Use of simple immuno-assay to detect illegal drugs and recreational drugs of abuse in wastewater

Research studies indicate that drugs of abuse are prevalent in the water system and if not properly treated, could impact the environment and future societies that use reclaimed water as a drinking source. Although they are not currently regulated by the Environmental Protection Agency (EPA), it would be beneficial for wastewater treatment facilities to begin testing for drugs of abuse to determine what concentrations are present and the facilities' removal rates. This data could be used to help the facility begin to plan for additional treatment methods when the EPA implements regulation. Most treatment facilities are government based and have limited funding. A method to detect illegal drugs in the wastewater that is cost and time effective that does not require gaining permits from the Drug Enforcement Administration (DEA) will allow municipalities to begin testing and preparing for drug removal. Detection by immuno-assay is more affordable, less time consuming and does not require permits through the DEA for drug standards as do conventional detection methods. Wastewater samples collected from Salt Lake County sewer lines and Central Valley Water Reclamation Facility were tested for caffeine, cocaine, cotinine, methamphetamine, nandrolone, oxycodone, and tetrahydrocannabinol (THC). The samples were processed and tested with Neogen Immuno-assay drug detection kits. The drugs caffeine, cocaine, methamphetamine, oxycodone, and THC were detected at concentration ranges similar to those in other studies. The concentrations for cotinine and nandrolone were undetectable. Immuno-assays proved to effectively detect drugs of abuse in wastewater.

USE OF SIMPLE IMMUNO-ASSAY TO DETECT ILLEGAL DRUGS AND RECREATIONAL DRUGS OF ABUSE IN WASTEWATER by Alex McKendra Fisher A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science Department of Civil and Environmental Engineering The University of Utah May 2013 Copyright © Alex McKendra Fisher 2013 All Rights Reserved The University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Alex McKendra Fisher has been approved by the following supervisory committee members: Otakuye Conroy-Ben , Chair 3/13/2012 Date Approved Steven Burian , Member 3/13/2012 Date Approved Andy Hong , Member 3/13/2012 Date Approved and by Chris Pantelides , Chair of the Department of Civil and Environmental Engineering and by Donna M. White, Interim Dean of The Graduate School. ABSTRACT Research studies indicate that drugs of abuse are prevalent in the water system and if not properly treated, could impact the environment and future societies that use reclaimed water as a drinking source. Although they are not currently regulated by the Environmental Protection Agency (EPA), it would be beneficial for wastewater treatment facilities to begin testing for drugs of abuse to determine what concentrations are present and the facilities' removal rates. This data could be used to help the facility begin to plan for additional treatment methods when the EPA implements regulation. Most treatment facilities are government based and have limited funding. A method to detect illegal drugs in the wastewater that is cost and time effective that does not require gaining permits from the Drug Enforcement Administration (DEA) will allow municipalities to begin testing and preparing for drug removal. Detection by immuno-assay is more affordable, less time consuming and does not require permits through the DEA for drug standards as do conventional detection methods. Wastewater samples collected from Salt Lake County sewer lines and Central Valley Water Reclamation Facility were tested for caffeine, cocaine, cotinine, methamphetamine, nandrolone, oxycodone, and tetrahydrocannabinol (THC). The samples were processed and tested with Neogen Immuno-assay drug detection kits. Theiv drugs caffeine, cocaine, methamphetamine, oxycodone, and THC were detected at concentration ranges similar to those in other studies. The concentrations for cotinine and nandrolone were undetectable. Immuno-assays proved to effectively detect drugs of abuse in wastewater. TABLE OF CONTENTS ABSTRACT .................................................................................................................. iii LIST OF FIGURES ....................................................................................................... vii LIST OF TABLES ....................................................................................................... viii ACKNOWLEDGMENTS .............................................................................................. ix Chapters 1 INTRODUCTION ........................................................................................................ 1 2 BACKGROUND .......................................................................................................... 3 Importance of Drugs of Abuse in Wastewater .............................................................. 3 Drugs of Abuse Tested ................................................................................................. 4 How do Drugs Enter the Wastewater System? ........................................................... 11 Possible Impacts from Drugs of Abuse in Wastewater ............................................... 12 Current Detection Methods ........................................................................................ 14 Current Treatment Methods ....................................................................................... 17 Use of Reclaimed Water and Drug Effects ................................................................. 18 3 MATERIALS AND METHODS ................................................................................ 26 Sample Collection ...................................................................................................... 26 Sample Processing ..................................................................................................... 29 Immuno-Assay .......................................................................................................... 29 4 RESULTS .................................................................................................................. 36 Introduction ............................................................................................................... 36 Caffeine ..................................................................................................................... 36 Cocaine ...................................................................................................................... 37 Cotinine ..................................................................................................................... 38 Methamphetamine ..................................................................................................... 38 Nandrolone ................................................................................................................ 39 Oxycodone ................................................................................................................ 39 vi THC........................................................................................................................... 40 Discussion ................................................................................................................. 40 5 CONCLUSION .......................................................................................................... 52 Final Remarks ............................................................................................................ 53 APPENDIX: METHOD DEVELOPMENT DETAILS .................................................. 54 REFERENCES.............................................................................................................. 66LIST OF FIGURES 1 - Chromatographic Process Schematic ........................................................................ 23 2 - Map of Central Valley Improvement Districts .......................................................... 32 3 - Central Valley Water Reclamation Facility Schematic.............................................. 33 4 - Color Variation Related to Concentration when K-Blue Substrate is Added ............. 34 5 - Color Variation Related to Concentration after Acid is Added .................................. 34 6 - Immuno-Assay Reaction Process ............................................................................. 35 7 - Caffeine Standard Curve .......................................................................................... 43 8 - Cocaine Standard Curve ........................................................................................... 44 9 - Cotinine Standard Curve .......................................................................................... 45 10 - Methamphetamine Standard Curve ......................................................................... 46 11 - Nandrolone Standard Curve ................................................................................... 47 12 - Oxycodone Standard Curve .................................................................................... 48 13 - THC Standard Curve .............................................................................................. 49 LIST OF TABLES 1 - Drug Physical and Chemical Properties .................................................................... 21 2 - Concentration Ranges of Illegal Drugs Detected in Recent Publications ................... 24 3 - Concentrations of Illegal Drugs Detected in Groundwater in Barcelona .................... 25 4 - Caffeine Concentration Results ................................................................................ 43 5 - Cocaine Concentration Results ................................................................................. 44 6 - Methamphetamine Concentration Results ................................................................. 46 7 - Oxycodone Concentration Results ............................................................................ 48 8 - THC Concentration Results ...................................................................................... 49 9 - Average, Maximum and Minimum Concentrations in Sewer Lines .......................... 50 10 - Mass Flux per Capita .............................................................................................. 50 11 - Percent Loss of Analytes due to Filtration .............................................................. 51 12 - Percent Removal from Wastewater Treatment ........................................................ 