UPTAKE AND ACCUMULATION OF PHARMACEUTICALS IN SURFACE AND OVERHEAD IRRIGATED LETTUCE  By  Gemini D. Bhalsod              A THESIS  Submitted to Michigan State University in partial fulfillment of the requirements for the degree of  Crop and Soil Sciences  Master of Science  2016  ABSTRACT  UPTAKE AND ACCUMULATION OF PHARMACEUTICALS IN SURFACE AND OVERHEAD IRRIGATED LETTUCE  By  Gemini D. Bhalsod   Crop irrigation with reclaimed water is becoming increasingly popular due to water shortages exacerbated by climate change and variability. Pharmaceuticals in reclaimed water may pose unintended food safety risks when they accumulate in crops. Understanding the uptake pathways and accumulation levels of pharmaceuticals in crops under typical irrigation practices is critical for accurate risk assessment of crop irrigation using reclaimed water. The objectives of this study were to investigate the uptake of pharmaceuticals by greenhouse-grown lettuce irrigated with pharmaceutical-contaminated water via overhead or surface irrigation. Eleven commonly used pharmaceuticals, including a fever reducer and pain reliever (acetaminophen), a stimulant (caffeine), an anticonvulsant (carbamazepine), and 8 antibiotics (sulfadiazine, sulfamethoxazole, carbadox, trimethoprim, lincomycin, oxytetracycline, monensin sodium, and tylosin) were selected. Lettuce plants were grown for 5 weeks in nursery pots in a greenhouse, and pharmaceutical concentrations in roots, shoots, soil, and irrigation water were analyzed to infer major uptake pathways. Results showed that pharmaceuticals with low lipophilicity, low molecular weight, and high water solubility (acetaminophen, caffeine, carbamazepine, sulfadiazine, sulfamethoxazole, and carbadox) had no significant difference in shoot concentration overtime between irrigation methods. Conversely, those pharmaceuticals with high lipophilicity, high molecular weight, and lower water solubility (monensin sodium and tylosin) showed a higher concentration in shoots of overhead-irrigated compared to surface-irrigated plants. iii ACKNOWLEDGEMENTS   I want to thank my major advisor, Dr. Wei Zhang for giving me guidance while working on this project. His time, commitment, and encouragement were greatly appreciated. In addition, I want to thank the other members of my committee, Dr. Hui Li and Dr. Elliot Ryser, for their valuable input and assistance. I also want to thank the Department of Plant, Soil and Microbial Sciences and the Horticultural Teaching Greenhouse for providing the support and space to complete this research. Thanks to the great administrative staff for helping me through my whole program. I want to acknowledge the immeasurable help and support of Sangho Jeon and Ya-Hui Chuang throughout all stages of this research. Additionally, I would like to thank all the members of our research group, past and present, for their feedback and guidance: Drs. Stephen Boyd, Wenjun Gui, Yingjie Zhang, Brett Sallach, Yuanbo Li along with Cheng-Hua Liu, Feng Gao, Shuai Zhang, Zeyou Chen, and all the visiting scholars and other students I have worked with throughout my time at MSU.  Thanks to my immediate family for their patience and my partner, Isaac Miller, for his never-ending praise and love. Additionally, I would like to thank my friends in Lansing who learned with me outside of the laboratory: Heather Kingsbury, Aida Amroussia, Noah Saperstein, Dee Jordan, Dr. Karen Chou, Pengchao Hao, Sowmya Surapur, and Carolyn Schulte.  iv TABLE OF CONTENTS   LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES  ......................................................................................................... vii KEY TO ABBREVIATIONS ............................................................................................ ix CHAPTER ONE: LITERATURE REVIEW .......................................................................1 INTRODUCTION ...............................................................................................................1 SOURCES OF PHARMACEUTICALS IN THE ENVIRONMENT .................................2 Livestock ..............................................................................................................................2 Human ..................................................................................................................................3 Aquaculture ..........................................................................................................................5 Antibiotic Use on Crops.......................................................................................................6 ENVIRONMENTAL DETECTION AND OCCURRENCE OF  PHARMACEUTICALS ......................................................................................................8 Soils and Sediments .............................................................................................................8 Surface Waters and Groundwater ........................................................................................8 POTENTIAL EXPOSURE AND RISKS TO HUMAN HEALTH ..................................10 Crop Uptake .......................................................................................................................10  CHAPTER TWO: UPTAKE AND ACCUMULATION OF PHARMACEUTICALS  IN SURFACE AND OVERHEAD IRRIGATED LETTUCE ..........................................13 INTRODUCTION .............................................................................................................13 MATERIALS AND METHODS .......................................................................................15 Experimental Design ..........................................................................................................15 Irrigation System Design ...................................................................................................16 Chemicals and Materials ....................................................................................................17 Soil and Lettuce Information .............................................................................................18 Irrigation Water Preparation ..............................................................................................19 Irrigation Schedule .............................................................................................................20 Water Sample Collection and Extraction ...........................................................................20 Plant and Soil Sample Preparation .....................................................................................21 Plant and Soil Extraction Method ......................................................................................22 LC-MS/MS Analysis .........................................................................................................23 Pharmaceutical Concentration and Mass Calculations ......................................................23 Statistical Analysis .............................................................................................................25 RESULTS AND DISCUSSION ........................................................................................25 Irrigation Water Analysis and Lettuce Biomass ................................................................25 Pharmaceutical Concentrations in Shoot Wash Water ......................................................26 Pharmaceutical Concentrations in Lettuce Shoot ..............................................................27 Pharmaceutical Concentrations in Lettuce Root ................................................................29 v Pharmaceutical Concentrations in Soil ..............................................................................30 Pharmaceutical Root Concentration Factors and Translocation Factors ...........................31 Pharmaceutical Mass Balance ............................................................................................33 IMPLICATIONS ...............................................................................................................35 APPENDIX ........................................................................................................................37  CHAPTER THREE: CONCLUSIONS AND FUTURE RECOMMENDATIONS .........77 CONCLUSIONS................................................................................................................77 FUTURE RECOMMENDATIONS ..................................................................................78  LITERATURE CITED ......................................................................................................79  vi LIST OF TABLES   Table 1. Physicochemical properties of pharmaceuticals used in this study ....................38  Table 2. Soil properties for Michigan loamy sand used in this study ...............................39   Table 3. Irrigation schedule and water volume in the 50-µg/L Trial ................................40  Table 4. Irrigation schedule and water volume in the 30-µg/L Trial ................................41  Table 5. The effect of Na2EDTA concentrations on the pharmaceutical extractions  from water ..........................................................................................................................42  Table 6. Pharmaceutical recovery in spiked and pharmaceutical-free soil extracted  with 150 mg/L Na2EDTA ..................................................................................................43  Table 7. Precursor ions, product ions, and mass spectrometer parameters used  in qualification and quantification of pharmaceuticals ......................................................44  Table 8. Pharmaceutical concentrations for overhead and surface irrigated plants  in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water) ..........45  Table 9. Pharmaceutical concentrations for overhead and surface irrigated plants  in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in irrigation water) ..........51  Table 10. Root concentration factors and translocation factors for pharmaceuticals  in the 30-µg/L trial .............................................................................................................56  Table 11. Mass balance for pharmaceuticals in the 50-µg/L trial .....................................58  Table 12. Mass balance for pharmaceuticals in the 30-µg/L trial .....................................60  vii LIST OF FIGURES   Figure 1. Schematic of automated irrigation system (Pump = P, Pressure Gauge = G,  and Valve = V) ...................................................................................................................62  Figure 2. Pharmaceutical concentrations in irrigation water over time in the 50 µg/L  Trial ....................................................................................................................................63  Figure 3. Pharmaceutical concentrations in irrigation water over time in the 30 µg/L  Trial ....................................................................................................................................64  Figure 4. Images of lettuce in the 50-µg/L and 30-µg/L trials at week 3 .........................65  Figure 5. Fresh and dry shoot biomass for Trial 1 and Trial 2 (nominal  pharmaceutical concentrations of 50 and 30 µg/L in irrigation water, respectively) ........66  Figure 6. Holm-Sidak two-tailed unpaired t-test showing significant difference  (p < 0.05) between plant biomass between Trial 1 and Tria 2 (Trial 1 = nominal  pharmaceutical concentration of 50 µg/L in irrigation water, Trial 2 = nominal  pharmaceutical concentration of 30 µg/L in irrigation water) ...........................................67  Figure 7. Pharmaceutical concentrations in shoot wash waters under overhead  and surface irrigation in Trial 1 (nominal pharmaceutical concentration of 50 µg/L  in irrigation water) .............................................................................................................68  Figure 8. Pharmaceutical concentrations in lettuce shoots for overhead and  surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L  in irrigation water) .............................................................................................................69  Figure 9. Pharmaceutical concentrations in lettuce shoots for overhead and surface  irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in  irrigation water) .................................................................................................................70  Figure 10. Pharmaceutical concentrations in lettuce roots for overhead and surface  irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in  irrigation water) .................................................................................................................71  Figure 11. Pharmaceutical concentrations in lettuce roots for overhead and surface  irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in  irrigation water) .................................................................................................................72  Figure 12. Pharmaceutical concentrations in soil for overhead and surface irrigated  plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in  irrigation water) .................................................................................................................73viii Figure 