.003 ND Zn ppm 4.000 Dar Alshefa 0.054 0.037 0.005 ND Ni ppm 0.020 Beir-zait 0.054 0.044 0.002 ND It has been observed during the examination of heavy metals for the three pharmaceutical companies the chromium was among the WHO acceptable maximum of (0.05 mg\L), while cadmium concentrations were found to be higher than the WHO acceptable maximum of 0.003 mg/L, so cadmium toxicity has been reported to cause food poisoning, mutation, hypertension, and cancer among others, long term exposure to cadmium has been found to cause serious damage to kidney, liver, bone and blood, (Anyakora,Ch., et al., 2011), but Zn concentration in the three companies 64 was lower than the WHO acceptable maximum of 3-5mg\L, and nickel heavy metal was not detected in the three companies . 4.2.4. HPLC Scanning of Ibuprofen, Amoxicillin, and Caffeine Concentrations The purpose of this task is for identification and quantification of amoxicillin, ibuprofen and caffeine in the pharmaceutical wastewater companies using HPLC, their retention time, and the wave length of the previous drugs, then adopted thesis wave lengths to measure the concentrations of drugs from soil lactates. The following table shows the retention time for ibuprofen, amoxicillin, and caffeine. Table (4.5): Retention time and peaks length and wave lengths of ibuprofen, amoxicillin, and caffeine in pharmaceutical wastewater companies. Company name Standard Alquds Dar Alshefa Beir –zait Amoxicillin Retention Time(minutes) 9.16 - - - Wave length 230nm Ibuprofen Retention Time(minutes) 2.00 - - - Wave length 254nm Caffeine Retention Time(minutes) 20.70 - - - Wave length 256nm 65 4.3 The Effect of Temperature on the Adsorption of (Amoxicillin, Ibuprofen, and Caffeine –Soil). Absorbance reading and calibration curve for amoxicillin, ibuprofen, and caffeine were recorded at 230 nm, 254 nm, and 256 nm respectively, in addition to measuring of their concentrations at different temperatures, as shown in the following tables and figures. Table (4.6): Amoxicillin standards at 230nm: Standard concentration (ppm) Absorbance 10 0.315 30 0.691 40 0.841 60 1.185 80 2.033 66 R² = 0.965 -0.5 0 0.5 1 1.5 2 2.5 0 20 40 60 80 100 A bs or ba nc e Standard conc.ppm Figure (4.1): Plot of amoxicillin calibration curve. Table (4.7): Amoxicillin concentration in supernatant and soil- adsorption at 10, 30, 60, 120,081 minutes: Concentration .ppm 10min 30min 60min 120min 150min At 15co Supernatant conc. 8.820 7.570 5.026 4.920 4.606 Soil adsorption 41.180 42.430 44.974 45.080 45.394 At 25co Supernatant conc. 5.206 4.670 3.020 4.350 3.942 Soil adsorption 44.794 45.330 46.980 45.650 46.058 At 35co Supernatant conc. 2.450 1.590 0.750 1.920 3.871 Soil adsorption 47.55 48.410 49.250 48.080 46.129 67 Table (4.8): Ibuprofen Standards at 254nm: Standard concentration (ppm) Absorbance 10 0.007 30 0.021 40 0.039 60 0.068 80 0.102 R² = 0.938 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 0 20 40 60 80 100 A b so rb an ce Standard conc.ppm Figure (4.2): Plot of Ibuprofen calibration curve 68 Table (4.9): Ibuprofen concentration in supernatant and soil- adsorption at 10, 30, 60, 120,081 minutes: Table (4.10): Caffeine Standards at 256nm: Standard concentration (ppm) Absorbance 10 0.406 30 0.903 40 1.401 60 2.061 80 2.537 Concentration .ppm 10min 30min 60min 120min 150min At 15co Supernatant conc. 9.785 8.732 7.020 7.047 7.120 Soil adsorption 40.215 41.268 42.980 42.953 42.880 At 25co Supernatant conc. 9.514 7.321 5.960 6.062 6.995 Soil adsorption 40.486 42.679 44.040 43.938 43.005 At 35co Supernatant conc. 7.020 5.880 3.820 4.016 3.910 Soil adsorption 42.980 44.120 46.180 45.984 46.090 69 Table (4.11): Caffeine concentration in supernatant and soil- adsorption at 10, 30, 60, 120,081 minutes: R² = 0.992 0 0.5 1 1.5 2 2.5 3 0 20 40 60 80 100 A bs or ba nc e