51 ACKNOWLEDGMENTS I must credit a number of people, for without their support and help, this thesis may not have been completed. First, I would like to acknowledge my parents, Keith and Stacy Fisher, who have given unconditional love and unlimited support over the years of my education. I am eternally grateful for the sacrifices they have made to make sure I achieve my personal, educational, and professional goals. My sister, Abby Fisher, has always provided emotional support and stress relief. The unlimited love and support from my grandparents, Larry and Sylvia Fisher, and Larry and Lynette Gatlin has always been motivating and inspirational. To my late grandfather, Stephen Sherrod, who taught me that I could have whatever I want in life, as long as I work hard and stay dedicated to my dreams. Dr. Otakuye Conroy-Ben was my academic advisor for my undergraduate career and influenced me to proceed into the graduate program. Dr. Conroy-Ben offered many research topics that provided me with endless learning opportunities, ultimately leading me to this research project. Dr. Conroy provided all the necessary funding for this research and has been a great resource. The staff at Central Valley Water Reclamation Facility was extremely helpful in sample collection. Christi Priest and Talena Walton in pretreatment contributed several x hours driving me to sewer collection sites and providing valuable information on the wastewater collection facilities. I would like to thank all of the people who have helped me in the lab. The relationships we have built have been very valuable and special to me. Finally, I would like to thank my boyfriend, Vince Willis, for standing by my side and not giving up on me even in the most stressful moments. CHAPTER 1 INTRODUCTION Pharmaceuticals and personal care products are a concern in wastewater treatment and environmental research. Endocrine disrupters and specific medications have been studied to determine how they impact the environment. Illegal and recreational drugs are of interest due to the high concentrations found in wastewater streams. The consequences from illegal drug exposure in the environment are not very well known, but it can be assumed that they will most likely have a negative impact as they will eventually be consumed by humans. Methods of detection and removal need to be implemented in wastewater treatment facilities to monitor removal rates and to reduce the concentrations being released into the environment. More precise monitoring of drugs in the wastewater will also be beneficial to forensics studies by having more accurate drug usage statistics which can be used by local law enforcement. Current detection methods for drugs in the wastewater system are expensive and time consuming. Municipal wastewater treatment facilities are not required to test for drugs, and are not properly equipped to do so. Methods for fast and inexpensive detection could be implemented at wastewater treatment facilities to determine an approximate concentration of drugs in the wastewater and an approximate removal rate throughout the 2 wastewater treatment plant. Using a method that is less expensive and can be performed at the site of sample collection would benefit the wastewater treatment plant operators and inspectors by supplying rapid and accurate results. Although the current detection methods are efficient and well known, obtaining permission to possess and test for drugs of abuse is difficult, as they are listed under Class II Controlled Substances with the Drug Enforcement Agency (DEA). Class II Controlled Substances are strictly regulated due to the potential for abuse leading to psychological or physical dependence.1 Permits are granted by registering with the DEA, under a state license, and the registration holder is required to abide by the state and federal rule pertaining to the controlled substance. Registration must be renewed every three years, and the cost varies depending upon the controlled substance. Strict storage regulations are enforced, and personnel with access to controlled substances are required to go through thorough training. Registration can be a time consuming and expensive process.1 As an alternative to gaining permits, the samples could be sent to a local laboratory that holds the proper permits for testing controlled substances, but this is costly and time consuming. This report discusses some important issues of drugs of abuse in wastewater, how they are currently detected, and how they are removed from wastewater using conventional wastewater treatment methods. Tests were performed using immuno-assays to determine if this method could be used to detect concentrations of specific drugs in wastewater in a timely and inexpensive manner. CHAPTER 2 BACKGROUND Importance of Drugs of Abuse in Wastewater The effects of drugs on people are fairly well known due to studies conducted on human safety. The effects of illegal drugs have been tested and evaluated to understand the dangerous effects on humans. Nicotine, which requires users to be at least 18 years of age, and oxycodone, which requires a prescription, are examples of legal drugs that are regulated to prevent abuse by users. However, little research has been done on the effects of drugs found in wastewater and the surrounding environment. Some studies have been performed on certain species, but these studies are very limited and do not represent the concentrations or the continuous exposure rates currently found in the environment. Some of the effects of drugs on humans may be relatively similar to the effects on the environmental species. These assumptions are considered throughout this report. A study in Europe that tested 19 drugs of abuse in wastewater systems proved that wastewater analysis would also be beneficial to forensic sciences. Most drug usage statistics are reported from surveys, which are highly dependent on consumers. Concentrations found in wastewater lead to more accurate data for drug usage in specific areas.2 4 Drugs of Abuse Tested The drugs used for this research are caffeine, cocaine, cotinine, methamphetamine, nandrolone, oxycodone, and tetrahydrocannabinol (THC). The chemical composition, properties, and effects of these drugs are discussed below. Table 1 displays the chemical properties of each drug. Caffeine Caffeine is a stimulant found naturally in beverages, like coffee and tea, and can be used as a pharmacological agent. Caffeine is one of the most commonly used drugs, consumed by 90% of the adult population, and is found in food, drinks, and supplements. An average of 120 mg/day of caffeine is consumed through beverages by Americans.3 A maximum of 12 μg / mL of caffeine can be excreted in urine with standard caffeine consumption.4 Concentrations up to 73 μg / L of caffeine have been found in wastewater in Europe.5 As the population continues to consume more caffeine, and it becomes more prominent in wastewater streams, the effects of caffeine on aquatic species need to be considered. Although caffeine is frequently used, there are some serious side effects if used in excess. Some studies have shown that the side effects to humans are similar to other species and plants. It can cause insomnia, nervousness, stomach irritation, and increased heart rate and respiration. Caffeine can also aggravate anxiety disorders and bleeding disorders. It has also been shown to increase blood pressure and weaken bones.6 Also, caffeine is a diuretic, which causes more frequent urination. 5 There have been some studies on the effects of caffeine on specific species. The northern leopard frog displayed behavioral and physiological effects when exposed to caffeine. Exposure to high concentrations of caffeine also caused a change in oxygen consumption by sea urchin fertilized eggs and impaired reproduction of a water flea species. Most studies conducted on the effects of caffeine on aquatic species have used high concentrations of caffeine that do not represent the environmental concentrations.7 The effects caused by long term exposure of lower concentrations need to be further studied to gain a better understanding. The chemical properties of caffeine are important to understand its environmental fate in aquatic systems. The log octanol-water partitioning coefficient (Log Kow) is the ratio of the molar concentration found in octanol verse water, which suggests the chemicals biodegradability and solubility. In general, the larger the Log Kow, the lower the solubility, the higher the log bioconcentration factor (Log BCF) and the water partition coefficient (Koc).8 The Log Kow for caffeine is -0.07, which indicates that caffeine is very soluble and biodegradable.9 This value, shown in Table 1, indicates that caffeine will not adsorb to solids present in an aquatic system. The Log BCF, refers to the uptake of a chemical from water by respiration or dermal contact. It is calculated using the Log Kow value, and values less than 3 are considered nonbioaccumulative, between 3 and 3.7 are considered bioaccumulative, and greater than 3.7 are considered very bioaccumulative. The Log BCF in aquatic organisms is low for caffeine, with a value of 3.10 6 Caffeine is removed with biological treatment and in rivers and streams. Activated sludge treatment has proven to be effective in removing caffeine since it is readily biodegradable.10 This study will compare the concentrations of caffeine before and after trickling filter biological treatment at Central Valley to determine if this facility correlates to other studies. Cocaine/Benzoylecgonine (BE) Cocaine is an alkaloid ester that is extracted from coca plants. Cocaine will increase the heart rates and blood pressure while constricting the arteries. This often leads to heart attack or causes arrhythmia. Constricted blood vessels in the brain cause strokes, seizures, and bizarre and violent behavior. Constriction of vessels in the stomach leads to ulcers or perforation of stomach or intestines. Cocaine has also been known to impair sexual function.11 Benzoylecgonine (BE) is the main metabolite formed when cocaine is consumed and contributes to many of the side effects of cocaine. The chemical properties of benzoylecgonine are displayed in Table 1. When cocaine is released into water, it is likely that it will adsorb to suspended solids or sediments due to its Log Kow value. The Log Kow for cocaine is 2.31, implying that cocaine is slightly water soluble and biodegradable. The Log BCF of cocaine is 3, which indicates it has a low bioconcentration in aquatic species. At a pH of 7, the cocaine half-life is 54 years.12 7 Cotinine Cotinine is the primary urinary metabolite of nicotine. It is detected in urine to determine the concentration of nicotine consumed by tobacco smokers, and is also found in urine of nonsmokers exposed to second hand smoke. Cotinine has a vapor pressure, shown in Table 1, which indicates it will be present in both vapor and particulate form in the atmosphere, compared to nicotine which is completely vaporized in the atmosphere. Thus, second hand smoke is more likely to contain particles of cotinine, which will show a higher presence in urine tests than nicotine.13 Studies have not shown any dangerous effects of cotinine in humans but the effects of nicotine have been proven to be quite dangerous.14 Nicotine can cause a faster and pounding heartbeat, extreme weakness, nausea, vomiting and wheezing. Tightness in the chest, stinging in the nose, throat and mouth, blisters in the mouth, and nose bleeds are also caused by nicotine. Life threatening coronary artery vasoconstriction and bronchospasms are dangerous symptoms of nicotine use.15 Although cotinine does not cause physical side effects, it does cause serious withdrawal symptoms, which makes it one of the most difficult addictions to conquer. There are drug detection kits available for nicotine detection, but cotinine detection was chosen because it is much more prominent in urine samples and easier to detect in diluted sources such as wastewater. Cotinine released into the environment by smoke vapor which will either degrade in the atmosphere or will be removed by wet or dry deposition. The Log Kow of