13. Pharmaceutical concentrations in soil for overhead and surface irrigated  plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in  irrigation water) .................................................................................................................74  Figure 14. Total percent of each pharmaceutical recovered for the 50 µg/L Trial ...........75  Figure 15. Total percent of each pharmaceutical recovered for the 30 µg/L Trial ...........76   ix KEY TO ABBREVIATIONS   CECs: chemicals of emerging concern WWTPs: wastewater treatment plants CAFOs: concentrated animal feeding operations IPM: integrated pest management FDA: Food and Drug Administration DI: deionized MeOH: methanol Na2SO4: anhydrous sodium sulfate PSA: primary-secondary amine Na2EDTA: disodium ethylenediaminetetraacetate NaCl: sodium chloride HLB: hydrophilic-lipophilic balance QuEChERS: quick, easy, cheap, effective, rugged, and safe  1  CHAPTER ONE: LITERATURE REVIEW INTRODUCTION Pharmaceuticals have been recognized as emerging contaminants or chemicals of emerging concern (CECs) (EPA, 2015), because of their widespread use, constant discharge into the environment, and possible risks to human and ecosystem health. Pharmaceuticals include antibiotics and other human and animal medicines for disease treatment or prevention. Antibiotics are an important class of pharmaceuticals, and are used in the treatment and prevention of bacterial infections by either killing bacteria or inhibiting their growth (Sarmah et al., 2006). They can occur naturally in the environment by soil microorganisms and fungi, or be introduced by anthropogenic activities. They are being released into the environment via two major pathways, human and animal waste. Since animals do not metabolize antibiotics fully, the remainder can end up on agricultural land where manure has been applied as fertilizer (Hirsch et al., 1999). They can also be introduced to the environment via land application of biosolids or effluents from wastewater treatment plants (WWTPs) (Yi et al., 2011; Estévez et al., 2012; Clarke and Porter, 2010).    2   SOURCES OF PHARMACEUTICALS IN THE ENVIRONMENT  Livestock. Antibiotics play an important role in modern agriculture, specifically in concentrated animal feeding operations (CAFOs) where low doses of antibiotics are given to livestock to prevent disease and promote growth. In 2015, 89.8 million cattle were raised for meat and milk production, generating an estimated $44 billion in economic impact (Beef USA, 2016). In the same year, 66.9 million swine (USDA, 2015a) and over 8 million poultry (USDA, 2015b) were produced. High production volume and close animal quarters can promote the spread of disease, resulting in the pervasive use of antibiotics in livestock production.  Antibiotics and other pharmaceuticals are commonly administered to animals via subcutaneous injection, oral injection, feed or drinking water (Sarmah et al., 2006). The most common pharmaceuticals, such as tetracyclines, sulfonamides, and ionophores (FDA, 2015), are used to treat or prevent diseases and infections, promote growth, and manage reproduction. Unfortunately, their widespread use has led to their transfer from agricultural areas into the environment through runoff or land application of manure. In a 2013 Food and Drug Administration (FDA) Summary Report, antimicrobials were divided into two categories as they pertain to humans: medically important and non-medically important. The domestic sale and distribution of non-medically important antimicrobials for livestock are reported to increase by 14% from 2009 through 2013 in domestic sales (FDA, 2015). During this time, the sale and distribution of medically important antimicrobials also increased by 20%, from about 7.6 to 9.1 million kg. Medically important antibiotics such as tetracyclines and penicillins accounted for 71% and 9% of sales to the livestock industry,  3  respectively. While sale and distribution data are helpful for making predictions about antibiotic use, actual use is unknown. For example, veterinarians are able to prescribe drugs for uses other than those specified on the label. Some antibiotics are poorly absorbed by animals. As much as 30-90% can be excreted in feces or urine (Sarmah et al., 2006). Metabolites can also be excreted and can potentially still be bioactive. Kim et al. (2011) found that excretion rates in cattle varied with antibiotic type, with 75% excretion for sulfamethazine and 50-100% for tylosin. Excretion rates also depend on species and route of application (Kemper, 2008). For example, sheep can excrete up to 20% of oxytetracycline when administered orally and cattle can excrete 17-75% of chlortetracycline (Montforts et al., 1999; Jjemba, 2002). These differences can result in varying concentrations of antibiotics in waste material and consequently in the environment from livestock production.   Pharmaceuticals are usually highly concentrated in manure and less likely than biosolids to be degraded quickly since there is virtually no processing (Carvalho et al., 2014). Some pharmaceuticals, such as monensin sodium and tylosin, have been shown to degrade during storage, but others, such as sulfadiazine and difloxacin in pig manure, have shown no decrease in concentration after 150 days of storage (Carvalho et al., 2014). Since pharmaceutical degradation in biosolids and manure can be slow, application onto arable land results in pharmaceutical loading.  Human. Widespread human use of antibiotics to treat bacterial infections has led to their introduction into the environment via municipal WWTPs. Through excretion in waste and improper disposal of unused medications, bioactive antibiotics can enter wastewater influents (Ternes, 1998).   4   Through screening, agitation, aeration, and chlorination technologies, WWTPs effectively remove over 90% of suspended solids and 99% of harmful bacteria (EPA, 1998; USGS, 2015). Unfortunately, the standard treatment process is not designed to filter out or deactivate pharmaceuticals, resulting in contaminated effluents. These effluents are also often referred to as reclaimed water if intended for beneficial use. Contaminated effluents are then released into surface waters or used for irrigation directly (Yi et al., 2011; Estévez et al., 2012).   In a 2010 survey of a Wastewater Treatment Facility at East Lansing, MI, Gao et al. (2012) analyzed the influents and effluents for the presence of various antibiotics. They found sulfamethoxazole, a sulfonamide antibiotic, at a concentration of 1566 ng/L in the raw influent and 178 ng/L in the final effluent. They also found that the average concentration of lincomycin, a lincosamide antibiotic, was 58 ng/L in the raw influent and 35 ng/L in the final effluent. In another study, Gros et al. (2010) analyzed effluents from seven WWTPs along the Ebro river in Northeastern Spain; they detected pharmaceuticals in 100% of their samples. Both studies highlight that pharmaceuticals are being emitted into water sources from WWTPs and that conventional methods of wastewater treatment are unable to remove all pharmaceuticals effectively.  Another example is provided by Yi et al. (2011), who describe how, driven by increasing population and water scarcity, reclaimed waste water use increased in many regions of China. China began using reclaimed water in the 1940s and since then has used it for everything from irrigation to industrial processes. As water scarcity becomes a global problem, reclaimed water may become a widely used source of water. These examples highlight that pharmaceuticals are increasingly contaminating water sources and reaching environments that are closely linked to food production.  5  Biosolids, or sewage sludge, is the solid portion of treated sewage from WWTPs (EPA, 2016). Biosolids are rich in nutrients and organic matter and are often contaminated with pharmaceuticals. It is common practice to recycle them and add them to agricultural lands as a fertilizer, which also unintentionally results in pharmaceutical loadings. Manure is used in a similar manner and can also be contaminated; both are used to improve or rebuild poor soils.   Aquaculture. Aquaculture, or fish farming, includes the breeding, rearing, and harvesting of plants and animals in all types of aquatic environments (NOAA, 2012). Aquaculture is practiced globally in both fresh and salt water systems. This type of farming produces vast amounts of commercial edible fish and eggs, bait fish, and ornamental fish. In the U.S., marine aquaculture primarily produces oysters, clams, mussels, shrimp, and salmon. On the other hand, freshwater aquaculture produces catfish, trout, tilapia, and bass. Both industries may overlap with natural environments, such as utilizing cages on the ocean floor or natural ponds, or take place inconstructed facilities, such as recirculating aquaculture systems. Demand for food and depletion of natural fish supplies has led to increasing popularity of global aquaculture and rapid growth of the industry (Cabello, 2006). Aquaculture practices include the use of large amounts of pharmaceuticals to manage disease and infection. Because of the proximity of natural fish environments and human-managed aquaculture, there is a significant risk of pharmaceuticals entering water sources. For example, in samples from shrimp aquaculture ponds in Vietnam, researchers detected high levels of trimethoprim (up to 2.03 mg/L in water and 734.61 mg/kg in mud), sulfamethoxazole (up to 5.57 mg/L in water and 820.49 mg/kg in mud), norfloxacin (up to 6.06 mg/L in water and 2615.96 mg/kg in mud) and oxolinic acid (up to 2.50 mg/L in water and 426.31 mg/kg in mud) and also found antibiotic residues in canals close to the ponds (Le and Munekage, 2004).  6  Fish are administered antibiotics through feed, the water, or injections (Cabello, 2006). Fish waste and unconsumed food that contain antibiotics can make contact with sediment and can potentially move into surrounding aquatic environments. Residual antibiotics can be consumed by non-targeted aquatic life, remain in sediments or water, and apply selective pressure to microorganisms.  Pharmaceuticals used in aquaculture contribute to environmental contamination. Since aquaculture production in the U.S. is relatively small compared to global aquaculture, antibiotics are thought to not presently play a large role in domestic environmental contamination, but data are scarce (Kümmerer, 2009).  Antibiotic Use on Crops. Antibiotics have been useful for controlling bacterial infections and diseases on crops and ornamental plants (Misra, 1986). Bacterial diseases of plants are less common than fungal or viral infections, and therefore, antibiotics are only utilized in well-defined circumstances (Vidaver, 2002). Historically, many antibiotics were commonly used to treat a variety of plant diseases. For example, penicillin was widely used to treat crown-gall bacterium and tetracycline was used against tomato canker (Misra, 1986). As of 2002, the two most common antibiotics used in crops are streptomycin and oxytetracycline. Antibiotics are most commonly utilized against Erwinia amylovora which causes fire blight in pear and apple, and Xanthomonas campestris, which causes bacterial spot on peach (Stockwell and Duffy, 2002). Since orchards are a large economic investment for growers, the potential loss resulting from pathogen infection warrants timed antibiotic sprays. Millions of kilograms of antibiotics are used in the U.S. annually, of which only about 0.1% are used in plant agriculture (Vidaver, 2002; Stockwell and Duffy, 2002). For example, in 2009, about 16,000 kg of antibiotics were used for orchards, which was only 0.12% of the total  7  antibiotics used in animal agriculture. Therefore, not much attention has been paid to antibiotic use on crops with respect to soil or water contamination although usage numbers are high. In another example, in 1999, 30% of total national pear orchard acres received about 2,700 kg of streptomycin and 40% of total pear orchard acres received about 5,400 kg of oxytetracycline as reported by the USDA (Vidaver, 2002). Apples received about 52,000 kg of streptomycin on about 20% of the total apple orchard acres or about 1,300 kg of oxytetracycline on 5% of apple orchard acres.  Bacterial resistance has occurred against antibiotics on crops; streptomycin resistant bacteria have been observed on tomatoes and peppers (Mirsa, 1986). Fire blight resistant to streptomycin resulted in limiting its use on orchards by incorporating more integrated pest management (IPM) strategies and adding new antibiotics (Stockwell and Duffy, 2002). One strategy of IPM used in orchards includes pruning and removing infected branches from orchards. Through effective management strategies, streptomycin resistance can be limited. The inclusion of IPM practices coupled with the relatively low percentage of antibiotics used on crops, means that there is relatively little attention paid to crop antibiotic use from an environmental perspective. Antibiotic residues have been detected on plant surfaces, but because the ones used specifically for crops are non-persistent and deactivated quickly by sunlight, residues on crops are not a large concern. Residue studies are also limited to streptomycin, and as of the 2000s, there have been no observations of toxicity to mammals or adverse effects to human health (Vidaver, 2002). Nonetheless, the persistence and spread of antibiotic resistant bacteria from plants remain unknown.   