Standard conc.ppm Figure (4.3): Plot of caffeine calibration curve. Concentration .ppm 10min 30min 60min 120min 150min At 15co Supernatant conc. 16.243 15.326 11.238 06..31 14.480 Soil adsorption 33.757 36.674 38.762 36.440 35.520 At 25co Supernatant conc. 15.515 14.293 12.860 12.071 16.240 Soil adsorption 34.485 35.707 37.140 37.161 33.760 At 35co Supernatant conc. 19.896 17.926 14.250 16.018 21.503 Soil adsorption 30.104 32.074 35.750 33.982 28.497 70 Table (4.12): Soil- adsorption of amoxicillin, ibuprofen, and caffeine at temperat re c . Figure (4.4): plot of concentrations of amoxicillin, ibuprofen and caffeine in soil at 0 c . Drugs-Soil Adsorption conc.ppm Time .minutes 10 30 60 120 150 Amoxicillin 41.180 42.430 44.974 45.080 45.394 Ibuprofen 40.215 41.268 42.980 42.953 42.880 Caffeine 33.757 36.674 38.762 36.440 35.520 30 32 34 36 38 40 42 44 46 48 0 30 60 90 120 150 180 C on c. p p m time in minutes Ibuprofen Amoxicilli n 71 a le ( . ): oil-a sorption o amoxicillin i pro en an ca eine at temperat re c . Drugs-Soil Adsorption conc. ppm Time .minutes 10 30 60 120 150 Amoxicillin 44.794 45.330 46.980 45.650 46.058 Ibuprofen 40.486 42.679 44.040 43.938 43.005 Caffeine 34.485 35.707 37.140 35.030 34.760 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 0 30 60 90 120 150 180 C on c. p p m time in minutes Ibuprofen Amoxicillin Caffeine Figure (4. 5): plot o concentrations o amoxicillin i upro en and ca eine in soil at c . 72 a le ( . ): oil- a sorption o amoxicillin i pro en an ca eine at temperat re c . Drugs-Soil Adsorption conc.ppm Time .minutes 10 30 60 120 150 Amoxicillin 47.550 48.410 49.250 48.080 46.129 Ibuprofen 42.980 44.120 46.180 45.984 46.090 Caffeine 30.104 32.074 35.750 33.982 28.497 Figure (4.6): plot of concentrations of amoxicillin, ibuprofen, and caffeine in soil at 35c °. To clarify the extent of the impact of temperature on the adsorption of drugs versus time on the soil, concentrations measured after different time intervals of incubation, the degradation of the selected pharmaceuticals was also influenced by microbial activities, oxygen status in the soil, soil type and compound characteristics. The study showed that the concentrations of 25 27 29 31 33 35 37 39 41 43 45 47 49 51 0 30 60 90 120 150 180 C on c. pp m time in minutes Ibuprofen Amoxicilli n 73 ibuprofen, amoxicillin in the supernatant are decreased with increasing in their adsorption on soil for the first hour, then started to decrease in their adsorption due to its starting degradation, for this reason it’s written on the medicine must be kept cool place, (Dwivedia, A., et al, 2011). While the more decreasing of soil adsorption for caffeine with increasing temperature after the first hour because it considered as hydrophilic compound and dissolves in water. 4.4. The Effect of pH on Drug-Soil Adsorption The extent to which adsorption was influenced by varying pharmaceutical ionization state and aquifer material surface properties with varying pH will be shown in following tables and figures: Table ( . ): moxicillin a sorption on soil at i erent p constant temperat re c an one o r. Amoxicillin pH 1.500 4.000 7.000 12.000 Supernatant concentration(ppm) 6.020 8.026 7.982 4.856 Soil adsorbed concentration (ppm) 43.98 41.974 42.018 45.144 74 Figure (4.7): plot of soil adsorption concentrations of amoxicillin in soil at different pH, temperature 25cº and one hour. Figure (4.8): plot of concentrations of amoxicillin in supernatant at different pH, temperature 25cە, and one hour. amoxicillin - soil adsorption concentration 41.5 42 42.5 43 43.5 44 44.5 45 45.5 02468101214 pH C o n c .p p m amoxicillin - supernatant concentration 0 1 2 3 4 5 6 7 8 9 02468101214 pH C o n c .p p m 75 Table (4.16): Ibuprofen adsorption on soil at different pH and constant temperat re c , and one ho