cotinine indicates it will be highly mobile in soil, but it will not be adsorbed by suspended particles in water systems. Instead, cotinine will most likely be biodegraded in the system. The Log BCF for cotinine is 3, which indicates that the bioconcentration in 8 aquatic species is low.13 A Log Kow of 0.07 for cotinine indicates that it is quite water soluble.16 Methamphetamine/MDMA Methamphetamine (meth) elevates dopamine levels in the body, up to 12 times higher than food or sexual intercourse. Meth destroys dopamine sensors, thus, making it impossible to feel pleasure. Meth also destroys tissues and blood vessels, causes acne, loss of elasticity and luster in skin and tooth decay by drying out salivary glands which allows acids in the mouth to eat away teeth enamel.17 MDMA, or 3, 4-methylenedioxy-N-methylamphetamine, is commonly known as ecstasy. It is a popular drug linked to raves and electronic music. MDMA binds to the serotonin transporter which, prolongs the serotonin signal. This causes excessive release of serotonin from neurons which causes heightened senses and euphoria. It can also cause confusion, depression, sleep problems, drug cravings, and severe anxiety. MDMA increases heart rate, blood pressure, causes muscle tension, involuntary teeth clenching, nausea, blurred vision, faintness, chills, sweating, and hypothermia.18 Meth has a wide range of Koc values, as seen in Table 1. These values are relatively low, indicating that meth is unlikely to be adsorbed by particles and sediments in water systems. The Log BCF for meth is 2, which means the bioconcentration in aquatic species is very low. The Log Kow is 2.07 meaning meth is somewhat hydrophobic.12 9 Nandrolone Nandrolone, also known as Deca-Durobolin, is an anabolic steroid which is produced naturally by the human body, but only in very small concentrations. Nandrolone is very similar to testosterone, except it is more anabolic than androgenic, which indicates nandrolone would be beneficial for muscle building without increasing male sexual characteristics. It has been used by athletes as a performance enhancer because it can increase muscle mass without an increase in body hair or aggressive behavior associated with using testosterone as a steroid. Nandrolone can still cause many problems if abused. Fluid retention, edema, congestive heart failure, and sexual problems may persist. Genitourinary effects including oligosperma, infertility, decreased ejaculatory volume, and an enlarged prostate can occur in men using nandrolone. Women may experience a deepening of the voice, hirsutism or excessive hair growth, acne, or clitomegaly, which is the enlarging of the clitoris.19 The International Olympic Committee set a limit of 2 ng/mL of urine as the maximum concentration produced naturally. Some athletes have had concentrations 100 times greater than this.20 Since nandrolone is a hormone, excessive concentrations in aquatic systems can have detrimental impacts on aquatic species. Studies have shown that exposure to some hormones in aquatic systems have direct effects on the gonads, reproductive systems, and sexual differentiation during early development. The effects of nandrolone have not been highly studied, but it can be assumed that the impacts caused by other androgenic hormones are a good indication that any hormone exposure is harmful to aquatic species.21 10 The Koc value of nandrolone assumes that nandrolone is likely to adsorb to particles and sediments in water bodies. The Log BCF of nandrolone is 21, indicating that the bioconcentration in aquatic species is low.13 Nandrolone is somewhat water soluble, with a Low Kow of 2.62.8 Oxycodone Oxycodone is a narcotic, or an opioid, pain reliever. Oxycodone, found in OxyContin, has become a highly abused prescription drug. The side effects of oxycodone include drowsiness, sedation, respiratory depression, apnea, intestinal obstruction, and anxiety. Withdrawal symptoms are also a common side effect of oxycodone. If too much is taken, hallucinations, psychosis, and slowing of the heartbeat can occur.22 Oxycodone is not expected to be adsorbed by suspended particles or sediments in water bodies due to the low Koc value. The Log BCF for oxycodone is 3, which means it has a low bioconcentration in aquatic species. A Log Kow of 0.7 implies that oxycodone is quite water soluble.13 THC Tetrahydrocannabinol, more commonly known as THC, is the main compound found in marijuana. It influences pleasure of memories and thinking, concentration, sensory and time perception and coordination. Chronic users show more impacts to memory loss that can last for weeks.23 Although there are risks, THC is becoming more popular in the medical industry as an antiemetic, which is a drug used to reduce nausea, and as an appetite stimulant. As more states begin to legalize marijuana, not just for 11 medical use but also for recreational use, it will become more prominent in the wastewater streams. If THC is released into a water system, it is expected to be adsorbed onto suspended particles and into sediments due to its high Koc value. THC is expected to volatilize from water surfaces because of its Henry's Law constant: 2.4∗10−7(𝑎𝑡𝑚−𝑚3)𝑚𝑜𝑙𝑒. The Log BCF for THC is very high, indicating that the bioconcentration in aquatic species is also very high.13 THC has a high Log Kow, 6.48, indicating that it is more hydrophobic than other drugs.24 How do Drugs Enter the Wastewater System? Only a portion of what is consumed is used by the human body. This includes all drugs. The components that remain unused are excreted in urine or feces in the parent or metabolized form. The amount excreted depends on the dose, the frequency of use, and the person's individual metabolism. Many drugs can be detected in urine post-ingestion for 1.5 to 4 days with just a single dose. Drugs can be detected for up to a week after the last dose in chronic users. Specifically, cocaine and THC can remain in the system for even longer periods of time. The limit of detection varies for each drug as well. The limit of detection of meth in urine is approximately 2.5 ng/ mL, while THC is 10 ng/mL.25 In 2010, it was estimated from a survey that 6.24% of Utah residents used illegal drugs in the past month, with 3.12% reporting the use of a drug other than marijuana. Almost 7000 Salt Lake County residents were admitted for substance abuse rehabilitation in 2009. In 2007, 546 people passed away from a drug-induced death, which is more than 12 the 320 who died from vehicle accidents and 253 from firearms in the same year.26, 27 The number of illicit drug users in the state is most likely higher than surveys suggest. Many residents will not fill out surveys, and others will not respond honestly about drug usage. The numbers shown in surveys suggest that there are still high volumes of drugs being used in Salt Lake County and they are entering the wastewater system. Using the concentrations found in wastewater would help local law enforcement determine a more accurate amount of drugs being used in Salt Lake County. These concentrations also help the wastewater treatment facility determine what further treatment is necessary to reduce the quantity of drugs entering the Jordan River. Possible Impacts from Drugs of Abuse in Wastewater Very little is known about the effects of drugs on aquatic species. The effects of specific drugs on humans are fairly well known, and it may be assumed that these effects will be similar to other species. Only a few studies have been performed on living specimens, with many being mammals. The few studies that have been conducted need to be considered when developing wastewater treatment methods to remove drugs from wastewater. A study performed by Castiglioni et al., on zebrafish is one of the few reports on the effects of illicit drugs on aquatic species.28 Zebrafish and zebrafish embryos were exposed to various drugs at different concentrations to determine the effects. When the zebrafish were exposed to 5 mg/L of cocaine, the fish would slowly circle at the bottom of the water column with their fins extended, which indicates arousal. The zebrafish 13 embryos were also exposed to THC, which reduced the amount of spontaneous tail muscle twitches that occur during development. At concentrations near 2.0 mg/L, the spine would curve and the tips of the tails would form a bulbous shape. At concentrations above 2.0 mg/L of THC, the embryo would die. Additionally, when zebrafish were exposed to 100 mg/L of nicotine for 3 minutes, they displayed a significant decrease in diving. When exposed to 50 mg/L for longer periods of time, the zebrafish would tend to dwell at the bottom of the water column.28 Studies on mammals have been conducted to determine how the drug will possibly affect humans. One study on primates showed that the effects on the serotonin nerves after a 4 day exposure to MDMA could last 6 to 7 years.18 Insects have also been exposed to drugs to understand the impacts. Walters performed a study on Drosophila melanogaster, a common fruit fly, using methamphetamine, to determine how it affects the species. The flies exhibited anorexic behavior when exposed to meth for several hours. The flies also displayed increased movement and activity, similar to humans, when exposed to meth.29 Although these studies were performed with high concentrations of drugs, the effects are critical to understanding how the concentrations seen in the environment can affect the ecology. The concentrations will continue to rise in the environment as the human population grows and more people use illegal drugs. Also, the species exposed to the drugs are continuously exposed, not for just short periods of time. These long term exposures can have even more detrimental effects to the surrounding environment. More 14 studies on the species specific to the areas of contamination, including plants and migratory birds, should be performed to determine long term effects of drug exposure. Current Detection Methods Chromatography is the main method used to detect drugs in liquid samples. The sample mixture is separated between two phases: a stationary phase and a mobile phase. The stationary phase is typically fixed in place while the mobile phase carries the mixture through the medium of the stationary phase. The mixture is controlled by the interactions between the mixture's components and the stationary and mobile phases. Some of the components will slow and interact with the stationary phase, while others will increase in speed and remain interacting with the mobile phase. The difference in the velocities controls the separation of different species in the mixture. The mobile phase will carry the separated species away from the stationary phase at different times, which can be measured to determine which species are present in the mixture. The stationary phase of chromatography is typically a substance coated on the interior walls of the column. There are different types of columns: open tubular, capillary, or packed column. The columns can have a variety of stationary phases and polarities and are chosen based upon the sample mixture and species that are being detected. The mobile phase is determined by the type of chromatography used and the column chosen. Figure 1 is an example of a chromatographic system. There are two types of chromatographic methods, gas-chromatography and liquid-chromatography. Both are commonly used to detect drugs in liquid samples. 