8  It is unlikely that new antibiotics will be added for use in plant agriculture because of their high costs, regulations, and health concerns, as well as the increase in biocontrol and genetically modified plants (Vidaver, 2002). ENVIRONMENTAL DETECTION AND OCCURRENCE OF PHARMACEUTICALS  Soils and Sediments. Application of manure, biosolids, and WWTP effluents has led to non-medically important and medically important antibiotics such as tetracyclines and sulphonamides, being detected in sediments and soils (Kümmerer, 2009). Sorption, leaching and degradation are three important processes that govern pharmaceutical movement in soil and water systems. These processes are driven by their chemical properties such as size and solubility (Sarmah et al., 2006). Many pharmaceuticals are persistent and have long half-lives in soils where they are bioavailable to bacteria and plants (Carvalho et al., 2014). Boxall et al. (2006) studied not only plant uptake, but also provided semi-quantitative evidence about antibiotic dissipation in soils. They showed that amoxicillin dissipated very quickly and florfenicol, enrofloxacin, and oxytetracycline could persist in the soil environment for over 6 months following application, highlighting the different behaviors among pharmaceuticals.  Surface Waters and Groundwater. Although low levels of pharmaceuticals are typically detected in surface and ground water, their constant addition to water systems makes them semi-persistent contaminants (Carvalho et al., 2014). Concentrations of pharmaceuticals in waters at or near discharge points from pharmaceutical manufacturers or hospitals are higher than overall environmental concentrations (Hirsch et al., 1999). For example, groundwater samples down from the Grindsted Landfill Site in Denmark, previously used for household and pharmaceutical production waste, contained a variety of sulfonamide antibiotics at  9  concentrations up to 5 mg/L (Holm et al., 1995). In another example, the ciprofloxacin concentration in effluent samples from a WWTP near Hyderabad, India that serves about 90 drug manufacturing plants was as high as 31 mg/L (Larsson et al., 2007). Discharge from WWTPs and run-off from fields are common ways for pharmaceuticals to enter surface waters. Run-off and leaching from livestock farms are the main pathways for veterinary pharmaceuticals to enter groundwater sources (Chiu and Westerhoff, 2010).  Calderón-Preciado et al. (2011) identified 40 emerging contaminants in surface waters from northeastern Spain. Concentrations were less than 5 naproxen and galaxoide were more frequently found than others. As expected, waterbodies in close proximity to WWTPs were impacted more heavily by treated wastewater effluents and had greater contaminant concentrations than those that were further removed.  Currently, pharmaceutical exposure through groundwater consumption is not considered a high risk to human health because of low concentrations and inconsistent detection, but more research is needed (Chiu and Westerhoff, 2010; Daughton, 2010). Chiu and Westerhoff (2010) examined 26 commonly used pharmaceuticals and personal care products in surface drinking water sources, a wastewater treatment plant, and groundwater in Arizona. Groundwater samples contained contaminants at 2 to 10 ng/L with only one single site detecting sulfamethoxazole and sucralose at 20 ng/L to 1 at concentrations ranging from 10 to 20 ng/L, and sucralose and oxybenzne at concentrations between 20 ng/L to 1 testosterone, progesterone, and ethinyl were not detected.    10  POTENTIAL EXPOSURE AND RISKS TO HUMAN HEALTH  Crop Uptake. In the future, as climate change progresses, some water-scarce areas will look towards new sources of water for irrigation (Pereira et al., 2002). Considering these pressures, an important question to ask is: how could using irrigation water or applying biosolids and manure contaminated with pharmaceuticals affect human and environmental health? It is clear that pharmaceuticals are being introduced into the environment and are detected in water sources, but the effects on human and environmental health are unclear. Studies show that antibiotics have been found in plants grown in soils treated with manure (Kang et al., 2013). Consumption of crops that have accumulated antibiotics is a potential pathway of human exposure. In uptake studies by Boxall et al. (2006) carrot roots and lettuce leaves took up florfenicol and trimethoprim. Interestingly, except for trimethoprim, a majority of the pharmaceuticals were associated with the outer layer of the carrot, giving evidence for the importance of food processing in the future. It has been suggested that for neutral chemicals, hydrophobicity is the most important property involved in uptake from soil to plants (Carter et al. 2014). Carter et al. (2014) grew radish and ryegrass in soil spiked with carbamazepine, diclofenac, fluoxetine, propranolol, sulfamethazine, and triclosan. Of the six chemicals, five were detected in plant tissues. Carbamazepine was taken up in the greatest ryegrass), likely due to its persistence in soils and high concentrations in soil pore water that resulted in its transport by mass flow.  Calderón-Preciado et al. (2013) evaluated the effect of relative humidity on foliar sorption of organic contaminants. The controlled experiment simulated overhead sprinkler irrigation of lettuce under 40 and 90% relative humidity. Lettuce leaves were sprayed with a  11  solution containing pharmaceuticals and other contaminants. Results showed that foliar sorption of compounds was similar at both relative humidities. Neutral and basic compounds predominated in the leaf compartment, while acidic compounds were partitioned between the leaf tissue and rinse water. They concluded that volatility and polarity play a large role in the final fate of the compound. This study shows that foliar sorption of contaminants through sprinkler irrigation is a viable and potentially important route of uptake into plants.  After reviewing risks of reclaimed water use for agricultural irrigation, Fatta-Kassinos et al. (2011) concluded that there are gaps in knowledge especially around risks to non-target organisms. Antibiotic availability to organisms can be reduced in the environment by biotic and abiotic factors such as degradation and adsorption (Kim et al., 2010). In the case of metals, stomatal accumulation has been described to play a role in plant uptake, which could also possibly apply to antibiotic uptake (Xiong et al., 2014). Calderón-Preciado et al. (2013) used models complemented by experimental samples, and concluded that human exposure to emerging contaminants through fruits and vegetables ranged from 1 to more than 461 ng per person per day. After reviewing the literature concerning pharmaceuticals in the environment, it is evident that there are wide gaps in knowledge around the environmental fate and mechanisms of uptake into plants. There is a wide diversity of plant uptake research techniques and subsequently a wide variation of pharmaceutical concentrations in plants. Results are dependent on factors such as plant species, soil type, growing conditions, and pharmaceutical concentrations in water, manure, biosolids, or soil. New human risks also emerge when considering crop irrigation methods, pharmaceutical residues in reclaimed water, and predicted fresh water scarcity. While pharmaceutical concentrations are low overall, the persistence of  12  pharmaceuticals, pervasive occurrence from non-point sources, and high concentrations at point-source areas means that more knowledge about the fate and uptake of pharmaceuticals is needed, and there is a growing concern for human and environmental health in the future.     13  CHAPTER TWO: UPTAKE AND ACCUMULATION OF PHARMACEUTICALS IN SURFACE AND OVERHEAD IRRIGATED LETTUCE INTRODUCTION Pharmaceuticals are considered chemicals of emerging concern because of their large use and frequent detection in the environment, and possible human health risks due to unintended exposure (EPA, 2015; Hirsch et al., 1999; Yi et al., 2011). Pharmaceuticals can enter the environment through many different pathways, particularly through use in animal agriculture and human medicine. Antibiotics are used for growth promotion and to fight infections, but the administered antibiotics often cannot be fully metabolized and utilized. For example, after administration of tetracycline and sulfamethoxazole in humans, 8090% and 1030% can be excreted and enter municipal WWTPs, and are eventually released to the environment through biosolids (i.e., sewage sludge) and wastewater effluents (Mathews and Reinhold, 2013). They can also be excreted by animals and end up in manure (Kim et al., 2011). Both biosolids and manure are often applied on agricultural land, resulting in contamination of soil and water.  14     15  Therefore, the objective of this study was to compare the accumulation of pharmaceuticals into lettuce irrigated with pharmaceutical-contaminated water via overhead or surface irrigation. We selected eleven commonly used pharmaceuticals, including a fever reducer and pain reliever (acetaminophen), a stimulant (caffeine), an anticonvulsant (carbamazepine), and 8 antibiotics (sulfadiazine, sulfamethoxazole, carbadox, trimethoprim, lincomycin, oxytetracycline, monensin sodium, and tylosin), because of their large use by humans and animals, and varying physicochemical properties such as molecular weight, water solubility, charge behaviors (pKa), and hydrophobicity (log KOW) (Table 1). Lettuce plants were greenhouse-grown for 5 weeks in nursery pots during which time pharmaceutical concentrations in roots, shoots, soil, and irrigation water were determined to infer major uptake pathways. MATERIALS AND METHODS  Experimental Design. This study was conducted at the Michigan State University Horticulture Teaching Greenhouses (East Lansing, MI, USA). Lettuce was seeded in sterile potting mix, watered with deionized (DI) water, fertilized, and transplanted to individual pots filled with air-dried and sifted field soil before each trial. Thirty-six lettuce plants were grown and irrigated with a constructed automatic overhead and surface irrigation system. Two trials were performed at two varying pharmaceutical concentration levels of 50 and 30 µg/L. Trial 1 was conducted from 4/20/15 to 5/25/15, and plants were irrigated with DI water spiked with 11 pharmaceuticals of approximately 50 µg/L for each pharmaceutical. Trial 2 was conducted from 9/17/15 to 10/22/15 and plants were irrigated with nutrient solution spiked with the same 11 pharmaceuticals of 30 µg/L for each pharmaceutical. The lettuce plants were harvested weekly, and then rinsed, separated into roots and shoots, and weighed. Shoot wash water was extracted and quantified for pharmaceuticals in the 50 µg/L trial, but not for the 30 µg/L trial, as minimal  16  pharmaceutical residues were found on the lettuce shoot surface. The soil in each pot was separated into top, middle, and bottom layers of 3-freezer (Northland, Greenville, MI, USA) before being dried, weighed, ground, extracted, and quantified by LC-MS/MS.   Irrigation System Design. An automatic irrigation system with overhead and surface irrigation emitters was designed and constructed by Sangho Jeon, and tested for accuracy and uniformity before the beginning of the experiments. Water amount was tested by measuring water amount at specific time intervals. The basic system design is shown in Figure 1. With this system, pump speed controlling water flow rate, and irrigation timing and duration were controlled via a control box. By connecting the control box to a laptop computer using an Arduino programming system (https://www.arduino.cc/), essential system functions could be controlled. A chipset microcontroller with a relay was used to control irrigation time. The system -240V, Portland, OR, USA). Main components of the system include a pump, pressure gauge, autovalve, controller, two water tanks with and without pharmaceuticals, distribution lines and emitters for overhead and surface irrigation. The pharmaceutical treatment also had an extra component: an accumulator tank to help evenly regulate irrigation and water amounts. The system accommodates 36 plants: 18 plants under overhead irrigation including 15 plants for pharmaceutical treatment and 3 plants for pharmaceutical-free treatment (i.e., control); 18 plants under surface irrigation including 15 plants for pharmaceutical treatment and 3 plants for pharmaceutical-free treatment. PC Woodpecker Dripper emitters (1.0 GPH, Netafim product # 01wpc4, Tel Aviv, Israel) were connected to anti-leak purple stoppers (28-13 psi, Dubois  17  Agrinovation product # IS 0340017-B, Quebec, Canada) to ensure that all the emitters stopped at the same time, before being connected to the main line.  The overhead emitters (3.2 GPH @60psi, NaanDan Jain product # NET-337, Jalgaon, India) were also connected to the anti-leak purple stopper. Irrigation duration was determined for drip emitters (y=1.16x+8.19, y=water amount (mL), x = time (s)) along with overhead emitters (y = 1.56x+7.07, y = water amount (mL), x = time (s)).   