15 Gas-chromatography (GC) uses a gas carrier as the mobile phase. When samples are injected into the GC, they are heated and converted into their vapor phase. The carrier gases commonly used are helium, argon, or nitrogen. The carrier gas will transfer the sample in vapor form to the stationary phase, which is typically a packed column. The individual species will interact with the stationary phase at different rates, as discussed above. When the gas exits the column, a detector will determine the species in the sample. There are many detectors available to be used with a GC. The most common detector used for drug detection is a mass spectrometer (MS). The GC will separate the compounds from each other, and the MS will identify each species by the fragmentation after chemical or electron ionization.30 Liquid-chromatography (LC) uses a liquid for the mobile phase. The liquid is chosen based on the stationary phase used, which is chosen based on the compounds being detected. Sodium chloride, methanol and water mixture, n-heptane, or ethanol are common examples of mobile phases that are used. A liquid sample that has been concentrated and resuspended in a compatible solution is injected into the LC. The sample and mobile phase liquids are transported through the LC column, interact with the stationary phase, and are then transported to the detector to determine which species are present. There are several detectors compatible with LC, but a tandem MS detector is the most commonly used for drug detection.31 Many studies have used other forms of GC and LC for drug detection to determine which method is most effective. High performance liquid chromatography (HPLC) has higher pressures within the column, forcing the solvent through and 16 completing detection at much faster rates. A high performance liquid chromatography system with tandem mass spectrometry (HPLC-MS/MS) is often used to detect drugs at higher concentrations. The high performance liquid chromatography-atmospheric pressure ionization (HPLC-API-MS) uses chemical ionization at atmospheric pressure to detect drugs. Solid phase extraction (SPE) is commonly coupled with HPLC-MS/MS for accurate drug detection in urine. Ultra performance liquid chromatography (UPLC) uses high pressures, similar to HPLC, coupled with increased sensitivity and resolution for even faster detection rates and high accuracy.32, 33, 34 Although the use of chromatography provides accurate results for drug concentrations in liquid samples, it is quite expensive and time consuming. A chromatography machine can cost from tens to hundreds of thousands of dollars. Many wastewater treatment facilities may have a chromatography systems, but new columns specific to each drug tested would need to be purchased. With multiple collection sites, the facility may want to use several machines at once to be more time efficient. The facility may want to consider investing in multiple detectors because the composition of wastewater may damage the machine. This can be a substantial initial cost for wastewater treatment facilities to begin detecting illegal drugs in wastewater. Another concern with detection of drugs in water and wastewater is the time for sample preparation. After samples are collected, they need to be filtered and concentrated. This process can take several hours per sample due to the high amount of suspended solids in raw wastewater. Running the sample through the GC or LC can take several hours as well. A lab technician would have to work several days continuously to 17 process and test one sample. It would be more convenient for wastewater treatment operators and inspectors to be able to test the samples and have accurate results within a couple hours of collection, rather than waiting a few days for results. Current Treatment Methods Although illegal drugs are currently not regulated by the U.S. Environmental Protection Agency (EPA), conventional wastewater treatment methods partially remove drugs from the system. Studies on the removal rates from various treatment methods have been examined and are discussed below. A research project in the United Kingdom tested wastewater from two rivers and two wastewater treatment facilities for amphetamines, cocaine, and BE. Using UPLC-MS, the study found that there were high concentrations of all three drugs present in the rivers. The cocaine metabolite, BE, was found in concentrations up to 10 times higher than the parent chemical. In both wastewater treatment facilities, activated sludge removed up to 100% of amphetamine, cocaine and BE, while the trickling filter only removed 95% of amphetamine, 25% of cocaine, and there was no noticeable removal of BE.35 Zuccato et al. examined removal rates in wastewater treatment facilities of illegal drugs. One study found that 85 to 99% removal of methamphetamine occurred while another study showed 60 to 98% removal, resulting in low concentrations (ng/L) in the effluent. MDMA was found to be removed at rates from 44 to 57%, with approximately 0.10 ng/L in the effluent. Cocaine and BE both were found to have approximately 10% of 18 the influent concentration remaining in the effluent after wastewater treatment. THC had the most extreme removal rate range, from 11 to 99% removal. Studies also showed that removal rates were higher in 2006 than in 2004. This is most likely due to improved treatment methods.36 Concentrations ranges detected in wastewater influent, effluent and receiving waters are displayed in Table 2. From the little research performed, it can be suggested that bioreactors can remove higher quantities of drugs from wastewater systems. Although a portion of the drugs are removed, the concentrations being released are still of concern due to possible effects on the environment. Use of Reclaimed Water and Drug Effects With growing populations and limited water resources, use of reclaimed water is gaining importance. If the reclaimed water is supplied from a municipal wastewater discharge area, such as a lake, river, or groundwater, drinking water may be contaminated with the drugs that are not removed during wastewater treatment. In Spain, the surface waters of the Ebro River and the tap water in Barcelona were tested for various drugs including methamphetamine, MDMA, THC, cocaine and BE. The most concentrated compound found was BE, at levels as high as 346 ng/L in the surface water. In drinking water, 130 ng/L of BE and 1.7 ng/L of meth was detected.37 Zuccato et al. also discussed drinking water treatment removal rates of specific drug compounds. Amphetamines were found to be almost completely removed during treatment by prechlorination, flocculation, and sand filtration. Granulated activated 19 carbon (GAC) removed 100% of cocaine, 88% of MDMA and 72% of BE. In 22 of the 24 samples taken from the treated drinking water, BE was found in concentrations ranging from 45 ng/L to 130 ng/L.36 A study in Barcelona proved that groundwater is being contaminated by drugs of abuse as well. Groundwater infiltration is not removing the drugs from the wastewater. Thirty-six groundwater samples from the Barcelona urban groundwater system were tested for cocaine, BE, THC, methamphetamine, and MDMA. The concentration ranges are shown in Table 3. Approximately 40% of the United States uses groundwater for drinking water. If groundwater recharge areas are supplied with treated wastewater contaminated with drugs, and the local municipalities use the groundwater as a culinary source, the drinking water could be at risk for drug contamination.38 Drinking water treatment methods need to be improved to ensure that the drugs are completely eliminated. It is alarming that traces of BE were found in all of the water samples. More research is necessary to determine what methods could be implemented to effectively remove BE from all of the water systems. The general public would be very concerned if the culinary drinking water had any traces of illegal drugs. As reclaimed water becomes more popular as a main drinking water source, the concentrations of illegal drugs need to be reduced to nonexistent levels to protect society from consumption. The previous information indicates that drugs are prevalent in the water system and if not properly treated, could impact the environment and future societies that use reclaimed water as a drinking source. Although not currently regulated by the EPA, it 20 would be beneficial for wastewater treatment facilities to begin testing for drugs of abuse to determine what concentrations are present and the facilities' removal rates. This data could be used to help the facility begin to plan for additional treatment methods when the EPA does implement regulation. As most treatment facilities are government based and have limited funding, a method to detect illegal drugs in the wastewater that is cost and time effective that does not require gaining permits from the DEA will allow municipalities to begin testing and preparing for drug removal. 21 Table 1 - Drug Physical and Chemical Properties Benzoylecgonine Caffeine Cocaine Cotinine Chemical Composition 𝐶16𝐻19𝑁𝑂4 𝐶8𝐻10𝑁4𝑂2 C17H21NO4 𝐶10𝐻12𝑁2𝑂 Chemical Structure Molecular Weight (g/mol) 289.33 194.1906 303.35294 176.21508 Vapor Pressure (mm Hg @ 25°C) 3.77x10-11 7.3x10-9 1.91x10-7 3.8x10-4 Koc 1.28 22 - 130 Henrys Constant (atm-m3/mole) 1.03x10-13 3.6x10-11 4.2x10-11 3.3x10-12 pKa 2.15 10.4 - - Half Life in Water - 0.8 days 54 yrs. at pH 7 5 yrs. at pH 8 - Log BCF 1.00 3 3 3 Log Kow -1.32 -0.07 2.31 0.07 Meth MDMA Nicotine Nandrolone Chemical Composition 𝐶10𝐻15𝑁 𝐶11𝐻15𝑁𝑂2 𝐶10𝐻14𝑁2 𝐶18𝐻26𝑂2 Chemical Structure Molecular Weight (g/mol) 149.2328 193.2423 162.23156 274.39784 Vapor Pressure (mm Hg @ 25°C) 1620 0.003 0.0038 3.5x10-8 Koc 9 to 22 - 100 630 Henrys Constant (atm-m3/mole) 7.34x10-3 2.75x10-9 3.0x10-9 2.7x10-9 22 Table 1 (cont) Meth MDMA Nicotine Nandrolone pKa 11 - - - Half Life in Water 1.0 Hrs. in river 3.9 days in lake - - - Log BCF 2 3 21 Log Kow 2.07 -0.32 1.2 2.62 Oxycodone THC Chemical Composition 𝐶18𝐻21𝑁𝑂4 𝐶21𝐻30𝑂2 Chemical Structure Molecular Weight (g/mol) 315.36364 314.4617 Vapor Pressure (mm Hg @ 25°C) 2.2x10-10 4.6x10-8 Koc 120 3.3x105 Henrys Constant (atm-m3/mole) - 2.4x10-7 pKa 8.3 - Half Life in Water - - Log BCF 3 5 Log Kow 0.7 6.48 23 Figure 1- Chromatographic Process Schematic Mobile Phase 24 Table 2 - Concentration Ranges of Illegal Drugs Detected in Recent Publications Drug Target Chemical Influent Concentration (ng/L)39, 40 Effluent Concentration (ng/L)41 Surface Water Concentration (ng/L)42 Oxycodone Opioid narcotic; pain reliever ND - 220 ND NA Cotinine Nicotine by-product; tobacco ND - 6820 ND - 2726 ND - 516 Cocaine Central nervous system stimulant 79-860 ND - 47 ND - 10 Caffeine Stimulant ND - 120000 ND - 22848 ND - 2991 THC Active chemical in marijuana NA NA NA MDMA Ecstasy 13.6 - 91 ND - 67 ND - 3.5 Nicotine Tobacco NA NA NA Benzoylecgonine Cocaine metabolic by-product 0.08 - 2800 ND - 928 ND - 111 Meth Stimulant ND - 2000 ND - 3.5 NA Nandrolone Anabolic Steroid ND - 2.4 ND - 1.2 ND - 0.60 25 Table 3 - Concentrations of Illegal Drugs Detected in Groundwater in Barcelona Drug Average Concentration ± Standard Deviation (ng/L)38 Max Concentration (ng/L)38 Cocaine 3.8±12.8 60.2 BE 1.5±4.5 19.6 THC - - Meth - - MDMA 3.9±6.6 36.8 CHAPTER 3 MATERIALS AND METHODS The objective of this study was to develop a cost and time effective method to detect drugs of abuse in wastewater. Research was conducted on sewage and treated wastewater from Central Valley Water Reclamation Facility and adjoining collection agencies in Salt Lake City, Utah. The samples were processed for testing with Neogen ELISA forensic drug detection kits. The Neogen kits were used to detect caffeine, cocaine/BE, cotinine, meth/MDMA, nandrolone, oxycodone, and THC. The materials and methods used are discussed below. Sample Collection Sewer Lines Samples were collected from the Salt Lake Valley wastewater sewer systems and Central Valley Water Reclamation Facility (CVWRF) in Salt Lake City, Utah. Eight sewer lines feed Central Valley from seven improvement districts: Cottonwood Improvement District, Granger-Hunter Improvement District, Kearns Improvement District, Murray City, Mt. Olympus Improvement District (formerly known as Salt Lake Suburban Sanitary District One, has two sewer lines), South Salt Lake City, and 27 Taylorsville-Bennion Improvement District. Figure 2 displays the boundaries of these improvement districts. Combined, these districts serve over 500,000 people and cover approximately 94 square miles.43 Each sewer line has a sampling station located before it merges with other districts' sewer lines and enters CVWRF. A permanent pump is located at each sampling station and samples are randomly taken daily for CVWRF testing. These pumps were used manually to collect the samples into 1 L amber glass bottles. The glass bottles were labeled, sealed, and stored in a cooler with ice packs. Wastewater Treatment Facility Central Valley Water Reclamation Facility Board was established in 1978 in response to the Clean Water Act. The members of the board represented the five original wastewater treatment facilities. Central Valley was designed to treat 75 million gallons of wastewater a day.43 Figure 3 is a schematic of Central Valley's wastewater treatment process. A conventional wastewater treatment plant has many components, as shown in Figure 3. Untreated wastewater enters the treatment facility through a network of sewer lines. The preliminary treatment includes bar screens, which capture any large debris that will be collected and removed. Sewage is then pumped into aerated grit chambers where smaller particles, like sand, are removed. Water is then transported to primary sedimentation, where the heavier solids are given time to settle to the bottom of the tank. During this process, the oils and fats are skimmed off of the surface, and 60% of the settable material has been removed. Next, wastewater is pumped to trickling filters, 28 where biological growth degrades pollutants. After the trickling filter, the water is transported to the solid contacts (aeration) tanks. In this process, the growth of microorganisms converts remaining pollutants into settable solids. The wastewater and biosolids flow into the secondary sedimentation tank, where solids form and settle to the bottom of the tank. At this point, 95% removal of pollutants is observed. Wastewater is then sent to disinfection, using chlorination to kill any disease causing bacteria. The water flows through aeration tanks, where dissolved oxygen is diffused into the water before it is discharged to Mill Creek. The sampling locations chosen within Central Valley were the trickling filter, solids contact, and final effluent. The trickling filter and solids contact were chosen because they have very high removal rates of most contaminants. The effluent was tested to make a comparison of the final concentration of drugs to those found in the sewage lines. Samples collected at the trickling filter were taken after the first filter, and those from the solids contact were taken from the second tank. Effluent was taken at the discharge point, before the water merges with Mill Creek. All samples collected within the plant were taken by using a bucket attached to a pole, which was rinsed with the water at the sampling location prior to collecting each sample. Samples were then poured into 1 L amber glass bottles, previously muffled at 550 °C for 6 hours, which were sealed, labeled, and stored in a cooler with ice packs. The samples were then refrigerated and processed within 24 hours of collection. 29 Sample Processing Samples were filtered using a 1.2 micrometer glass fiber filter within 24 hours of collection. At least 500 mL of sample was filtered. Organics from the filtrate were concentrated on a hydrophobic resin (C-18, octadecylsilane, Empore). Organics were eluted from the C-18 disk with 20 mL of ethanol, after which the volume was reduced and the analytes were re-constituted in water or buffer. Appendix A displays instructions for sample processing for each drug kit. Immuno-Assay Current analytical methods used to detect drugs in the wastewater are expensive and very time consuming. Affordable products are available for drug testing from human urine, blood, and saliva that are relatively quick to perform and are very efficient. Testing wastewater samples with the assay could greatly reduce time and costs for treatment facilities to begin monitoring and reporting drugs of abuse. Drug kits used for this experiment were purchased from Neogen Corporation. The kits cost $160 each and only take a couple hours to perform. Each kit contains 96 reactions, at $1.67 per well on the assay plate. Including the standard curve, a sample can be analyzed for under $30. On the other hand, quantification of opioid drugs in 1L of water costs $800-900 as quoted by the U.S. Geological Survey.44 Enzyme-linked immunosorbent assays (ELISA) use antibodies, antigens and enzymes to determine the concentrations of drugs. The 96-well assay plates are coated with immobilized antibodies that are specific to the antigen of the drug being tested. 30 There are a limited number of antibodies on the plates, controlling the number of antigen binding sites. This limitation restricts the concentration ranges that can be detected.45 The wastewater sample and drug conjugate were added to the wells and incubated for a specified amount of time. A metalloenzyme from the root of horseradish, horseradish peroxidase, is used as the drug conjugate. Horseradish peroxidase uses hydrogen peroxide to oxidize organic and inorganic compounds. When reacted with specific substrates, bright colors are produced. Using various wavelengths, "transparent" proteins that are bound to an enzyme substrate can be detected. For the Neogen test kits, the drug-horseradish peroxidase conjugate competes with the drugs in the sample to bind to the antibodies that are immobilized to the plate during incubation. The higher concentration of drug present reduces the amount of enzyme conjugates that can bind to the plated antibodies.46 After incubation, the wells are washed to remove any unreacted conjugate and sample. A K-Blue Substrate of 3, 3', 5, 5' Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) is then used to react with the unconjugated enzyme for color development. This reaction produces a blue pigment in the solution. The higher concentration of horseradish peroxidase, the brighter the solution becomes. After 30 minutes of substrate interaction with enzyme conjugate, acid, typically sulfuric acid, is added to each well to stop the reaction. The absorbance is then read to determine the concentration of the drugs compared to the concentration of conjugate in each well. Samples that have high concentrations of drugs will have lower absorbance because there is less reaction occurring between the enzymes and substrates. Figure 4 displays the color 31 variation due to concentration ranges when K-Blue Substrate has reacted. The dark blue wells show little to zero concentration of drug, while the light blue to clear wells indicate high concentrations of drug. Figure 5 displays a plate with a different chromogenic substrate that has reacted with acid. The dark yellow color indicates low concentrations while the clear wells indicate high concentrations of drug detected. Figure 6 is an example of the process used for the Neogen Kits.47 32 Legend Cottonwood Heights Improvement District Granger-Hunter Improvement District Kearns Improvement District Mt. Olympus Improvement District Murray City South Salt Lake City Taylorsville-Bennion Improvement District Figure 2- Map of Central Valley Improvement Districts 33 Figure 3 - Central Valley Water Reclamation Facility Schematic 34 Figure 4 - Color Variation Related to Concentration when K-Blue Substrate is Added Figure 5 - Color Variation Related to Concentration after Acid is Added 35 Testing is done in a micro-well containing pre-plated antibodies responsive to specific drug. Sample and Enzyme/ Substrate conjugate are added along with buffer Competition-binding onto antibody site proceeds for 1 hour. Wells are washed with buffer. K-Blue Substrate (yellow) is added, reacts with enzyme/ substrate complex. The presence of more enzyme/ substrate converts the dye blue. Color is measure using a spectrophotometer. Figure 6 - Immuno-Assay Reaction ProcessCHAPTER 4 RESULTS Introduction A standard curve was developed for each drug kit used. The standard curves were used to determine the concentration of drug at each sampling location. The results for each drug are discussed below. Caffeine Wastewater samples used for caffeine testing were filtered and diluted two-fold. An unfiltered sample was processed to compare to the filtered results. Samples were tested in triplicate. Figure 7 shows the standard curve developed for caffeine. The standard curves were developed by running tests using the positive (approximately 100 μg/L) and negative controls supplied in the Neogen kit. The ratio of the wavelength of known concentration over the wavelength of the negative sample were found and used to scale the y-axis. Thus, at higher concentrations, the ratio would be a smaller value. To develop a more precise standard curve, an exact caffeine standard would be more appropriate. The standard curve was developed for each using the positive and negative 37 controls supplied to determine if it would provide the necessary results to establish the approximate drug concentrations. Table 4 displays the approximate concentrations found in the sewage and wastewater samples. The concentrations found in the samples from the sewage lines that were filtered through 1.2 μm glass fiber range from 70 to 168 μg/L. The samples that were unfiltered had a concentration range of 90 μg/L to 194 μg/L. Caffeine levels decreased after the trickling filter (1 μg/L), solids contact (1.5 μg/L) and effluent (0.5 μg/L). Cocaine Samples for cocaine/BE analysis were processed without being diluted or concentrated. The concentration ranges found in wastewater fell within the limits of the Neogen detection kits. The samples for cocaine testing were filtered and ran in triplicate. Figure 8 displays the standard curve developed for cocaine. A 300 μg/L urine cutoff calibrator supplied with the Neogen kit was used to develop the standard curve. The concentrations of cocaine found in wastewater are listed in Table 5. Concentrations ranged from 1 μg/L to 51 μg/L in the 1.2 μm glass fiber filtered samples from the sewer lines, while raw samples showed concentrations ranges of 42 μg/L to 73 μg/L. The concentrations after treatment showed a slight decrease. The trickling filter had a concentration of 44 μg/L for the filtered sample, and 65 μg/L for the raw sample. The solids contact had concentrations of 37 μg/L and 40 μg/L for the unfiltered sample. The effluent concentration of the filtered sample was 20 μg/L and the