The pressure gauge (Super Pro® Pool Filter 0-60 Pressure Gauge 1/4" Fitting, Moorooka, Australia) helped regulate the SHURflo® Revolution Water Pump (Product # 4008-101-E65 3.0, Pentair, Costa Mesa, CA, USA), when the pressure reached about 50 psi, the pump automatically turns off as per an internal regulatory mechanism in the pump. Pump speed was controlled by a knob and each water tank was also attached to a strainer (Shurflo® (255-313) 1/2" Twist-On Pipe Strainer) to prevent debris from clogging the pump.   Chemicals and Materials. Eleven analytical standard pharmaceuticals: acetaminophen (101.3% purity), caffeine (100.5%), carbamazepine (100%), carbadox (100%), lincomycin (>90%), monensin sodium (90-95%), oxytetracycline (95%), sulfadiazine (99%), sulfamethoxazole (99.2%), trimethoprim (99%), and tylosin (analytical grade) were used (Sigma-Aldrich, St. Louis, MO, USA). Pharmaceuticals were chosen based on varying structures and physiochemical properties as shown in Table 1. Chemicals were dissolved in HPLC-grade methanol (MeOH) to prepare stock solutions at concentrations ranging from 10 to 1000 mg/L. Acetonitrile and anhydrous sodium sulfate (Na2SO4) were purchased from EMD Chemicals (Gibbstown, NJ, USA). Ceramic homogenizers, C18 and primary-secondary amine (PSA) were purchased from Agilent Technologies (Santa Clara, CA, USA). Disodium  18  ethylenediaminetetraacetate (Na2EDTA), formic acid, and sodium chloride (NaCl) were purchased from J.T. Baker (Phillipsburg, NJ, USA). All chemicals used were of analytical grade or better. Oasis® hydrophilic-lipophilic balance (HLB) 6cc (200mg) Extraction Cartridges were purchased from Waters Corporation (Milford, MA, USA).   Soil and Lettuce Information. The soil used was a Michigan loamy sand collected from Charlotte, MI. This soil represents a high water use scenario since sandy soils have lower water holding capacity and loamy sand is a common soil type in California, where much of U.S lettuce is produced. The soil was air-dried for 12 weeks and then sieved to 2 mm and stored in a greenhouse. Selected soil properties are provided in Table 2. Nursery pots of 14.6-cm diameter and 10.8-cm height were uniformly packed with the air-dried soil to 9-cm soil depth and approximately 1455 g, resulting in a bulk density of 1.35 g/cm3.  Concurrently, 4-6 Burpee® Black Seeded Simpson Lettuce (Burpee, Warminster, PA, USA) seeds were planted in sterile potting mix for approximately two weeks before the start of each trial. Burpee® Black Seeded Simpson Lettuce is a commonly grown lettuce type. Seedlings were watered with DI water and Peters Professional® water soluble 20-20-20 general purpose fertilizer (Scotts, Marysville, OH, USA) as needed. After one week, seedlings were thinned and transplanted into individual soil pots. Before transplanting, excess potting mix was separated from transplants and soil pots were saturated with DI water. Plants were left to acclimate for about 2 days before beginning each trial. Soil pots were randomly placed under each emitter.  Overhead irrigated plants had a transparent screen with holes for air flow placed around the pot to minimize water loss from overhead spray. Surface irrigated pots also had a screen in the 30 µg/L trial.   19   Irrigation Water Preparation. Pharmaceutical stock solutions for Trial 1 (i.e., the 50 µg/L trial) were prepared at 100 mg/L, with the exception of carbadox at 10 mg/L. The stock solutions for Trial 2 (i.e., the 30 µg/L trial) were prepared at 1000 mg/L, except for sulfadiazine at 100 mg/L and carbadox at 10 mg/L. These stock solutions were used to prepare pharmaceutical-spiked irrigation water with the exception of carbadox in Trial 1 and the standards for LC/MS-MS analyses. For Trial 1, carbadox was added to irrigation water from pharmaceutical powder. Irrigation water for the pharmaceutical water tank in Trial 1 (Figure 2) was prepared at a concentration of 50 µg/L by combining 20 mL of each stock solution (excluding carbadox), 2 mg powdered carbadox, and 39.8 L of DI water. It was assumed that there were no chemical reactions between chemicals in the water tanks. The methanol concentration in the final tank was 0.5%, which would not negatively affect plant growth (Li et al., 2010). Tanks were cleaned out and refilled on 5/3/15 and 5/21/15.  The irrigation water in the pharmaceutical water tank for Trial 2 (Figure 3) was prepared at a concentration of 30 µg/L by combining 1.2 mL of each stock solution (excluding sulfadiazine and carbadox), 12 mL of 100 mg/L sulfadiazine stock solution, 120 mL of 10 mg/L carbadox stock solution, and 39 L of DI water. The methanol concentration in the final tank was 0.357%. Fertilizer was added to the tank water to obtain a final nitrogen concentration of 125 mg/L (i.e., 25 g of 20-20-20 added). Tanks were cleaned out and refilled on 10/2/15 and 10/13/15. The non-pharmaceutical water tank (i.e., control water tank) contained DI water in Trial 1 and fertilizer solution with 125 mg/L nitrogen in Trial 2. The non-pharmaceutical treatment was used to examine if there was any phytotoxic effect of pharmaceuticals to the lettuce plants, and  20  to obtain matrix background matching water, plant, and soil samples for the LC/MS-MS analysis. The water in both tanks was used to irrigate plants until water level reached the tank outlet, after which the tanks were cleaned out and refilled twice before the end of experiments.   Irrigation Schedule. Plants in Trial 1 were irrigated with 25 to 100 mL of irrigation water each day, and plants in Trial 2 were irrigated with 25 to 125 mL irrigation water. Amounts of irrigation water were determined by using temperature, humidity, soil water content, and evaporation data. Temperature and humidity were measured in the greenhouse, soil water content and evaporation were determined using weight measurements. A more detailed irrigation schedule and irrigation amounts can be found in Tables 3 and 4. Water Sample Collection and Extraction. Water samples (20 mL) collected from both the pharmaceutical and control tanks were taken daily for Trial 1 and 23 times a week during Trial 2 and stored in amber glass vials with polyurethane caps. To extract pharmaceuticals from the water samples, 1 mL of 300 or 3000 mg/L Na2EDTA was added to each 20 mL sample and vortexed. Although different concentrations of Na2EDTA were used during the experiments, Na2EDTA concentration did not greatly affect the recovery of any chemical except for oxytetracycline as indicated by the high relative standard deviation. For the Na2EDTA and pharmaceutical-water extraction recovery test, 20 mL of both 50-µg/L and 30-µg/L pharmaceutical water was prepared, replicating the experimental irrigation water preparation procedure outlined above. Triplicate irrigation water samples were extracted with both Na2EDTA concentrations (300 and 3000 mg/L) using the same process as described below. Mass of each pharmaceutical was determined, and percent recovery and relative standard deviation were calculated. Results from this test can be found in Table 5.  21  HLB cartridges were used with a PrepSep 12-Port Vacuum Manifold (Fischer Scientific, Waltham, MA, USA). The cartridges were conditioned with 5 mL MeOH followed by elution with 5 mL DI water. After conditioning, the water samples were loaded by passing the water sample and Na2EDTA mixture through the cartridge. The extracted pharmaceuticals were then eluted with 5 mL of MeOH into clean amber vials that were stored in a 20°C freezer until analysis. Extraction procedure was followed from Chuang et al., 2015.  Plant and Soil Sample Preparation. Three overhead and surface irrigated lettuce plants were harvested weekly and control plants were harvested periodically throughout the trials. Shoots were cut at the base of each plant using scissors that were washed with DI water and MeOH between plants. The shoots were washed in 200 mL of DI water. The shoot wash water in Trial 1 was saved for later analysis. Because negligible pharmaceutical residues were washed off the shoots in Trial 1, the shoot wash water in Trial 2 was discarded. Lettuce shoots were dried with a paper towel, weighed, placed in a labeled beaker, and stored soil pots were overturned, and each soil core was cut into 3 vertical soil layers of 3-cm depth using a sterilized knife, referred to as top, middle, and bottom layers. Lettuce roots were separated from each soil layer by washing and removing excess soil. Keeping each soil layer separate, lettuce roots were collected, washed, towel-dried, weighed, and placed in a labeled beaker and held  homogenized, placed in a labeled beaker, and frozen at   After thorough freezing, plants were freeze-dried using a Vitris Sentry freeze mobile lyophilizer (Gardiner, New York, USA), weighed, ground using a Smartgrind electric grinder (Black & Decker, Middleton, WI, USA) or hand-operated mortar and pestle. Ground plant material was stored in foil packets in a freezer until extraction and analysis.  22   Plant and Soil Extraction Method. Plant extraction was performed following a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method (Chuang et al., 2015). Initially, 0.25 or 0.50 g of the freeze-dried and ground plant or soil sample was placed into 50-mL centrifuge tubes. After adding 2 mL of 150 mg/L Na2EDTA, the centrifuge tube was vortexed for 1 minute. Thereafter two ceramic homogenizers and 5 mL of acetronitrile/MeOH (65/35) were added to each tube and vortexed for 1-2 minutes. After adding 2 g Na2SO4 and 0.5 g NaCl packets, the tube was vortexed for 1.5 minutes, and centrifuged in a Sorvall® RC 6 Centrifuge for 10 minutes using the size SS-34 rotor at 5000 g, 5 accelerations, and 5 decelerations (Thermo Scientific, Waltham, MA, USA,). Thereafter, 1.3 mL of supernatant was transferred into 1.5 mL centrifuge tubes that were prefilled with 12.5 mg of C18, 12.5 mg of PSA and 225 mg of Na2SO4. The small centrifuge tubes were vortexed for 1 minute and then centrifuged in an Eppendorf 5415D centrifuge (Hauppague, NY, USA) for 5 minutes at 10,000 g. Finally, 0.9 mL of supernatant was transferred into a 1 mL amber LC vial containing 0.1 mL of MeOH and stored in the freezer until analysis. Using this procedure pharmaceutical recovery in plant samples ranged from about 72-96% as reported in Chuang et al. (2015). The extraction procedure for soil samples was similar to the procedure for plant samples after method development and recovery testing. A QuEChERS method was adapted for the soil extraction (Chuang et al., 2015). To compare the pharmaceutical recovery as influenced by Na2EDTA concentration, 150 mg/L of Na2EDTA and 300 mg/L of Na2EDTA were used. Four replicate soil samples (0.25 g each) for each Na2EDTA treatment were spiked with 0.2 mL of 500 µg/L stock solution (triplicate spiked soil samples were used for lincomycin and pharmaceutical-free soil for sulfamethoxazole because of outliers). The spiked and pharmaceutical-free soils were extracted using the QuEChERS method with either 150 or 300  23  mg/L of Na2EDTA with 150 mg/L Na2EDTA chosen based on recovery between 15% for oxytetracycline and 96% for sulfadiazine as provided in Table 6.   LC-MS/MS Analysis. The extracts were analyzed for pharmaceutical concentrations using a Shimadzu Prominence high performance liquid chromatograph (Colombia, MD, USA) coupled with an Applied Biosystems Sciex QTrap® 4500 triple quadrapole mass spectrometer (Foster City, CA, USA). An Agilent Eclipse Plus C18 Column (2.1 mm × 50 mm, particle size 5 µm) was used for separation. The mobile phase consisted of phase A (0.3% formic acid in DI water) and phase B, acetonitrile/methanol (1/1) with 0.3% formic acid. The flow rate was 0.35 mL/min and the sample injection volume was 10 µL. Pharmaceuticals were quantified using a matrix-based calibration curve. Precursor ions and product ions for qualification and quantification, along with mass spectrometer parameters can be found in Table 7.   Pharmaceutical Concentration and Mass Calculations. Pharmaceutical concentrations in the irrigation water and the amount of pharmaceutical applied to each plant were calculated as follows for all pharmaceuticals except carbadox and oxytetracycline. After LC-MS/MS analysis, the pharmaceutical concentrations in the MeOH extract was divided by 4 to obtain the concentrations in the irrigation water. This was done because during the extraction, the water sample was concentrated by a factor of four. Carbadox and oxytetracycline concentrations in the irrigation water were inaccurate because of the extraction method. For oxytetracycline, Na2EDTA concentration extraction generally underestimated oxytetracycline. For carbadox, using powder rather than stock solution in the 50-µg/L trial underestimated carbadox after extracting irrigation water. It is unclear why, but the 30-µg/L trial concentrations for carbadox were also inaccurate and much lower than the other pharmaceuticals. Therefore, for both chemicals, the nominal concentrations (i.e., 50 or 30 µg/L) were used. The amount of  24  pharmaceutical applied to each plant was calculated by multiplying the concentrations in the irrigation water by the amount of water applied to each plant on each day.   The pharmaceutical concentrations in the shoot wash water were calculated by dividing LC-MS/MS concentration by eight, because the sample was concentrated by a factor of eight during extraction.   