raw sample was 33 38 μg/L. These results show that cocaine and the metabolite BE are not effectively removed during wastewater treatment. Cotinine The standard curve for cotinine was developed using the urine standards provided with the Neogen kit. The cotinine standard curve is shown in Figure 9. Although wastewater samples were concentrated 250:1, concentrations were not detected by the Neogen plate. Utah has the lowest percent of cigarette smokers in the nation at 9.3%. The low percentage of smokers and high biodegradability of cotinine likely contribute to undetectable concentrations. Methamphetamine Samples for methamphetamine detection were diluted two-fold. Glass fiber filtered samples were run in triplicate and raw samples were run individually. The standard curve for meth, shown in Figure 10, was developed using the 500 μg/L urine cut-off calibrator. Methamphetamine concentrations in the sewer lines were much higher than the treated effluent. The concentrations range was 86 μg/L to 460 μg/L for the 1.2 μm glass fiber filtered samples. All of the raw samples displayed higher meth concentrations, 308 μg/L to 966 μg/L, indicating that the methamphetamine compounds have likely sorbed onto the biosolids. The filtered organic particles would need to be tested to determine if some of the meth compounds were removed by filtration. The trickling filter showed concentrations of 32 μg/L and 54 μg/L for the filtered and unfiltered samples, 39 respectively. Solids contact showed a higher concentration, 58 μg/L and 64 μg/L. There are some chemicals in the wastewater that may be converted into methamphetamine during the solids contacts, such as MDMA. Any MDMA in the wastewater may be converting to meth, thus increasing the concentrations detected by the immuno-assay. The effluent of the filtered sample showed a concentration of 12 μg/L, while the unfiltered effluent sample had a concentration of 54 μg/L (Table 6). Nandrolone The standard curve for nandrolone was developed using the positive control provided by Neogen. Figure 11 displays the standard curve developed for nandrolone. The wastewater samples for nandrolone were concentrated 25:1, which proved to be too low to detect nandrolone in raw sewage and wastewater. Oxycodone The Neogen oxycodone testing kit provided a 100 μg/L urine cut-off calibrator, which was used to develop the standard curve shown in Figure 12. The samples used for oxycodone detection were concentrated 25:1. Table 7 displays the concentrations found. The concentrations in the sewer lines ranged from 0.04 μg/L to 0.13 μg/L. The wastewater treatment showed a decrease in concentration in all three sampling locations: trickling filter (0.08 μg/L), solids contact (0.07 μg/L), effluent (0.04 μg/L). 40 THC The THC standard curve was developed using the urine cutoff calibrator supplied in the Neogen Kit (Figure 13). Samples collected for THC testing were concentrated 25:1. THC concentrations varied in the sewer lines from 0.005 μg/L to 1.42 μg/L, as displayed in Table 8. The concentration from the trickling filter was 0.03 μg/L. There were no detectable traces of THC found in the samples from the solids contact and effluent, indicating that the majority of THC was likely removed during solids contact due to its high biodegradability. Discussion The drug test kits for caffeine, cocaine, methamphetamine, oxycodone, and THC detected concentrations that are within the ranges found from the literature review, in Table 2. Although the concentrations were on the lower end of the ranges, it can be assumed that the Neogen kits can be effective at detecting illegal drugs in wastewater. To verify the concentrations found, analysis with another detection method, such as GC-MS or LC-MS/MS, would be beneficial. Drug standards that are regulated by the DEA would be required for chromatographic detection methods, thus, comparisons were not made with this research. After comparing concentrations found using the Neogen kits versus another method, the practicality of using Neogen kits for testing would be determined. The kits for cotinine and nandrolone did not have low enough detection limits for this analysis. It can be assumed that either the concentrations in the wastewater in Salt 41 Lake County are low enough to not be of concern at this time or use of immuno-assay is inefficient for detection in wastewater. Utah has the lowest rate of cigarette smokers in the nation, so it may be possible that cotinine levels are just too low for detection. The highest concentration of samples came from the raw sewage samples from various districts. Table 9 shows that Kearns Improvement District had the highest concentrations of caffeine and cocaine, Taylorsville-Bennion had the highest of methamphetamine, Granger-Hunter was highest for oxycodone and Cottonwood had the highest for THC. Drug statistic data is published per county, not city, so it is difficult to know if these values correlate to true consumption of drugs in each of these areas. Volumetric flow rates were taken at the time of sampling. These flow rates, used with population of districts, were used to calculate the mass flux per capita. These values (Table 10) can be used with forensic data to obtain more accurate estimates of populations of users. Murray City has the highest mass flux for caffeine, cocaine, and methamphetamine, while Kearns has the highest flux for oxycodone and Cottonwood Heights has the highest flux for THC. It appears that Murray City is a high drug abuse region. Results also determined that there are losses of drug analytes during glass fiber filtration. Table 11 displays the percent losses of drug analytes due to filtration. The percent loss ranges from 22% to 92%. The total concentration of filtered samples could be verified by collecting the solids from the glass fiber filter and using solid phase extraction, followed by detection using the Neogen kit, GC-MS or LC-MS. 42 Wastewater treatment processes proved to remove a portion of the drugs in wastewater, as shown in Table 12. The removal rates varied from 43% of cocaine to 100% removal of THC. Cocaine showed an increase in concentrations in the trickling filter and solids contact, rather than removal. The drug test kit used for cocaine detects concentrations of BE more accurately than that of cocaine. It is possible that any cocaine in the wastewater was metabolized during the trickling filter and solids contact, thus showing higher concentrations in the drug detection kits. Samples from more locations within the wastewater treatment plant could be used to verify this hypothesis. For further research, other types of drugs could be tested in Salt Lake County's wastewater, such as opioids and hallucinogens. Further testing at each process in the wastewater treatment plant, surface waters and groundwater that are impacted by wastewater, and drinking water sources using immune-assays, would further confirm whether it is a sufficient method for illegal drug detection. Immuno-assay detection coupled with chromatographic detection in future experiments will determine the efficiency of immuno-assay concentration detection. 43 Figure 7 - Caffeine Standard Curve Table 4 - Caffeine Concentration Results Caffeine Concentration from Raw Samples (μg/L) Caffeine Concentration from Filtered Samples (μg/L) Cottonwood 194 70 ± 32.22 Granger - Hunter 90 98 ± 24.56 Kearns 192 168 ± 9.96 Murray 124 160 ± 2.50 Mt. Olympus East 160 126 ± 10.74 Mt. Olympus South 124 126 ± 1.28 South Salt Lake 132 118 ± 6.48 Taylorsville-Bennion 136 146 ± 2.70 Trickling Filter 3 1 ± 0.59 Solids Contact 2.5 1.5 ± 0.34 Effluent 1 0.5 ± 0.02 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0.1 1 10 100 1000 Percent Relative Caffeine Concentration Caffeine Concentration Caffeine Standard Curve44 Figure 8 - Cocaine Standard Curve Table 5 - Cocaine Concentration Results Cocaine Concentration from Raw Samples (μg/L) Cocaine Concentration from Filtered Samples (μg/L) Cottonwood 42 31 ± 10.51 Granger - Hunter 50 42 ± 11.46 Kearns 73 10 ± 2.19 Murray 45 51 ± 6.05 Mt. Olympus East 63 40 ± 13.66 Mt. Olympus South 48 50 ± 14.30 South Salt Lake 67 45 ± 5.24 Taylorsville-Bennion 27 41 ± 3.65 Trickling Filter 65 44 ± 9.36 Solids Contact 40 37 ± 4.43 Effluent 33 20 ± 1.67 0% 20% 40% 60% 80% 100% 120% 0.1 1 10 100 1000 Percent Relative Cocaine Concentation Cocaine Concentration Cocaine Standard Curve45 Figure 9 - Cotinine Standard Curve 0% 20% 40% 60% 80% 100% 120% 0.1 1 10 100 1000 10000 Percen t Ralative Cotinine Concentration Cotinine Concentration Cotinine Standard Curve46 Figure 10 - Methamphetamine Standard Curve Table 6 - Methamphetamine Concentration Results Methamphetamine Concentration from Raw Samples (μg/L) Methamphetamine Concentration from Filtered Samples (μg/L) Cottonwood 308 86 ± 11.17 Granger - Hunter 540 372 ± 15.59 Kearns 966 422 ± 39.53 Murray 624 414 ± 42.24 Mt. Olympus East 616 174 ± 17.66 Mt. Olympus South 454 176 ± 4.82 South Salt Lake 588 286 ± 24.79 Taylorsville-Bennion 940 460 ± 37.62 Trickling Filter 54 32 ± 0.71 Solids Contact 64 58 ± 4.61 Effluent 54 12 ± 0.36 0% 20% 40% 60% 80% 100% 120% 0.1 1 10 100 1000 Percent Relative Methamphetamine Concentration Methamphetamine Concentration Methamphetamine Standard Curve47 Figure 11 - Nandrolone Standard Curve -20% 0% 20% 40% 60% 80% 100% 120% 0.001 0.01 0.1 1 10 Percent Relative Nandrolone Concentration Nandrolone Concentration Nandrolone Standard Curve48 Figure 12 - Oxycodone Standard Curve Table 7 - Oxycodone Concentration Results Oxycodone Concentration from Filtered Samples (μg/L) Cottonwood 0.11 ± 0.012 Granger - Hunter 0.13 ± 0.010 Kearns 0.13 ± 0.016 Murray 0.10 ± 0.004 Mt. Olympus East 0.04 ± 0.004 Mt. Olympus South 0.08 ± 0.010 South Salt Lake 0.09 ± 0.012 Taylorsville-Bennion 0.04 ± 0.009 Trickling Filter 0.08 ± 0.010 Solids Contact 0.07 ± 0.006 Effluent 0.04 ± 0.009 0% 20% 40% 60% 80% 100% 120% 0.01 0.1 1 10 100 Percent Relative Oxycodone Concentration Oxycodone Concentratin Oxycodone Standard Curve49 Figure 13 - THC Standard Curve Table 8 - THC Concentration Results THC Concentration (μg/L) Cottonwood 1.42 ± 0.294 Granger - Hunter 0.68 ± 0.052 Kearns 0.92 ± 0.120 Murray 0.32 ± 0.045 Mt. Olympus East 0.38 ± 0.069 Mt. Olympus South 0.78 ± 0.202 South Salt Lake 0.03 ± 0.042 Taylorsville-Bennion 0.005 ± 0.015 Trickling Filter 0.03 ± 0.014 Solids Contact 0.0 ± 0.006 Effluent 0.0 ± 0.017 0% 20% 40% 60% 80% 100% 120% 0.1 1 10 100 Percent Relative THC Concentration THC Concentration THC Standard Curve50 Table 9 - Average, Maximum and Minimum Concentrations in Sewer Lines NA indicates that raw samples were not tested for detection of that particular drug. Drug Average Raw Concentration (μg/L) Average Filtered Concentration (μg/L) Maximum Concentration (μg/L) Minimum Concentration (μg/L) Caffeine 144 127 168 - Kearns 70 - Cottonwood Cocaine 52 35 51 - Kearns 1 - Cottonwood Cotinine NA ND ND ND Methamphetamine 630 299 460 - Taylorsville - Bennion 86 - Cottonwood Nandrolone NA ND ND ND Oxycodone NA 0.09 0.134 - Granger 0.041 - Taylorsville THC NA 0.57 1.42 - Cottonwood 0.005 - Taylorsville Table 10 - Mass Flux per Capita Mass Flux Per Capita (μg / person - day) Population Flow Rate (MGD) Caffeine Cocaine Meth Oxycodone THC Cottonwood Heights 129000 12.78 26165 378 32145 41 531 Granger-Hunter 120000 36.72 29987 12852 113827 40 208 Kearns 40000 15.90 66775 3975 175681 52 366 Mt. Olympus (Combined) 49.32 Murray City 46000 21.88 75610 24100 195641 47 151 South Salt Lake City 24000 12.04 59145 22555 143353 45 15 Taylorsville-Bennion 70000 22.37 46661 13103 147015 13 1.6 51 Table 11 - Percent Loss of Analytes due to Filtration % Loss in Drug from Filtering vs. Raw Samples Caffeine Cocaine Methamphetamine Average for Sewer Lines 87.8% 67.5% 47.5% Trickling Filter 33.3% 67.7% 59.3% Solids Contact 60.0% 92.5% 90.6% Effluent 50.0% 60.6% 22.2% Table 12 - Percent Removal from Wastewater Treatment (Cotinine and Nandrolone were not detected) % From Trickling Filter % From Solids Contact % Total Removal Caffeine 99.2% 98.8% 99.2% Cocaine -25.7% -5.7% 42.9% Cotinine - - - Methamphetamine 89.3% 80.6% 96.0% Nandrolone - - - Oxycodone 11.1% 22.2% 55.6% THC 94.7% 100% 100% CHAPTER 5 CONCLUSION This study proved that the use of immuno-assays could be used to detect some illegal and recreational drugs of abuse in wastewater in a timely and cost effective manner. Cotinine and nandrolone analytes were not detected by the ELISA kits. The five remaining drugs tested were all detected within the limits of the Neogen ELISA kits used. Caffeine, methamphetamine, and THC showed a high percentage of removal (99.2% - 100%) through the wastewater treatment process. Cocaine showed a 42.9% removal and oxycodone showed a 55.6% removal during treatment. To improve the studies on the use of immuno-assays for illegal drug detection in wastewater, ELISA tests should be performed with GC or LC methods to confirm detection accuracy. The biosolids removed during filtration with glass fiber filters could also be tested for illegal drugs to determine an accurate percentage of analytes lost during filtration. To determine more precise removal rates, activated sludge samples could also be tested. To further research, the effluent and water body of discharge, the Jordan River, could also be detected for the illegal drugs to determine the removal rates by environmental factors. 53 Final Remarks The EPA has not put into place discharge limits on drugs of abuse. Illegal drug usage rates are continually rising and the effects on the environment are unknown. European countries have begun tests to determine the impacts, but there is little research effort in the United States. This may be due to DEA restrictions, or costs of detection methods. As more societies begin using reclaimed water as a drinking water source, illegal drug concentrations will become an even larger concern. When the EPA begins enforcing discharge concentrations, wastewater facilities will need to incorporate new detection and treatment methods. Immuno-assays may be a cost and time effective method to begin detection. APPENDIX METHOD DEVELOPMENT DETAILS Glass Fiber Filtering 1. 1.2 μm glass fiber filters are recommended. 2. Rinse filtering flask, glass frit, and filter funnel with millique water. 3. Pass 100mL of millique water through assembled filter holder. 4. Add glass fiber filter to assembly and rinse filter with 100 mL of millique water. 5. Filter sample. 6. Collect filtrate for testing and for C-18 processing. C-18 Concentration 1. Rinse all glassware with millique water followed by ethanol. 2. Assemble filter holder and pass ethanol through glass frit. 3. Reassemble with C-18 membrane in place. 4. Soak for 1 minute in 20% ethanol, apply vacuum, and pull through. 5. Repeat with 60% ethanol and 100% ethanol. 6. Add 15 mL ethanol and soak for 1 minute. Do not allow disk to run dry at this point. 7. Pull ethanol through until 2-3 mm above membrane surface. 8. Add 15 mL Nanopure water. Pull through until 2-3 mm above membrane surface. 9. Add 500 mL of sample to be concentrated. Do not allow disk to run dry until entire volume has been extracted. 10. Transfer filter assembly to smaller vacuum flask. 11. Add 10 mL ethanol, soak for 1 minute. Pull through. Repeat. 12. Transfer sample to 40 mL (muffled) vial. Dry under N2. 13. Re-suspend with provided buffer solution in ELISA kits.48 Caffeine Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 2. Dilute sample two-fold using EIA Buffer solution provided with Neogen Caffeine/ Pentoxifylline Detection Kit. 3. Use the instructions included with the Neogen Caffeine/Pentoxifylline Detection Kit with some alterations as shown in test procedure steps a through l listed below: a) Determine the number of wells to be used. 56 b) Prepare the enzyme conjugate by diluting the 180X enzyme conjugate stock 1 to 180 in the EIA buffer provided. Mix the solution by inversion. Do not vortex. c) Add 20 μL of sample, laboratory calibrators, and Neogen controls to the appropriate wells in triplicate. DO NOT dilute Neogen's positive and negative controls. d) Add 180 μL of diluted drug-enzyme conjugate to each well. Use 8-channel pipetter for rapid addition. e) Mix by gently shaking plate. f) Cover plate with plate cover and incubate at room temperature for 45 minutes. g) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. h) Once incubation is complete, dump the liquid from the wells. Tap the plate on a clean lint-free towel to remove any remaining liquid in the wells. i) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. j) Add 150 μL of the K-Blue Substrate to each well. k) Incubate at room temperature for 30 minutes. 57 l) Add 50 μL of Neogen's Red Stop Solution to each well to stop enzyme reaction. Mix gently before measuring absorbance. Measure the absorbance at a wavelength of 630 nm. Wells should be read within 2 hours of stopping reaction. Cocaine/ Benzoylecgonine Test Procedures 1. Filter sample with 1.2 μm glass fiber filter (Do not dilute sample for cocaine tests). 2. Use the instructions included with the Neogen Cocaine/ Benzoylecgonine Detection Kit with some alterations as shown in test procedure steps a through k listed below: a) Determine the number of wells to be used. b) Gently mix the ready to use conjugate solution by inversion. Do not vortex. c) Add 10 μL of sample, Neogen calibrators to the appropriate wells in triplicate. d) Add 180 μL of ready to use drug-enzyme conjugate to each well. Use 8-channel pipetter for rapid addition. e) Mix by gently shaking plate. f) Cover plate with plate cover and incubate at room temperature for 45 minutes. g) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. 58 h) Once incubation is complete, dump the liquid from the wells. Tap the plate on a clean lint-free towel to remove any remaining liquid in the wells. i) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. j) Add 100 μL of the K-Blue Substrate to each well. k) Incubate at room temperature for 30 minutes. l) Add 100 μL of Acid Stop (1N H2SO4) to each well to stop enzyme reaction. Mix gently before measuring absorbance. Measure the absorbance at a wavelength of 450 nm. Wells should be read within 2 hours of stopping reaction. Cotinine Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 2. Perform C-18 Concentration to obtain a 250:1 concentrated ratio, re-suspended in provided dilution buffer. 3. Use the instructions included with the Neogen Cotinine Detection Kit with some alterations as shown in test procedure steps a through k listed below: a) Allow at least 60 minutes for all test kit components and samples to reach room temperature (20-23ºC). 59 b) Prior to using a new kit, prepare fully diluted enzyme by adding exactly 150 μL of cotinine enzyme conjugate to the enzyme diluent. Gently invert 15 times. c) Determine the number of wells to be used. d) Pipette 20 μL of sample and standards into wells in triplicate. e) Add 100 μL of prepared cotinine enzyme conjugate to each well. Invert the conjugate several times before adding to wells. f) Cover plate with plate cover and incubate at room temperature for 30 minutes. Tap side of plate 3 to 4 times throughout incubation. g) Dump the solution from the wells and tamp onto lint free towel. Add 350 μL of deionized water to each well. Dump the deionized water and tamp plate again lint free towel. Repeat the wash step two more times. h) Without allowing the wells to dry out, add 100 μL of K-Blue Substrate to each well. i) Incubate at room temperature for 30 minutes, tapping sides of the plate 3 to 4 times throughout incubation. j) Add 100 μL of Stop Solution to each well to stop enzyme reaction. Mix gently before measuring absorbance. k) Measure the absorbance at a wavelength of 450 nm. Wells should be read within 2 hours of stopping reaction. Methamphetamine/ MDMA Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 60 2. Dilute sample two-fold using EIA Buffer solution provided with Neogen Methamphetamine/ MDMA Detection Kit. 3. Use the instructions included with the Neogen Methamphetamine/ MDMA Detection Kit with some alterations as shown in test procedure steps a through l listed below: a) Determine the number of wells to be used. b) Gently mix the ready to use conjugate solution by inversion. Do not vortex. c) Add 10 μL of sample, Neogen calibrators to the appropriate wells in triplicate. d) Add 100 μL of ready to use drug-enzyme conjugate to each well. Use 8-channel pipetter for rapid addition. e) Mix by gently shaking plate. f) Cover plate with plate cover and incubate at room temperature for 45 minutes. g) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. h) Once incubation is complete, dump the liquid from the wells. Tap the plate on a clean lint-free towel to remove any remaining liquid in the wells. i) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After 61 completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. j) Add 100 μL of the K-Blue Substrate to each well. k) Incubate at room temperature for 30 minutes. l) Add 100 μL of Acid Stop (1N H2SO4) to each well to stop enzyme reaction. Mix gently before measuring absorbance. Measure the absorbance at a wavelength of 450 nm. Wells should be read within 2 hours of stopping reaction. Nandrolone Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 2. Perform C-18 Concentration to obtain a 25:1 concentrated ratio, re-suspended in provided EIA Buffer. 3. Use the instructions included with the Neogen Nandrolone Detection Kit with some alterations as shown in test procedure steps a through n listed below: a) Allow at least 60 minutes for all test kit components and samples to reach room temperature (20-23ºC). b) Determine the number of wells to be used. c) Prepare the enzyme conjugate by diluting the 180X enzyme conjugate stock 1 to 180 in the EIA Buffer provided. Mix the solution by inversion. d) Add 20 μL of sample and standards into wells in triplicate. e) Add 180 μL of diluted enzyme conjugate to each well. f) Mix gently by shaking plate. 62 g) Cover plate with plate cover and incubate at room temperature for 60 minutes. h) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. i) Dump the solution from the wells and tap onto lint free towel. j) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. k) Add 150 μL of K-Blue Substrate to each well. l) Incubate at room temperature for 30 minutes, tapping sides of the plate three to four times throughout incubation. m) Add 50 μL of Neogen's Red Stop Solution to each well to stop enzyme reaction. Mix gently before measuring absorbance. n) Measure the absorbance at a wavelength of 630 nm. Wells should be read within 2 hours of stopping reaction. Oxycodone/ Oxymorphone Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 2. Perform C-18 Concentration to obtain a 25:1 concentrated ratio, re-suspended in provided EIA Buffer Solution provided. 