To calculate the pharmaceutical concentrations in the shoots, the concentrations measured by LC-MS/MS were multiplied by 10/9 to obtain the concentrations in the MeOH extract, which were then converted to the concentrations in the dry shoots by multiplying by 0.005 L and then dividing by the dry weight of the shoot sample. The total mass of pharmaceuticals in the shoots was then calculated by multiplying the dry-weight concentrations by the dry weight of the total shoot. Fresh weight concentrations were calculated by dividing the total mass of the pharmaceutical by the total fresh weight of the shoot. Pharmaceutical concentrations in the root were calculated using the same procedure as described for the shoots.  To calculate dry-weight and fresh-weight concentrations and total mass of pharmaceuticals in the soil, the procedure as described above was followed. In Trial 1, the acetaminophen concentration in top soil from one replicate, caffeine concentration in middle soil from one replicate, and lincomycin concentration in bottom soil from one replicate were outliers and excluded from further analysis because their concentrations were at least 10 times greater than the average value. The concentrations from the other two replicates were therefore used. Background pharmaceutical concentration for caffeine, carbamazepine, trimethoprim, oxytetracycline, and tylosin, which was detected in pharmaceutical-free soil samples (Table 6), was subtracted from analyzed soil samples to get final soil pharmaceutical concentration.   25  Root concentration factors were calculated by dividing the concentration of each pharmaceutical in roots by the concentration in the soil for the same treatment pot at each harvest point. Translocation factors were calculated by dividing the concentration of each pharmaceutical in shoots by the concentration in the roots for the same plant. Each factor was averaged for each treatment for each week and standard deviations were also calculated.  Pharmaceutical mass balance was calculated cumulatively for each week. The amount of pharmaceutical applied was added together for each week. Next, the average mass of pharmaceuticals in the shoots, roots, and soils for each week were combined.  In Trial 2, the pharmaceutical concentrations in the irrigation water were measured every few days, and the average concentrations for the two neighboring samplings and measurements were used for days within that duration. Other calculations were the same as those in Trial 1, except no soil sample measurements were excluded. No samples were corrected for recovery for either trial.  Statistical Analysis. All statistical analyses were conducted using GraphPad PRISM 7. Plant biomass comparisons by trials was analyzed as grouped unpaired t-tests. Statistical significance was determined using the Holm-Sidak method, with alpha = 0.05. Each week was analyzed individually, without assuming a consistent standard deviation. Plant biomass comparisons of each irrigation treatment method within trials were analyzed in the same manner. RESULTS AND DISCUSSION  Irrigation Water Analysis and Lettuce Biomass. Pharmaceutical concentrations in irrigation water for the 50 and 30 µg/L trials are shown in Figures 2 and 3. While the nominal concentration of all spiked pharmaceuticals was either 50 or 30 µg/L in these two trials, the measured concentrations ranged from 31.06 µg/L to 83.69 µg/L in the 50 µg/L Trial and from  26  10.65 µg/L to 52.41 µg/L in the 30 µg/L Trial, respectively. The differences between the measured and nominal concentrations might be due to analytical errors or degradation (Chaung et al., 2015). The difference between the 50and 30-µg/L trial was the starting concentration for pharmaceuticals in irrigation water. Lettuce in the 50-µg/L trial was stunted and showed major necrosis during the experiment (Figure 4), even with periodic fertilizer application. It was determined that growth was limited due to stress because of being exposed to a high concentration of pharmaceutical over most of the growth cycle. For example, previous studies have shown that a decline in plant growth is possible with oxytetracycline exposure (Boxall et al., 2006; Jjemba, 2002). Therefore, for the next trial, plants were given fertilizer throughout the whole growing period and the initial concentration of pharmaceuticals was lowered. Fresh and dry shoot biomass in the 50 and 30-µg/L trials were measured after each week (Figure 5). A Holm-Sidak two-tailed unpaired t-test (p < 0.05) revealed that average dry shoot biomass in the 30-µg/L trial was greater than that harvested in the 50-µg/L trial (Figure 6), likely due to the change in pharmaceutical concentrations of irrigation water and fertilizer application. However, the fresh weight of shoots under overhead irrigation had no significant difference compared to surface irrigated shoots with the exception of week 2 in the 30-µg/L trial for which fresh weight under surface irrigation was greater than that under overhead irrigation (p = 0.006).    Pharmaceutical Concentrations in Shoot Wash Water. During harvest in both trials, shoots were washed to simulate consumer washing of lettuce before consumption. Shoot wash water was analyzed for the 50-µg/L trial, but not for the 30-µg/L trial due to low concentrations observed in the first trial (Figure 7); average concentrations for all pharmaceuticals were 2.5  27  µg/L or below for plants harvested in weeks 2-5. Pharmaceutical concentration data can be found in Table 8 for the 50-µg/L trial. Pharmaceutical concentrations in the shoot wash water ranged in 08.60 µg/L for overhead irrigation and 05.37 µg/L for surface irrigation. Greater concentrations of acetaminophen, caffeine, carbamazepine, sulfamethoxazole, trimethoprim, lincomyacin, monensin sodium, and tylosin were observed for 3 or more weeks on overhead irrigated shoots as compared to surface irrigated shoots (Figure 7). Surface-irrigated lettuce concentrations were low compared to overhead-irrigation concentrations because overhead-irrigated lettuce came into direct foliar contact with irrigation water. As water evaporated from lettuce leaves, pharmaceutical residues remaining on lettuce that were removed during washing. These results indicate the importance of consumer processing techniques when handling crops irrigated with reclaimed water. Although concentrations were low in both treatments, improper washing could lead to unintended exposure to some pharmaceuticals. Oxytetracycline, sulfadiazine, and carbadox concentrations were similar in the shoot wash water regardless of irrigation treatment for 2 or more weeks, differences between pharmaceutical concentrations that were observed were inconclusive because of overlapping standard deviation.    Pharmaceutical Concentrations in Lettuce Shoot. Results for each pharmaceutical concentration in shoots from the 50 and 30-µg/L trials are shown in Figures 8 and 9. For pharmaceuticals of lower lipophilicity, lower molecular weight, and higher water solubility including acetaminophen, caffeine, carbamazepine, sulfadiazine, sulfamethoxazole, carbadox, and oxytetracycline, conclusive difference in their concentrations in the shoot between overhead irrigation and surface irrigation was observed overtime (Figures 8 and 9). Pharmaceutical  28  concentration data can be found in Tables 8 and 9 for the 50-µg/L and 30-µg/L trial, respectively. However, for pharmaceuticals of high lipophilicity, high molecular weight, and lower water solubility including monensin sodium and tylosin, their shoot concentrations were consistently greater for overhead irrigation as compared to surface irrigation (Figures 8 and 9). Previous studies have also shown that tylosin cannot be taken up by corn, cabbage, and onion, possibly due to its large molecular size (Kang et al., 2013). Therefore, it is likely that the high concentration observed in our experiment was due to overhead irrigation. There were exceptions for trimethoprim and lincomycin which have a relatively larger molecular weight, higher water solubility, and lower log KOW (Figures 8 and 9). In this case, the trimethoprim concentration in overhead irrigated shoots was greater than that for surface irrigation (Figures 8 and 9), and the lincomycin concentration in overhead irrigated shoots was greater than that for surface irrigation only in the 30 µg/L trial (Figure 9). Irrigation water pH was approximately 7 for both trials. Due to their pKa values (7.12 and 7.6 respectively), trimethoprim and lincomycin are mostly in neutral and cation form and are likely to either diffuse into the waxy leaf cuticle (Calderón-Preciado et al., 2013) or bind with negatively charged leaf surface under overhead irrigation. It is unclear why this trend did not occur for lincomycin in the 50 µg/L trial, but it is suspected to be due to low biomass and poor plant growth.  29    Pharmaceutical Concentrations in Lettuce Root. Results for each pharmaceutical concentration in roots for the 50 and 30-µg/L trials are shown in Figures 10 and 11. Generally, there was no statistically significant difference in pharmaceutical concentration in the roots using the two irrigation treatments based on standard deviation indicating that irrigation method does not play a large role in root accumulation of pharmaceuticals in lettuce. In the 50 µg/L trial, the root concentrations of acetaminophen, carbamazepine, trimethoprim, and tylosin appeared to be greater for overhead as compared to surface irrigated lettuce. However, no such differences were observed in the 30-µg/L trial. It was speculated that the difference observed between trials was  30  likely due to differences in growing conditions. Also, due to the low root biomass in the 50-µg/L trial, all of the roots collected from the three pots were combined. Consequently, no statistical significance can be inferred. Pharmaceutical concentration data can be found in Tables 8 and 9 for the 50-µg/L and 30-µg/L trial, respectively. In surface-irrigated plants, chemicals are taken up via soil pore water (Boxall et al., 2006). Root uptake of organic chemicals is related to the Kow of the chemical and uptake into roots is greater for hydrophobic compounds, whereas polar compounds are accumulated less. Root concentration trends were observed in the 30 µg/L trial overtime regardless of irrigation method (Figure 11). There was an increase in root concentration overtime for carbamazepine, sulfadiazine, carbadox, trimethoprim, lincomycin, and oxytetracycline, indicating that these pharmaceuticals are able to be taken up by lettuce roots. In a study using alfalfa, oxytetracycline uptake by roots was concluded as being both an active and passive process, treated alfalfa had a liner increase in oxytetracycline uptake over 4 hours (Kong et al. 2007). There was a decrease in root concentration overtime for acetaminophen. This result is supported by a hydroponic study by Bartha et al. (2010), which found higher concentrations of acetaminophen roots than shoots after 24 hours, however a steep decrease in both tissues concentrations was seen after one week. Caffeine, sulfamethoxazole, monensin sodium, and tylosin had relatively stable root concentrations overtime.  Pharmaceutical Concentrations in Soil. Results for each pharmaceutical concentration in the soil layers for the 50 and 30-µg/L trials are shown in Figures 12 and 13. All pharmaceutical concentrations in soil were similar regardless of irrigation method in either trial, which was expected since soil was not covered for overhead-irrigated lettuce (i.e., simulating field irrigation conditions). Thus, soil was exposed to pharmaceutical irrigation water in both  31  treatments. Pharmaceutical concentration data can be found in Tables 8 and 9 for the 50-µg/L and 30-µg/L trial, respectively. Caffeine, sulfadiazine, sulfamethoxazole, lincomycin, oxytetracycline and monensin sodium showed no patterns in concentrations between the different layers. Sulfadiazine has been shown to dissipate in soil. When Boxall et al. (2006) used a loamy sand soil spiked with pharmaceuticals, 50% of the sulfadiazine was dissipated in the soil in less than 103 days, and 90% by 103 days. Wang et al. (2012) found that cation exchange was the primary mechanism for lincomycin sorption into soil. Since it competes with potassium and calcium for cation exchange sites, having these ions in solution decreases lincomycin sorption. There is also less sorption of lincomycin in soil that have a lower cation exchange capacity. Thus, lincomycin was able to leach downward and was evenly distributed. Acetaminophen concentration was lower in soil compared to the other pharmaceuticals in the 50-µg/L trial and was not detected in the 30-µg/L trial. Because of its low logKow (0.46), partitioning into soil organic matter is predicted to be minor. Therefore, it was likely that acetaminophen was degraded in soil. Carbamazepine, trimethoprim, and tylosin concentrations increased in the top layer of soil overtime in the 50-µg/L trial (Figure 12), in addition to carbadox in the 30- µg/L trial (Figure 13), suggesting strong sorption to soils. It is unclear why a different trend was observed for carbadox between trials. Carbamazepine has been reported as being persistent in soil, with dissipation reported over 40 days (Carter et al. 2014). Pharmaceutical Root Concentration Factors and Translocation Factors. Pharmaceutical root concentration factors and translocation factors can be found in Table 10 for the 30-µg/L trial. Factors were not calculated for the 50-µg/L trial since samples had to be  32  combined before extractions because of low biomass and because plants were unhealthy compared to 30-µg/L trial plants. Root concentration factors were not available when