63 3. Use the instructions included with the Neogen Oxycodone/ Oxymorphone Detection Kit with some alterations as shown in test procedure steps a through l listed below: a) Determine the number of wells to be used. b) Gently mix the ready to use conjugate solution by inversion. Do not vortex. c) Add 10 μL of sample, Neogen calibrators to the appropriate wells in triplicate. d) Add 100 μL of ready to use drug-enzyme conjugate to each well. Use 8-channel pipetter for rapid addition. e) Mix by gently shaking plate. f) Cover plate with plate cover and incubate at room temperature for 45 minutes. g) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. h) Once incubation is complete, dump the liquid from the wells. Tap the plate on a clean lint-free towel to remove any remaining liquid in the wells. i) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. j) Add 100 μL of the K-Blue Substrate to each well. 64 k) Incubate at room temperature for 30 minutes. l) Add 100 μL of Acid Stop (1N H2SO4) to each well to stop enzyme reaction. Mix gently before measuring absorbance. Measure the absorbance at a wavelength of 450 nm. Wells should be read within 2 hours of stopping reaction. THC Test Procedures 1. Filter sample with 1.2 μm glass fiber filter. 2. Perform C-18 Concentration to obtain a 25:1 concentrated ratio, re-suspended in provided EIA Buffer Solution provided. 3. Use the instructions included with the Neogen THC Detection Kit with some alterations as shown in test procedure steps a through l listed below: a) Determine the number of wells to be used. b) Gently mix the ready to use conjugate solution by inversion. Do not vortex. c) Add 10 μL of sample, Neogen calibrators to the appropriate wells in triplicate. d) Add 100 μL of ready to use drug-enzyme conjugate to each well. Use 8-channel pipetter for rapid addition. e) Mix by gently shaking plate. f) Cover plate with plate cover and incubate at room temperature for 45 minutes. 65 g) During conjugation period, dilute concentrated wash buffer ten-fold with deionized water. Mix thoroughly. h) Once incubation is complete, dump the liquid from the wells. Tap the plate on a clean lint-free towel to remove any remaining liquid in the wells. i) Wash each well with 300 μL of diluted wash buffer. Repeat for a total of three washings, invert and tap dry the plate following each step. After completing the last step wipe the bottom of the wells with a lint-free towel to remove any liquid on the outside of the wells. j) Add 100 μL of the K-Blue Substrate to each well. k) Incubate at room temperature for 30 minutes. l) Add 100 μL of Acid Stop (1N H2SO4) to each well to stop enzyme reaction. Mix gently before measuring absorbance. Measure the absorbance at a wavelength of 450 nm. Wells should be read within 2 hours of stopping reaction. REFERENCES 1. Rannazzisi, J. T.; Caverly, M. W. Practitioner's Manual: An Informational Outline of Controlled Substances Act. United States Department of Justice: Drug Enforcement Administration: Office of Diversion of Control: Springfield, VA, 2006. 2. Thomas, K. V. et al. Comparing Illicit Drug Use in 19 European Cities through Sewage Analysis. Science of the Total Environment. 2012, 432, 432-439. 3. Knight, C. A.; Knight, I.; Michel, D. C.; Zepp, J. E. Beverage Caffeine Intake in US Consumers and Subpopulations of Interest: Estimates from the Share of Intake Panel Survey. Food and Chemical Toxicology. 2004, 42, 1923-1930. 4. Birkett, D. J.; Miners, J. O. Caffeine Renal Clearance and Urine Caffeine Concentrations during Steady State Dosing. Implications for Monitoring Caffeine Intake during Sports Events. British Journal of Clinical Pharmacology. 1991, 31, 401-408. 5. Buerge, I. J.; Poiger, T.; Muller, M. D.; Buser, H. Caffeine, an Anthropogenic Marker for Wastewater Contamination of Surface Waters. Environmental Science Technology. 2003, 37, 691-700 6. WebMD, LCC. Find a Vitamin or Supplement: Caffeine. http://www.webmd.com/vitamins-supplements/ingredientmono-979-CAFFEINE.aspx?activeIngredientId=979&activeIngredientName=CAFFEINE (accessed Aug 21, 2012). 7. Rodriguez del Rey, Z.; Granek, E. F.; Buckley, B. A. Expression of HSP70 in Mytilus Californianus Following Exposure to Caffeine. Ecotoxicology. 2011, 20, 855-861. 8. International Program on Chemical Safety (INCHEM). Chemical Safety Information from Intergovernmental Organizations. http://www.inchem.org/ (accessed Dec 5, 2012). 67 9. TEVA Pharmaceuticals USA Inc. Oxycodone Hydrochloride. http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=2d044a8e-8fdb-4299-a3facd8761d9ad58 (accessed Nov 21, 2012). 10. Sui, Q.; Huang, J.; Deng, S.; Yu, G.; Fan, Q. Occurrence and Removal of Pharmaceuticals, Caffeine and DEET in Wastewater Treatment Plants of Beijing, China. Water Research. 2010, 44, 417-426. 11. WebMD, LCC. Mental Health Center: Cocaine Use and Its Effects. http://www.webmd.com/mental-health/cocaine-use-and-its-effects (accessed Aug 21, 2012). 12. Sangster, James. Octanol-Water Partition Coefficients of Simple Organic Compounds. Journal of Physical and Chemical Reference Data. 1989, 18, 1111-1227. 13. National Center for Biotechnology Information, U.S. National Library of Medicine (NCBI). The PubChem Project. http://pubchem.ncbi.nlm.nih.gov/ (accessed Sept 18, 2012). 14. University of California, San Francisco (UCSF). Lifecycle of Cotinine. http://www.teensmokingstudy.com/cotinine.html (accessed Aug 21, 2012) 15. Drugs.com: Drug Information Online. Nicotine Side Effects. http://www.drugs.com/sfx/nicotine-side-effects.html (accessed Nov 25, 2012). 16. Bones, J.; Thomas K. V.; Paull, B. Using Environmental Analytical Data to Estimate Levels of Community Consumption of Illicit Drugs and Abused Pharmaceuticals. Supplementary Material (ESI) for Journal of Environmental Monitoring. 2007, 7, 701-701. 17. Frontline, WGBH Educational Foundation. The Meth Epidemic: How Meth Destroys the Body. http://www.pbs.org/wgbh/pages/frontline/meth/body/ (accessed Aug 8, 2012). 18. National Institute on Drug Abuse: The Science of Drug Abuse & Addiction. DrugFacts: MDMA (Ecstasy). http://www.drugabuse.gov/publications/drugfacts/mdma-ecstasy (accessed July 7, 2012). 19. Drugs.com: Drug Information Online. Nandrolone Side Effects. http://www.drugs.com/sfx/nandrolone-side-effects.html (accessed Nov 25, 2012). 68 20. May, P. School of Chemistry, University of Bristol. Molecule of the Month-October 2000: Nandrolone. http://www.chm.bris.ac.uk/motm/nandrolone/nandh.htm (accessed Sept 18, 2012). 21. Lange, I. G.; Daxenberger, A.; Schiffer, B.; Writters, H.; Ibarreta, D.; Meyer, H. H. Sex Hormones Originating from Different Livestock Production Systems: Fate and Potential Disrupting Activity in the Environment. Analytica Chimica Acta, 2002, 473, 27-37. 22. Drugs.com: Drug Information Online. Oxycodone Side Effects. http://www.drugs.com/sfx/oxycodone-side-effects.html (accessed Nov 25, 2012) 23. National Institute on Drug Abuse: The Science of Drug Abuse & Addiction. DrugFacts: Marijuana. http://www.drugabuse.gov/publications/drugfacts/marijuana (accessed July 7, 2012). 24. Swartz, M. E. Ultra Performance Liquid Chromatography (UPLC): An Introduction. Separation Science Redefined [Online] 2005, p 8-14. Chromatography Online. http://www.chromatographyonline.com/lcgc/data/articlestandard/lcgc/242005/164646/article.pdf (accessed Nov 21, 2012). 25. Verstraete, A. G. Detection Times of Drugs of Abuse in Blood, Urine, and Oral Fluid. The Drug Monit. 2004, 26, 200-2005. 26. Utah Drug Control Update: 2010; Office of National Drug Control Policy Programs. Salt Lake City, UT, 2010. 27. Division of Substance Abuse and Mental Health 2009 Annual Report; Department of Human Services; Department of Human Services: Salt Lake City Utah, 2009. 28. Castiglioni, S.; Zuccato, E.; Fanelli R. Illicit Drugs in the Environment; Occurrence, Analysis and Fate Using Mass Spectrometry; John Wiley & Sons, Inc. New Jersey, pp 251-274. 29. Walters Jr. K. R.; Rupassara, S. I.; Marketz, R. C.; Leakey, A. D.; Muir, W. M.; Pittendrigh, B. R. Methamphetamine Causes Anorexia in Drosophila Melanogaster, Exhausting Metabolic Reserves and Contributing to Mortality. The Journal of Toxicological Science. 2012, 37, 773-790. 30. Library 4 Science: Chromatography-Online.org. Liquid Chromatography. http://www.chromatographyonline.org/4/contents.html (accessed Dec 14, 2012). 69 31. Doucette, W. J. Octanol/water Partition Coefficient. Presented at Introduction to Environmental Organic Chemistry, Utah State University, Logan, UT, November 2012. 32. Dyke, T. M.; Sams, R.A. Detection and Determination of Theobromine and Caffeine in Urine after Administration of Chocolate-Coated Peanuts to Horses. Journal of Analytical Toxicology. 1998¸ 22, 112-116. 33. ChemGuide. High Performance Liquid Chromatography-HPLC. http://www.chemguide.co.uk/analysis/chromatography/hplc.html (accessed Dec 14, 2012). 34. U.S. Department of Justice: Drug Enforcement Administration; Office of Diversion Control. Controlled Substances Schedules. http://www.deadiversion.usdoj.gov/schedules/index.html#define (accessed Dec 03, 2012). 35. Kasprzyk-Hordern, B.; Dinsdale, R. M.; Guwy, A. J. The Removal of Pharmaceuticals, Personal Care Products, Endocrine Disruptors and Illicit Drugs during Wastewater Treatment and its Impact on the Quality of Receiving Waters. Water Research. 2009, 43, 363-380. 36. Zuccato, E.; Castiglioni, S. Illicit Drugs in the Environment. Philosophical Transactions of the Royal Society A. 2009, 367, 3965-3978. 37. Valcarcel, Y. et al. Drugs of Abuse in Surface and Tap Waters of the Tagus River Basin: Heterogeneous Photo-Fenton Process is Effective in their Degradation. Environment International. 2012, 41, 35-43. 38. Jurado, A.; Mastroianni, N.; Vazquez-Sune, E.; Carrera, J.; Tubau, I.; Pujades, E.; Postigo, C.; Lopez de Alda, M.; Barcelo, D. Drugs of Abuse in Urban Groundwater. A case study: Barcelona. Sciences of the Total Environment. 2012, 424, 280-288. 39. Utah Department of Human Services. Substance Abuse and Mental Health. http://www.dsamh.utah.gov/substanceabusetreatment.htm (accessed Nov 25, 2012). 40. Zuccato, E.; Chiabranod, C.; Castiglioni, S.; Bagnati, R.; Fanelli, R. Estimating Community Drug Abuse by Wastewater Analysis. Environmental Health Perspective. 2008, 116.8, 1027-1032. 70 41. Chiaia A. C.; Banta-Green, C.; Field, J. Eliminating Solid Phase Extraction with Large Volume LC/MS/MS: Analysis of Illicit and Legal Drugs and Human Urine Indicators in US Wastewaters. Environmental Science Technology. 2008, 42(23), 8841-8848. 42. Boleda R. M.; Galceran M. T.; Ventura F. Trace Determination of Cannabinoids and Opiates in Wastewater and Surface Waters by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry. Journal of Chromatography A. 2007, 1175, 38-48. 43. Central Valley Water Reclamation Facility: A Natural Solution. http://www.cvwrf.org/brochure/page2.php (accessed June 28, 2012). 44. Utah Water Science Center. U.S. Geological Survey, West Valley, UT. Personal communication, 2012. 45. Neogen Corporation. Neogen Toxicology: ELISA Assay Principal. http://www.neogen.com/Toxicology/Assay_Principle.html (accessed May 5, 2012). 46. University of California, Davis (UC-Davis). Instrumental Analysis: Chromatography. http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography (accessed Dec 14, 2012). 47. University of California, Los Angeles (UCLA). Gas Chromatography. http://www.chem.ucla.edu/~bacher/General/30BL/gc/theory.html (accessed Nov 1, 2012). 48. Conroy, O. Estrogenic and Anti-estrogenic Activity Present in Wastewater Effluent and Reclaimed Water. Ph.D. Dissertation, University of Arizona, Tucson, AZ, 2006.