pharmaceuticals were not detected in soils as seen with acetaminophen over the whole growing period (weeks 1-5) and trimethoprim for weeks 1-3. Root concentration factors for all pharmaceuticals was similar between treatments for most weeks. This was expected since all plants received the same mass of pharmaceuticals each week regardless of treatment and because soil was not covered for overhead-irrigated lettuce, allowing for pharmaceutical contaminated water to reach soils and be taken up through lettuce roots. Translocation factors below 1 generally indicate that pharmaceuticals are not readily transported from roots to shoots in plants (Eggen et al., 2012). Translocation factors above 1 generally indicate that pharmaceuticals are readily transported from roots to shoots and can accumulate in shoots (Eggen et al., 2012). Pharmaceuticals that did not show a difference in concentration between irrigation methods had similar translocation factors for both overhead-irrigated and surface-irrigated lettuce overtime (acetaminophen, caffeine, carbamazepine, sulfadiazine, sulfamethoxazole, carbadox, and oxytetracycline), meaning irrigation method does not play a large role in pharmaceutical accumulation overtime. Carbamazepine had a consistently high translocation factor (>1) for both treatments indicating that it can be easily transported from lettuce roots to shoots and can also accumulate in plants, supporting previous conclusions on pharmaceutical concentration and trends overtime. Trimethoprim and lincomycin showed differences in translocation factors between treatments for weeks 1 and 2, with overhead-irrigated lettuce having higher concentration factors than surface-irrigated. While surface-irrigated lettuce translocation factors clearly indicate a transfer of pharmaceuticals from roots to shoots, overhead-irrigated lettuce translocation factors  33  do not because of direct foliar contact between pharmaceutical contaminated water and lettuce shoots. Differences between the two translocation factors can indicate that surface-irrigated plants have a lower transference from roots to shoots than overhead-irrigated because of the direct contact. Both pharmaceuticals had higher concentrations in overhead-irrigated lettuce than surface-irrigated lettuce. Because their translocation factors started out as different and converged overtime, we can conclude that irrigation method can play a difference in pharmaceutical concentration, but because both pharmaceuticals are also more easily transported from roots to shoots because of water solubility and molecular weight, the differences between irrigation methods can diminish overtime.  Monensin sodium and tylosin both had much higher translocation factors in overhead-irrigated lettuce than surface-irrigated. This difference indicates that these pharmaceuticals are not readily transferred from roots to shoots in lettuce, but that overhead irrigation can result in higher transference to shoots because of foliar contact. These results support the previous conclusions that overhead-irrigated lettuce will have higher concentrations of these pharmaceuticals than surface-irrigated.  Pharmaceutical Mass Balance. Pharmaceutical mass balance information can be found in Tables 11 and 12 for the 50-µg/L and 30-µg/L trials, respectively. Acetaminophen, caffeine, carbamazepine, and oxytetracycline all had a higher mass in shoots compared to roots for both treatments. Trimethoprim, monensin sodium, and tylosin had a higher mass in overhead-irrigated shoots than roots for both trials, but no difference was seen between roots and shoots in surface-irrigated samples. Sulfamethoxazole and trimethoprim also had a higher mass in overhead-irrigated shoots than roots, but only for the 50-µg/L trial and lincomycin had the same trend  34  except in the 30-µg/L trial, indicating that pharmaceuticals can transfer to the edible portion of lettuce and could lead to human exposure through consumption.  Monensin sodium and tylosin had higher mass in overhead compared to surface-irrigated shoots for all weeks during both trials. Sulfamethoxazole, lincomycin, and oxytetracycline showed the same trend, but just in one trial; the 50-µg/L trial for sulfamethoxazole and 30-µg/L for the other two pharmaceuticals, indicating that irrigation method can play a role in final pharmaceutical levels in lettuce shoots.  Carbamazepine, monensin sodium, and tylosin had increasing mass in shoots overtime in both trials: carbamazepine in both irrigation treatments, and monensin sodium and tylosin in the overhead irrigated lettuce. Carbadox showed the same trend in the 30-µg/L trial, indicating that shoots can accumulate pharmaceuticals overtime, and harvest time could play a role in final levels of pharmaceuticals in lettuce. Sulfadiazine had very low mass in roots and shoots for both trials and sulfamethoxazole in the 30-µg/L trial. Oxytetracycline had a low amount in roots for the 50-µg/L trial, whereas, carbamazepine increased in roots overtime in the 30-µg/L trial. All pharmaceuticals had a higher mass in soil compared to shoots or roots in both trials for a majority of weeks with the exception of acetaminophen and oxytetracycline in the 30-µg/L trial, indicating that soil sorption can be significant and pharmaceuticals can be persistent in soils (Boxall et al., 2006). The time needed to dissipate the oxytetracycline level by 50% in soil was less than 103 days and by 90% was greater than 152 days (Boxall et al., 2006). Our finding is in agreement with other studies showing that oxytetracycline sorbs strongly to soils (Christian et al., 2003). Tetracyclines can form complexes with cations, like calcium, in soil and partition into soil organic matter. The oxytetracycline level in soil was likely low because of the low recovery  35  and soil recovery method (Table 6.), so final amounts reported may be underestimated. Acetaminophen was not detected in soils in the 30-µg/L trial. Trimethoprim, tylosin and carbamazepine had an increased mass in soil overtime in the 50-µg/L trial and carbamazepine also exhibited the same trend in the 30-µg/L trial indicating that these pharmaceuticals can sorb to soils. Tylosin has been reported to dissipate in soils by 50and 90% in less than 103 and 103 days respectively (Boxall et al., 2006). Pharmaceutical recovery percentages for both trials can be found in Figures 14 and 15. Carbamazepine had the highest recovery in both trials (over 40%) among all weeks. All other pharmaceuticals had low overall recovery, possibly due to plant metabolism, bacterial metabolism, photodegradation, or irreversible soil sorption. Taking acetaminophen as an example, Bartha et al. (2010) found that uptake and concentration of acetaminophen increased in the first 72 hours, but decreased thereafter. The highest concentrations of acetaminophen were found in roots and leaves after 24 hours. Due to the decrease after week 1 of acetaminophen treatment, the authors suggest the presence of both a plant independent pathway for acetaminophen sorption or degradation and a plant dependent acetaminophen metabolism pathway. Their LC-MS/MS analysis also detected two acetaminophen metabolites in plant tissues, indicating that metabolism could play a large role in low recovery of acetaminophen as seen in both trials. Other pharmaceuticals, such as caffeine, have been shown to degrade through photolysis in laboratory experiments (Bruton et al., 2011) IMPLICATIONS Our findings may have interesting implications on utilizing reclaimed water to irrigate vegetable crops. Despite the wide use of overhead sprinkler systems, their use in vegetable production should be discouraged when using reclaimed water for irrigation due to greater  36  concentration and mass of some pharmaceuticals (specifically monensin sodium and tylosin) in overhead as opposed to surface-irrigated lettuce shoots. Our study showed the ubiquitous accumulation of pharmaceuticals in lettuce from irrigation water, demonstrating the need for further assessing the environmental and food safety risks associated with using pharmaceutical-contaminated water for irrigation.     37            APPENDIX     38  Table 1. Physicochemical properties of pharmaceuticals used in this study (Chuang et al., 2015).  Pharmaceutical Molecular Weight (g/mol) Chemical Structure Water Solubility (mg/L) pKa logKow acetaminophen 151.16  14000 9.38 0.46 caffeine 194.19  21600 10.4 -0.07 carbamazepine 236.27  18 2.3, 13.9 2.45 sulfadiazine 250.28  77 2.01, 6.99 -0.09 sulfamethoxazole 253.25  610 1.6, 5.7 0.89 carbadox 262.22  1755 1.8, 10.5 -1.22 trimethoprim 290.32  400 7.12 0.91 lincomycin 406.54  927 7.6 0.2 oxytetracycline 460.43  313 3.57, 7.49, 9.44 -0.9 monensin sodium 692.87  Slightly Soluble 4.3 5.43 tylosin 916.10  5 7.73 3.27  39  Table 2. Soil properties for Michigan loamy sand used in this study.  Sand 81.3% Silt 10.5% Clay 8.2% Soil pH 7.4 Phosphorus 71 mg/kg Potassium 50 mg/kg Magnesium 126 mg/kg Calcium 1298 mg/kg Cation Exchange Capacity 7.0 meq/100 g Organic Matter 2.5%     40  Table 3. Irrigation schedule and water volume in the 50-µg/L Trial.  Date Irrigation Amounts (mL) Notes 04/20/15 50  04/21/15 25  04/22/15 50  04/23/15 50  04/24/15 75  04/25/15 75  04/26/15 75  04/27/15 75 Plants harvested this date received 25 mL of irrigation water before harvest. 04/28/15 75  04/29/15 75  04/30/15 75  05/01/15 75  05/02/15 75  05/03/15 75 New pharmaceutical tank water (50 µg/L) 05/04/15 75 Plants harvested this date received 25 mL of irrigation water before harvest 05/05/15 50  05/06/15 50  05/07/15 75  05/08/15 75  05/09/15 75  05/10/15 75  05/11/15 75 Plants harvested this date received 25 mL of irrigation water before harvest 05/12/15 75  05/13/15 75  05/14/15 75  05/15/15 75  05/16/15 75  05/17/15 75  05/18/15 75 Plants harvested this date received 25 mL of irrigation water before harvest 05/19/15 75  05/20/15 75  05/21/15 100 New pharmaceutical tank water (50 µg/L) 05/22/15 100  05/23/15 100  05/24/15 100  05/25/15 25 Plants harvested this date received 25 mL of irrigation water before harvest 04/20/15 50   41   Table 4. Irrigation schedule and water volume in the 30-µg/L Trial.  Date Irrigation Amounts (mL) Notes 09/17/15 25  09/18/15 50  09/19/15 75  09/20/15 75  09/21/15 75  09/22/15 75  09/23/15 100  09/24/15 100 Plants harvested this date received 25 mL of irrigation water before harvest 09/25/15 125  09/26/15 125  09/27/15 125  09/28/15 100  09/29/15 100  09/30/15 100  10/01/15 100 Plants harvested this date received 25 mL of irrigation water before harvest 10/02/15 150 New pharmaceutical tank water (30 µg/L) 10/03/15 125  10/04/15 125  10/05/15 125  10/06/15 100  10/07/15 100  10/08/15 100 Plants harvested this date received 25 mL of irrigation water before harvest 10/09/15 100  10/10/15 100  10/11/15 100  10/12/15 125  10/13/15 150 New pharmaceutical tank water (30 µg/L) 10/14/15 125  10/15/15 125 Plants harvested this date received 25 mL of irrigation water before harvest 10/16/15 125  10/17/15 125  10/18/15 125 New pharmaceutical tank water (30 µg/L) 10/19/15 125  10/20/15 125  10/21/15 125  10/22/15 25 Plants harvested this date received 25 mL of irrigation water before harvest  42  Table 5. The effect of Na2EDTA concentrations on the pharmaceutical extractions from water.  Pharmaceutical Pharmaceutical water concentration (µg/L) Concentration of Na2EDTA (mg/L) Percent Recovery of Pharmaceutical (%) Relative Standard Deviation (%) Acetaminophen 50 3000 102.9 0.812 Acetaminophen 50 300 105.5 2.577 Acetaminophen 30 3000 118.8 1.582 Acetaminophen 30 300 113.8 4.159 Caffeine 50 3000 101.9 0.881 Caffeine 50 300 109.6 2.542 Caffeine 30 3000 121.4 5.166 Caffeine 30 300 121.3 2.717 Carbamazepine 50 3000 113.0 1.175 Carbamazepine 50 300 113.1 1.990 Carbamazepine 30 3000 143.1 2.813 Carbamazepine 30 300 135.5 1.227 Sulfadiazine 50 3000 105.1 1.209 Sulfadiazine 50 300 104.8 3.741 Sulfadiazine 30 3000 126.5 3.015 Sulfadiazine 30 300 117.9 2.887 Sulfamethoxazole 50 3000 107.1 1.124 Sulfamethoxazole 50 300 102.6 4.764 Sulfamethoxazole 30 3000 115.7 3.930 Sulfamethoxazole 30 300 107.0 3.077 Carbadox 50 3000 121.6 1.480 Carbadox 50 300 107.9 7.012 Carbadox 30 3000 130.6 5.021 Carbadox 30 300 120.6 4.048 Trimethoprim 50 3000 104.0 2.059 Trimethoprim 50 300 107.0 6.553 Trimethoprim 30 3000 122.7 4.275 Trimethoprim 30 300 117.2 5.649 Lincomycin 50 3000 110.2 0.930 Lincomycin 50 300 109.4 3.310 Lincomycin 30 3000 133.3 3.353 Lincomycin 30 300 131.1 1.459 Oxytetracycline 50 3000 81.1 17.746 Oxytetracycline 50 300 77.0 9.267 Oxytetracycline 30 3000 75.8 52.486 Oxytetracycline 30 300 82.8 6.013 Monensin Sodium 50 3000 88.4 2.421 Monensin Sodium 50 300 96.8 1.729 Monensin Sodium 30 3000 114.6 6.321 Monensin Sodium 30 300 110.2 5.692 Tylosin 50 3000 93.4 1.529 Tylosin 50 300 94.2 3.973 Tylosin 30 3000 101.4 5.223 Tylosin 30 300 94.0 3.045  43  Table 6. Pharmaceutical recovery in spiked and pharmaceutical-free soil extracted with 150 mg/L Na2EDTA.  Pharmaceutical Percent Recovery in spiked soil (%) Relative Standard Deviation in spiked soil (%) Average concentration in pharmaceutical-free soil samples (µg/L) Acetaminophen 84.7 3.981 N/A Caffeine 76.7 7.563 0.3762 Carbamazepine 86.3 4.630 0.0106 Sulfadiazine 85.3 9.419 N/A Sulfamethoxazole* 76.2 4.278 N/A Carbadox 92.7 3.750 N/A Trimethoprim 90.2 3.760 0.0387 Lincomycin** 83.7 4.483 N/A Oxytetracycline 16.1 3.704 0.2879 Monensin Sodium 77.7 2.348 N/A Tylosin 80.0 2.234 0.3025 * Triplicate used for pharmaceutical-free soil ** Triplicate used for spiked soil    44  Table 7. Precursor ions, product ions, and mass spectrometer parameters used in qualification and quantification of pharmaceuticals.  Pharmaceutical Precursor Ion (m/z) Product Ion (m/z) DP (V) EP (V) CE (V) CXP (V) Acetaminophen 151.931 110 60 10 20 8   93 60 10 30 6 Caffeine 194.975 138 60 10 30 10   110 60 10 30 6 Carbamazepine 236.961 193.7 80 10 30 10   192 80 10 30 12 Sulfadiazine 250.907 156 60 10 20 12   108 60 10 30 8 Sulfamethoxazole 253.958 155.9 60 10 20 8   107.9 60 10 30 6 Carbadox 262.92 230.9 60 10 20 12   144.8 60 10 30 8 Trimethoprim 291.044 261 80 10 30 12   230 100 10 30 12 Lincomycin 407.122 126 60 10 30 8   359.1 80 10 30 6 Oxytetracycline 460.982 426.1 60 10 30 8   283.1 60 10 50 8 Monensin Sodium 694.227 676.3 120 10 50 6   480 120 10 70 6 Tylosin 916.323 173.8 100 10 40 10   83 60 10 100 4 Curtain gas (psi) = 20 ionspray voltage (V) = 5000 ion source temperature = 700 ion source gas pressure = 60 & 90 Precursor ion is used for qualification and product ions are used for quantification    45  Table 8. Pharmaceutical concentrations for overhead and surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water).  Pharmaceutical Sample Type Harvest Week Average Overhead Concentration (µg/kg) Standard Deviation (µg/kg) Average Surface Concentration (µg/kg) Standard Deviation (µg/kg) Acetaminophen Shoot Wash 1 1.034 1.069 N/A N/A  Shoot Wash 2 0.750 0.475 N/A N/A  Shoot Wash 3 0.923 0.082 0.036 N/A  Shoot Wash 4 0.872 0.510 0.178 N/A  Shoot Wash 5 0.839 0.716 0.134 0.171  Shoot 1 99.803 N/A 111.049 N/A  Shoot 2 17.918 N/A 41.769 N/A  Shoot 3 16.877 8.361 19.152 6.506  Shoot 4 18.922 15.411 14.240 5.048  Shoot 5 29.428 16.488 18.624 8.355  Root 1 1.977 N/A 0.000 N/A  Root 2 49.383 N/A 7.580 N/A  Root 3 31.139 N/A 24.091 N/A  Root 4 39.377 N/A 13.614 N/A  Root 5 40.583 N/A 0.000 N/A  Soil - Top Layer 1 5.241 2.993 8.598 N/A  Soil - Top Layer 2 0.542 N/A 2.264 1.454  Soil - Top Layer 3 5.324 N/A 5.206 N/A  Soil - Top Layer 4 6.691 4.667 4.177 2.495  Soil - Top Layer 5 7.993 1.652 5.740 4.048  Soil - Middle Layer 1 3.118 2.441 1.023 0.097  Soil - Middle Layer 2 4.284 N/A 5.148 1.908  Soil - Middle Layer 3 3.752 2.202 1.340 1.044  Soil - Middle Layer 4 4.495 2.722 1.409 N/A  Soil - Middle Layer 5 3.410 1.331 1.392 N/A  Soil - Bottom Layer 1 5.123 N/A 2.470 N/A  Soil - Bottom Layer 2 4.875 1.542 2.463 2.874  Soil - Bottom Layer 3 6.107 3.422 4.215 5.309  Soil - Bottom Layer 4 2.976 2.942 1.238 0.984  Soil - Bottom Layer 5 0.407 N/A 3.664 1.535 Caffeine Shoot Wash 1 8.597 12.436 5.374 7.494  Shoot Wash 2 1.013 0.444 N/A N/A  Shoot Wash 3 1.354 0.248 0.026 N/A  Shoot Wash 4 1.310 0.342 N/A N/A  Shoot Wash 5 1.139 0.862 0.055 N/A  Shoot 1 104.606 N/A 57.346 N/A  Shoot 2 143.374 N/A 11.037 N/A  Shoot 3 65.443 46.750 121.271 56.514  Shoot 4 70.595 76.561 62.257 52.063  Shoot 5 74.691 43.123 58.465 71.643  Root 1 668.121 N/A 229.720 N/A  Root 2 27.862 N/A 100.341 N/A  Root 3 67.759 N/A 126.059 N/A  Root 4 178.860 N/A 41.603 N/A  Root 5 16.626 N/A 365.954 N/A  Soil - Top Layer 1 30.847 34.148 80.056 56.564  Soil - Top Layer 2 69.952 92.526 41.720 45.862  Soil - Top Layer 3 24.805 6.059 35.166 10.765  Soil - Top Layer 4 33.322 23.203 34.931 11.659  Soil - Top Layer 5 24.248 17.610 112.559 164.666  Soil - Middle Layer 1 1.559 1.680 14.487 14.538  Soil - Middle Layer 2 5.072 7.173 10.476 9.865  Soil - Middle Layer 3 28.274 5.925 25.020 23.235  Soil - Middle Layer 4 52.408 51.954 40.055 22.553  46  Table 8)  Soil - Middle Layer 5 18.114 4.153 27.655 6.389  Soil - Bottom Layer 1 14.259 12.442 7.390 9.143  Soil - Bottom Layer 2 8.710 11.814 12.743 7.288  Soil - Bottom Layer 3 71.045 102.470 31.398 19.887  Soil - Bottom Layer 4 13.364 10.031 23.439 1.240  Soil - Bottom Layer 5 19.038 15.104 2.415 2.634 Carbamazepine Shoot Wash 1 2.082 0.801 0.039 0.010  Shoot Wash 2 1.834 0.574 0.094 0.105  Shoot Wash 3 2.103 0.293 0.144 0.083  Shoot Wash 4 2.327 0.680 0.200 0.068  Shoot Wash 5 1.638 0.864 0.208 0.057  Shoot 1 71.273 N/A 28.495 N/A  Shoot 2 274.891 N/A 40.085 N/A  Shoot 3 140.603 108.492 179.412 168.385  Shoot 4 86.226 38.494 152.191 119.655  Shoot 5 152.127 32.282 159.028 43.062  Root 1 1068.001 N/A 26.065 N/A  Root 2 142.020 N/A 9.832 N/A  Root 3 54.251 N/A 7.101 N/A  Root 4 169.495 N/A 42.218 N/A  Root 5 20.492 N/A 26.896 N/A  Soil - Top Layer 1 8.783 5.315 104.912 127.703  Soil - Top Layer 2 67.585 62.281 34.945 15.217  Soil - Top Layer 3 53.952 18.728 65.609 23.308  Soil - Top Layer 4 86.441 23.076 109.056 21.822  Soil - Top Layer 5 136.472 25.845 132.407 7.640  Soil - Middle Layer 1 12.151 21.046 0.000 0.000  Soil - Middle Layer 2 9.234 7.563 11.105 12.480  Soil - Middle Layer 3 21.989 5.905 19.089 9.219  Soil - Middle Layer 4 30.689 16.272 17.141 9.018  Soil - Middle Layer 5 18.528 2.891 17.664 4.847  Soil - Bottom Layer 1 0.000 0.000 3.307 5.728  Soil - Bottom Layer 2 0.000 0.000 6.806 11.788  Soil - Bottom Layer 3 6.820 11.813 2.721 3.427  Soil - Bottom Layer 4 7.513 10.964 5.107 4.432  Soil - Bottom Layer 5 21.291 11.286 39.762 44.714 Sulfadiazine Shoot Wash 1 5.501 7.072 1.399 1.758  Shoot Wash 2 0.715 0.356 N/A N/A  Shoot Wash 3 0.813 0.085 N/A N/A  Shoot Wash 4 0.514 0.128 N/A N/A  Shoot Wash 5 0.179 0.222 0.123 N/A  Shoot 1 0.000 N/A 0.000 N/A  Shoot 2 0.000 N/A 0.000 N/A  Shoot 3 0.040 0.070 0.334 0.578  Shoot 4 0.317 0.496 0.000 0.000  Shoot 5 0.019 0.033 1.595 2.762  Root 1 0.000 N/A 1.355 N/A  Root 2 0.235 N/A 0.000 N/A  Root 3 0.964 N/A 0.383 N/A  Root 4 1.550 N/A 0.696 N/A  Root 5 0.595 N/A 0.768 N/A  Soil - Top Layer 1 0.396 0.686 2.120 1.934  Soil - Top Layer 2 1.929 3.082 0.258 0.299  Soil - Top Layer 3 1.548 2.046 0.683 0.594  Soil - Top Layer 4 5.058 0.501 3.001 0.727  Soil - Top Layer 5 1.275 0.774 3.249 3.445  Soil - Middle Layer 1 0.035 0.060 0.941 1.630  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.235 0.407 1.551 1.677  Soil - Middle Layer 4 1.936 2.861 10.115 15.927  Soil - Middle Layer 5 0.446 0.773 0.744 1.161  47  Table 8. ()  Soil - Bottom Layer 1 5.342 9.253 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.145 0.251  Soil - Bottom Layer 3 0.000 0.000 3.537 1.830  Soil - Bottom Layer 4 0.233 0.403 1.715 2.618  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Sulfamethoxazole Shoot Wash 1 1.195 0.326 0.099 0.054  Shoot Wash 2 0.805 0.381 N/A N/A  Shoot Wash 3 1.012 0.075 0.703 N/A  Shoot Wash 4 0.708 0.204 0.002 N/A  Shoot Wash 5 0.336 0.281 0.011 N/A  Shoot 1 34.561 N/A 3.350 N/A  Shoot 2 1.801 N/A 0.000 N/A  Shoot 3 37.461 64.884 30.214 52.332  Shoot 4 1.279 2.216 0.557 0.964  Shoot 5 5.118 8.864 0.000 0.000  Root 1 60.340 N/A 19.635 N/A  Root 2 12.151 N/A 6.225 N/A  Root 3 4.467 N/A 3.612 N/A  Root 4 36.767 N/A 1.295 N/A  Root 5 2.918 N/A 8.768 N/A  Soil - Top Layer 1 11.570 20.040 0.000 0.000  Soil - Top Layer 2 0.000 0.000 76.496 132.495  Soil - Top Layer 3 0.000 0.000 0.000 0.000  Soil - Top Layer 4 45.595 61.571 0.940 1.628  Soil - Top Layer 5 0.000 0.000 41.065 71.127  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 13.407 23.221  Soil - Middle Layer 4 2.780 4.815 18.635 32.277  Soil - Middle Layer 5 0.000 0.000 0.000 0.000  Soil - Bottom Layer 1 0.005 0.008 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 57.763 100.049 0.000 0.000  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 75.659 131.045 0.000 0.000 Carbadox Shoot Wash 1 0.605 N/A N/A N/A  Shoot Wash 2 0.083 N/A N/A N/A  Shoot Wash 3 N/A N/A N/A N/A  Shoot Wash 4 N/A N/A N/A N/A  Shoot Wash 5 N/A N/A N/A N/A  Shoot 1 11.505 N/A 7.708 N/A  Shoot 2 2.526 N/A 0.000 N/A  Shoot 3 1.339 1.760 10.891 11.213  Shoot 4 0.000 0.000 0.000 0.000  Shoot 5 0.000 0.000 0.000 0.000  Root 1 41.103 N/A 32.856 N/A  Root 2 3.716 N/A 4.717 N/A  Root 3 4.203 N/A 5.385 N/A  Root 4 5.047 N/A 0.000 N/A  Root 5 16.178 N/A 7.817 N/A  Soil - Top Layer 1 0.000 0.000 1.309 1.683  Soil - Top Layer 2 1.282 1.829 0.000 0.000  Soil - Top Layer 3 0.000 0.000 2.839 2.465  Soil - Top Layer 4 3.679 3.187 4.871 3.320  Soil - Top Layer 5 1.683 2.915 4.446 2.522  Soil - Middle Layer 1 0.000 0.000 0.092 0.160  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 0.000 0.000  Soil - Middle Layer 4 39.320 49.058 0.000 0.000  Soil - Middle Layer 5 1.113 1.928 0.000 0.000  Soil - Bottom Layer 1 3.200 5.542 0.000 0.000  48  Table 8)  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 2.869 4.969  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Trimethoprim Shoot Wash 1 2.422 2.828 1.126 N/A  Shoot Wash 2 0.740 0.206 N/A N/A  Shoot Wash 3 1.265 0.106 0.050 N/A  Shoot Wash 4 1.490 0.155 N/A N/A  Shoot Wash 5 1.240 0.690 N/A N/A  Shoot 1 88.132 N/A 1.416 N/A  Shoot 2 33.520 N/A 1.096 N/A  Shoot 3 60.262 27.379 10.469 10.997  Shoot 4 33.429 25.903 2.241 0.689  Shoot 5 30.915 13.131 1.302 0.761  Root 1 15.935 N/A 9.781 N/A  Root 2 18.662 N/A 6.984 N/A  Root 3 16.688 N/A 5.473 N/A  Root 4 24.421 N/A 5.446 N/A  Root 5 23.027 N/A 14.167 N/A  Soil - Top Layer 1 5.383 2.233 14.921 3.834  Soil - Top Layer 2 17.377 6.854 21.402 4.767  Soil - Top Layer 3 17.062 5.148 40.424 15.787  Soil - Top Layer 4 35.308 10.048 94.069 62.678  Soil - Top Layer 5 60.885 12.821 102.634 23.960  Soil - Middle Layer 1 0.725 0.825 0.000 0.000  Soil - Middle Layer 2 2.890 3.160 3.944 2.838  Soil - Middle Layer 3 3.250 2.780 10.641 6.479  Soil - Middle Layer 4 6.655 5.413 4.250 3.774  Soil - Middle Layer 5 55.636 90.641 5.785 4.942  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.889 1.540  Soil - Bottom Layer 3 0.981 1.700 0.631 0.979  Soil - Bottom Layer 4 0.836 1.448 4.680 6.633  Soil - Bottom Layer 5 4.233 5.617 8.660 10.968 Lincomycin Shoot Wash 1 0.224 0.152 0.147 N/A  Shoot Wash 2 0.278 0.291 N/A N/A  Shoot Wash 3 1.045 0.103 0.010 N/A  Shoot Wash 4 0.778 0.108 N/A N/A  Shoot Wash 5 0.739 0.483 N/A N/A  Shoot 1 24.739 N/A 61.012 N/A  Shoot 2 35.321 N/A 4.675 N/A  Shoot 3 171.134 206.070 324.612 252.785  Shoot 4 18.546 6.461 23.161 37.021  Shoot 5 12.298 3.880 5.916 9.027  Root 1 41.944 N/A 351.732 N/A  Root 2 532.451 N/A 1092.944 N/A  Root 3 15.839 N/A 107.499 N/A  Root 4 181.828 N/A 15.878 N/A  Root 5 14.728 N/A 13.729 N/A  Soil - Top Layer 1 25.435 24.585 24.902 15.922  Soil - Top Layer 2 41.480 21.230 11.917 2.871  Soil - Top Layer 3 10.298 3.430 14.558 4.800  Soil - Top Layer 4 22.144 7.289 184.537 296.013  Soil - Top Layer 5 5.865 2.456 12.461 4.214  Soil - Middle Layer 1 4.865 0.638 39.692 58.747  Soil - Middle Layer 2 10.142 5.321 6.730 2.057  Soil - Middle Layer 3 7.430 3.164 18.459 18.659  Soil - Middle Layer 4 43.944 68.189 7.147 1.455  Soil - Middle Layer 5 4.182 1.552 10.599 8.181  Soil - Bottom Layer 1 3.963 1.118 21.310 31.733  Soil - Bottom Layer 2 4.561 2.563 38.414 25.766  49  Table 8)  Soil - Bottom Layer 3 7.651 3.191 4.871 0.882  Soil - Bottom Layer 4 10.276 9.279 4.583 1.930  Soil - Bottom Layer 5 3.190 1.173 6.342 5.475 Oxytetracycline Shoot Wash 1 0.085 0.029 0.105 0.044  Shoot Wash 2 0.107 0.006 0.131 0.070  Shoot Wash 3 0.078 0.025 0.071 0.010  Shoot Wash 4 0.064 0.004 0.092 0.032  Shoot Wash 5 0.106 0.040 0.064 0.004  Shoot 1 1.520 N/A 0.808 N/A  Shoot 2 0.915 N/A 0.638 N/A  Shoot 3 2.849 3.314 2.447 2.660  Shoot 4 1.033 0.321 8.613 12.558  Shoot 5 1.464 0.402 7.001 8.097  Root 1 0.183 N/A 0.000 N/A  Root 2 0 N/A 0.000 N/A  Root 3 0.000 N/A 0.000 N/A  Root 4 0.000 N/A 0.000 N/A  Root 5 3.585 N/A 0.000 N/A  Soil - Top Layer 1 26.421 6.049 23.012 2.924  Soil - Top Layer 2 36.264 9.520 24.077 3.242  Soil - Top Layer 3 32.581 7.102 28.958 10.104  Soil - Top Layer 4 21.356 0.480 22.138 2.488  Soil - Top Layer 5 28.259 6.004 28.709 15.047  Soil - Middle Layer 1 27.692 6.537 22.314 4.377  Soil - Middle Layer 2 25.000 9.348 27.214 6.856  Soil - Middle Layer 3 24.707 9.413 36.273 19.607  Soil - Middle Layer 4 62.929 45.518 50.161 47.301  Soil - Middle Layer 5 22.920 1.918 28.941 4.851  Soil - Bottom Layer 1 22.498 3.329 27.358 7.278  Soil - Bottom Layer 2 22.684 1.751 26.140 8.510  Soil - Bottom Layer 3 25.014 8.333 25.275 3.220  Soil - Bottom Layer 4 20.464 1.922 32.615 3.002  Soil - Bottom Layer 5 32.368 15.804 23.846 5.586 Monensin Sodium Shoot Wash 1 1.100 0.147 0.041 0.016  Shoot Wash 2 1.224 0.165 0.016 0.020  Shoot Wash 3 1.807 0.195 0.116 N/A  Shoot Wash 4 1.460 0.328 0.022 0.016  Shoot Wash 5 1.169 0.916 0.062 0.028  Shoot 1 22.161 N/A 19.317 N/A  Shoot 2 23.451 N/A 0.029 N/A  Shoot 3 18.565 7.335 5.679 4.597  Shoot 4 25.106 12.817 1.134 0.210  Shoot 5 26.417 3.529 3.180 N/A  Root 1 5.120 N/A 9.634 N/A  Root 2 3.479 N/A 9.345 N/A  Root 3 7.698 N/A 4.164 N/A  Root 4 23.758 N/A 0.126 N/A  Root 5 2.331 N/A 1.614 N/A  Soil - Top Layer 1 3.134 1.427 15.216 4.560  Soil - Top Layer 2 0.000 0.000 1.328 1.875  Soil - Top Layer 3 0.000 0.000 11.832 12.294  Soil - Top Layer 4 13.499 14.523 5.659 6.875  Soil - Top Layer 5 1.889 2.488 5.729 2.801  Soil - Middle Layer 1 31.314 54.237 0.434 0.439  Soil - Middle Layer 2 0.000 0.000 5.562 9.633  Soil - Middle Layer 3 0.401 0.694 0.000 0.000  Soil - Middle Layer 4 15.987 25.889 7.585 12.129  Soil - Middle Layer 5 2.942 2.765 1.539 2.666  Soil - Bottom Layer 1 0.000 0.000 10.124 17.536  Soil - Bottom Layer 2 0.000 0.000 3.131 5.424  Soil - Bottom Layer 3 0.000 0.000 6.177 10.699  50  Table 8)  Soil - Bottom Layer 4 0.350 0.606 0.000 0.000  Soil - Bottom Layer 5 3.550 3.990 3.547 6.144 Tylosin Shoot Wash 1 0.334 0.060 N/A N/A  Shoot Wash 2 0.233 0.174 N/A N/A  Shoot Wash 3 1.231 0.048 0.030 0.006  Shoot Wash 4 0.611 0.262 0.012 N/A  Shoot Wash 5 0.401 0.165 0.095 N/A  Shoot 1 9.946 N/A 6.167 N/A  Shoot 2 12.141 N/A 0.000 N/A  Shoot 3 21.045 6.375 0.068 0.117  Shoot 4 14.337 8.577 0.174 0.124  Shoot 5 16.070 4.436 0.337 0.298  Root 1 5.340 N/A 2.076 N/A  Root 2 3.868 N/A 1.183 N/A  Root 3 5.627 N/A 0.523 N/A  Root 4 14.596 N/A 3.697 N/A  Root 5 7.465 N/A 4.499 N/A  Soil - Top Layer 1 0.000 0.000 6.638 2.837  Soil - Top Layer 2 9.585 8.647 10.313 3.119  Soil - Top Layer 3 6.792 0.255 15.704 15.171  Soil - Top Layer 4 25.035 10.490 32.765 12.831  Soil - Top Layer 5 34.353 20.286 47.330 1.716  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.000 0.000 0.565 0.978  Soil - Middle Layer 3 1.521 2.634 2.959 3.428  Soil - Middle Layer 4 1.827 3.165 0.000 0.000  Soil - Middle Layer 5 0.000 0.000 4.566 7.909  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.397 0.688 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000     51  Table 9. Pharmaceutical concentrations for overhead and surface irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in irrigation water).  Pharmaceutical Sample Type Harvest Week Average Overhead Concentration (µg/kg) Standard Deviation (µg/kg) Average Surface Concentration (µg/kg) Standard Deviation (µg/kg) Acetaminophen Shoot 1 15.219 6.000 8.964 0.276  Shoot 2 2.321 3.120 1.724 1.431  Shoot 3 3.878 1.617 25.062 38.892  Shoot 4 2.322 0.339 1.229 1.132  Shoot 5 7.345 2.216 5.386 2.804  Root 1 141.998 12.413 138.831 29.171  Root 2 60.514 1.429 35.778 6.878  Root 3 37.553 25.222 33.044 20.876  Root 4 24.987 17.303 28.122 18.506  Root 5 20.910 12.316 11.447 7.895  Soil - Top Layer 1 0.000 0.000 0.000 0.000  Soil - Top Layer 2 0.000 0.000 0.000 0.000  Soil - Top Layer 3 0.000 0.000 0.000 0.000  Soil - Top Layer 4 0.000 0.000 0.000 0.000  Soil - Top Layer 5 0.000 0.000 0.000 0.000  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 0.000 0.000  Soil - Middle Layer 4 0.000 0.000 0.000 0.000  Soil - Middle Layer 5 0.000 0.000 0.000 0.000  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Caffeine Shoot 1 12.096 7.090 1.368 0.760  Shoot 2 5.217 0.212 1.505 0.761  Shoot 3 3.522 1.445 4.029 0.979  Shoot 4 5.103 1.859 0.492 0.112  Shoot 5 11.577 6.222 2.921 1.503  Root 1 2.477 3.945 0.289 0.403  Root 2 2.293 2.782 1.633 0.134  Root 3 1.921 2.011 1.167 0.809  Root 4 0.224 0.388 0.132 0.229  Root 5 0.761 0.750 1.409 2.149  Soil - Top Layer 1 7.300 2.916 0.323 0.560  Soil - Top Layer 2 13.342 11.586 93.699 146.385  Soil - Top Layer 3 24.594 11.511 14.391 3.824  Soil - Top Layer 4 32.063 29.446 17.150 5.098  Soil - Top Layer 5 32.313 1.715 11.036 2.528  Soil - Middle Layer 1 0.000 0.000 2.357 4.082  Soil - Middle Layer 2 0.000 0.000 6.862 8.230  Soil - Middle Layer 3 0.000 0.000 11.239 10.743  Soil - Middle Layer 4 0.000 0.000 0.000 0.000  Soil - Middle Layer 5 3.311 5.735 0.000 0.000  Soil - Bottom Layer 1 26.369 45.673 2.748 4.760  Soil - Bottom Layer 2 1.888 3.271 33.028 57.206  Soil - Bottom Layer 3 0.000 0.000 2.800 2.492  Soil - Bottom Layer 4 14.756 25.558 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Carbamazepine Shoot 1 41.137 3.122 10.776 2.807  Shoot 2 86.377 7.478 52.265 21.247  Shoot 3 94.579 10.591 129.768 19.440  Shoot 4 166.533 32.160 224.616 38.441  52  Table 9)  Shoot 5 326.507 99.080 344.673 139.161  Root 1 5.341 2.386 2.082 0.374  Root 2 7.220 2.334 10.393 4.636  Root 3 10.683 3.198 14.505 2.214  Root 4 15.667 3.708 21.203 2.433  Root 5 36.961 5.758 31.289 3.062  Soil - Top Layer 1 17.659 5.688 12.137 3.537  Soil - Top Layer 2 24.434 4.004 26.869 3.306  Soil - Top Layer 3 39.180 5.601 42.427 7.508  Soil - Top Layer 4 59.613 21.045 61.619 4.898  Soil - Top Layer 5 74.184 1.768 56.051 14.513  Soil - Middle Layer 1 4.016 2.266 2.958 1.952  Soil - Middle Layer 2 9.979 4.607 20.303 27.605  Soil - Middle Layer 3 7.603 1.236 12.285 2.203  Soil - Middle Layer 4 17.252 5.937 12.983 6.113  Soil - Middle Layer 5 19.634 6.414 14.952 15.010  Soil - Bottom Layer 1 1.715 0.884 7.207 9.999  Soil - Bottom Layer 2 1.734 0.554 2.014 1.714  Soil - Bottom Layer 3 2.294 0.980 1.900 1.314  Soil - Bottom Layer 4 3.521 1.559 1.691 0.868  Soil - Bottom Layer 5 5.110 4.553 1.089 0.641 Sulfadiazine Shoot 1 0.119 0.058 0.035 0.061  Shoot 2 0.168 0.054 0.148 0.249  Shoot 3 0.077 0.074 0.026 0.045  Shoot 4 0.061 0.014 1.544 2.636  Shoot 5 0.300 0.300 0.047 0.042  Root 1 0.520 0.281 0.551 0.256  Root 2 0.775 0.269 0.672 0.133  Root 3 0.725 0.167 0.900 0.169  Root 4 0.699 0.244 0.801 0.034  Root 5 1.242 0.296 1.050 0.241  Soil - Top Layer 1 2.065 0.697 0.864 0.350  Soil - Top Layer 2 1.588 0.204 1.589 0.199  Soil - Top Layer 3 2.738 0.300 1.651 0.301  Soil - Top Layer 4 2.575 0.544 2.500 0.371  Soil - Top Layer 5 3.216 0.113 2.348 0.781  Soil - Middle Layer 1 0.392 0.369 1.576 2.253  Soil - Middle Layer 2 0.694 0.204 0.556 0.388  Soil - Middle Layer 3 0.581 0.169 0.674 0.233  Soil - Middle Layer 4 0.761 0.104 0.764 0.173  Soil - Middle Layer 5 0.850 0.088 0.595 0.539  Soil - Bottom Layer 1 0.381 0.154 0.237 0.252  Soil - Bottom Layer 2 0.184 0.142 0.269 0.243  Soil - Bottom Layer 3 0.252 0.063 0.175 0.153  Soil - Bottom Layer 4 0.858 1.049 0.252 0.116  Soil - Bottom Layer 5 0.464 0.198 0.093 0.081 Sulfamethoxazole Shoot 1 0.000 0.000 0.153 0.264  Shoot 2 0.000 0.000 0.000 0.000  Shoot 3 0.035 0.060 0.008 0.007  Shoot 4 0.019 0.033 0.000 0.000  Shoot 5 0.572 0.072 0.061 0.105  Root 1 1.272 1.228 0.607 0.091  Root 2 0.932 0.206 0.743 0.167  Root 3 4.684 5.518 1.294 0.199  Root 4 2.320 1.793 2.121 1.355  Root 5 1.790 0.413 1.728 0.522  Soil - Top Layer 1 0.000 0.000 0.000 0.000  Soil - Top Layer 2 0.000 0.000 0.000 0.000  Soil - Top Layer 3 41.968 72.691 2.801 4.851  Soil - Top Layer 4 0.000 0.000 2.158 3.738  Soil - Top Layer 5 2.387 4.135 6.781 11.746  53  Table 9)  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.027 0.047 0.000 0.000  Soil - Middle Layer 3 0.463 0.114 0.250 0.254  Soil - Middle Layer 4 0.620 0.442 0.555 0.119  Soil - Middle Layer 5 1.156 0.529 0.947 0.537  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 9.343 16.183  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.088 0.153 0.000 0.000  Soil - Bottom Layer 5 0.231 0.400 0.000 0.000 Carbadox Shoot 1 0.874 0.208 0.718 0.386  Shoot 2 1.334 0.208 0.576 0.275  Shoot 3 1.096 0.379 1.371 0.153  Shoot 4 2.346 0.980 2.381 0.716  Shoot 5 2.235 0.444 3.310 1.241  Root 1 1.803 0.863 0.468 0.434  Root 2 3.086 1.089 2.093 1.209  Root 3 5.053 1.852 3.028 1.048  Root 4 5.189 1.036 6.269 0.935  Root 5 7.100 3.738 7.940 2.141  Soil - Top Layer 1 1.198 0.207 0.403 0.217  Soil - Top Layer 2 2.330 3.107 0.536 0.430  Soil - Top Layer 3 9.169 6.694 10.262 4.167  Soil - Top Layer 4 18.541 17.229 14.414 2.767  Soil - Top Layer 5 10.173 8.069 9.700 6.739  Soil - Middle Layer 1 0.392 0.080 0.187 0.138  Soil - Middle Layer 2 0.034 0.059 1.371 1.219  Soil - Middle Layer 3 0.111 0.107 1.961 1.773  Soil - Middle Layer 4 0.579 0.513 1.063 1.301  Soil - Middle Layer 5 0.790 0.697 2.330 3.737  Soil - Bottom Layer 1 1.273 1.762 0.247 0.221  Soil - Bottom Layer 2 0.960 0.858 1.495 2.392  Soil - Bottom Layer 3 0.211 0.224 1.490 2.207  Soil - Bottom Layer 4 0.379 0.336 0.035 0.060  Soil - Bottom Layer 5 0.024 0.042 0.919 0.754 Trimethoprim Shoot 1 0.789 0.145 0.049 0.057  Shoot 2 0.420 0.221 0.010 0.005  Shoot 3 0.155 0.056 0.021 0.008  Shoot 4 0.168 0.126 0.010 0.001  Shoot 5 0.128 0.026 0.025 0.013  Root 1 1.276 0.953 0.320 0.338  Root 2 2.220 0.987 2.403 1.929  Root 3 3.163 0.309 6.283 1.124  Root 4 5.505 2.300 4.437 1.449  Root 5 8.589 3.145 7.467 2.142  Soil - Top Layer 1 0.000 0.000 0.000 0.000  Soil - Top Layer 2 0.000 0.000 0.000 0.000  Soil - Top Layer 3 0.253 0.437 3.297 5.711  Soil - Top Layer 4 31.990 36.166 8.791 1.825  Soil - Top Layer 5 24.791 3.969 4.670 4.088  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 0.000 0.000  Soil - Middle Layer 4 0.000 0.000 0.000 0.000  Soil - Middle Layer 5 0.000 0.000 0.000 0.000  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Lincomycin Shoot 1 2.311 0.347 0.866 0.209  54  Table 9)  Shoot 2 3.773 0.887 0.929 0.273  Shoot 3 3.031 0.312 1.668 0.489  Shoot 4 5.584 1.897 3.169 0.418  Shoot 5 5.800 1.439 3.867 1.138  Root 1 1.621 0.441 0.438 0.075  Root 2 3.009 0.207 2.613 1.147  Root 3 4.057 0.423 3.062 0.802  Root 4 4.158 0.980 3.267 0.097  Root 5 5.699 2.312 3.840 0.833  Soil - Top Layer 1 2.663 1.273 65.561 112.342  Soil - Top Layer 2 4.937 0.081 6.488 0.697  Soil - Top Layer 3 117.727 188.556 9.673 2.763  Soil - Top Layer 4 14.053 7.741 12.674 1.395  Soil - Top Layer 5 7.887 6.923 10.832 2.133  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.157 0.273 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 2.775 4.807  Soil - Middle Layer 4 2.081 1.961 1.301 1.175  Soil - Middle Layer 5 0.786 1.361 1.547 2.680  Soil - Bottom Layer 1 11.888 20.590 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 12.720 22.032  Soil - Bottom Layer 3 0.000 0.000 2.443 4.231  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 0.000 0.000 1.869 3.237 Oxytetracycline Shoot 1 1.071 0.075 0.698 0.112  Shoot 2 2.613 2.687 1.157 0.678  Shoot 3 1.223 0.084 0.978 0.108  Shoot 4 3.065 1.261 1.683 0.159  Shoot 5 3.341 0.635 5.807 5.070  Root 1 2.647 1.210 1.362 0.389  Root 2 1.611 0.013 1.900 0.980  Root 3 1.813 0.276 2.815 0.609  Root 4 2.579 0.170 2.928 0.610  Root 5 3.019 0.629 4.613 2.473  Soil - Top Layer 1 0.000 0.000 0.000 0.000  Soil - Top Layer 2 0.000 0.000 0.573 0.993  Soil - Top Layer 3 0.032 0.056 0.000 0.000  Soil - Top Layer 4 0.264 0.457 0.000 0.000  Soil - Top Layer 5 1.397 1.720 0.000 0.000  Soil - Middle Layer 1 0.000 0.000 0.000 0.000  Soil - Middle Layer 2 0.000 0.000 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 0.000 0.000  Soil - Middle Layer 4 0.000 0.000 0.000 0.000  Soil - Middle Layer 5 0.000 0.000 0.000 0.000  Soil - Bottom Layer 1 0.000 0.000 0.307 0.532  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.000 0.000 0.249 0.432  Soil - Bottom Layer 5 0.000 0.000 0.000 0.000 Monensin Sodium Shoot 1 6.327 1.397 0.000 0.000  Shoot 2 4.593 1.601 0.000 0.000  Shoot 3 5.020 1.394 0.000 0.000  Shoot 4 7.081 3.904 0.000 0.000  Shoot 5 9.137 1.453 0.731 1.153  Root 1 0.698 0.123 0.391 0.260  Root 2 1.416 1.511 0.710 0.444  Root 3 0.776 0.330 1.882 1.192  Root 4 0.228 0.198 0.552 0.325  Root 5 1.079 0.159 1.436 1.052  Soil - Top Layer 1 10.883 1.070 7.296 1.662  55  Table 9)  Soil - Top Layer 2 6.844 0.820 6.428 1.198  Soil - Top Layer 3 6.107 0.651 5.980 1.502  Soil - Top Layer 4 5.892 3.210 4.759 0.783  Soil - Top Layer 5 5.196 0.774 5.127 1.124  Soil - Middle Layer 1 4.495 0.521 4.196 0.268  Soil - Middle Layer 2 3.607 0.161 8.178 8.038  Soil - Middle Layer 3 3.111 0.084 4.388 0.985  Soil - Middle Layer 4 9.457 10.663 3.711 0.309  Soil - Middle Layer 5 4.315 1.642 9.715 10.635  Soil - Bottom Layer 1 3.938 0.457 3.325 0.219  Soil - Bottom Layer 2 4.406 2.305 3.511 1.027  Soil - Bottom Layer 3 2.923 0.120 4.209 2.406  Soil - Bottom Layer 4 3.038 0.251 2.911 0.098  Soil - Bottom Layer 5 3.696 0.696 3.556 0.620 Tylosin Shoot 1 1.328 0.133 0.027 0.026  Shoot 2 1.478 0.272 0.106 0.165  Shoot 3 1.272 0.339 0.111 0.060  Shoot 4 1.495 0.666 0.042 0.017  Shoot 5 1.434 0.119 0.121 0.084  Root 1 0.371 0.203 0.174 0.016  Root 2 0.478 0.143 0.732 0.330  Root 3 0.581 0.042 1.377 0.635  Root 4 0.589 0.311 0.512 0.112  Root 5 0.803 0.233 0.700 0.284  Soil - Top Layer 1 5.789 1.215 2.003 1.179  Soil - Top Layer 2 4.707 1.458 11.219 3.315  Soil - Top Layer 3 14.799 3.246 17.490 4.938  Soil - Top Layer 4 27.261 18.275 21.132 0.764  Soil - Top Layer 5 22.323 2.774 14.209 2.447  Soil - Middle Layer 1 0.003 0.006 0.000 0.000  Soil - Middle Layer 2 0.913 1.582 0.000 0.000  Soil - Middle Layer 3 0.000 0.000 0.787 1.364  Soil - Middle Layer 4 1.340 1.407 1.505 2.608  Soil - Middle Layer 5 0.435 0.754 3.201 5.545  Soil - Bottom Layer 1 0.000 0.000 0.000 0.000  Soil - Bottom Layer 2 0.000 0.000 0.000 0.000  Soil - Bottom Layer 3 0.000 0.000 0.000 0.000  Soil - Bottom Layer 4 0.000 0.000 0.000 0.000  Soil - Bottom Layer 5 0.146 0.254 0.000 0.000     56  Table 10. Root concentration factors and translocation factors for pharmaceuticals in the 30-µg/L trial.    Overhead Surface Pharmaceutical Harvest Week Average Root Concentration Factor Root Concentration Factor Standard Deviation Average Translocation Factor Translocation Factor Standard Deviation Average Root Concentration Factor Root Concentration Factor Standard Deviation Average Translocation Factor Translocation Factor Standard Deviation Acetaminophen 1 n/a n/a 0.108 0.043 n/a n/a 0.066 0.013  2 n/a n/a 0.042 0.049 n/a n/a 0.053 0.044  3 n/a n/a 0.139 0.079 n/a n/a 1.646 2.736  4 n/a n/a 0.183 0.196 n/a n/a n/a n/a  5 n/a n/a 0.396 0.153 n/a n/a 0.758 0.625 Caffeine 1 0.143 0.120 66.551 94.294 n/a n/a n/a n/a  2 0.347 0.384 22.678 35.326 0.210 0.317 0.945 0.510  3 0.253 0.217 2.803 1.401 0.125 0.077 5.111 3.860  4 0.010 0.017 n/a n/a 0.034 0.058 n/a n/a  5 0.058 0.047 20.405 10.006 0.324 0.487 n/a n/a Carbamazepine 1 0.688 0.233 8.477 2.630 0.360 0.210 5.145 0.591  2 0.623 0.287 12.651 3.260 0.677 0.140 5.077 0.272  3 0.649 0.138 9.168 1.632 0.787 0.215 8.950 0.039  4 0.614 0.244 11.326 4.517 0.844 0.179 10.673 2.144  5 1.121 0.138 8.689 1.526 1.374 0.428 10.892 3.592 Sulfadiazine 1 0.274 0.216 0.274 0.216 0.129 0.224 0.129 0.224  2 0.235 0.098 0.235 0.098 0.181 0.302 0.181 0.302  3 0.108 0.118 0.108 0.118 0.033 0.056 0.033 0.056  4 0.092 0.024 0.092 0.024 1.972 3.371 1.972 3.371  5 0.213 0.192 0.213 0.192 0.045 0.040 0.045 0.040 Sulfamethoxazole 1 n/a n/a n/a n/a n/a n/a 0.298 0.516  2 n/a n/a n/a n/a n/a n/a n/a n/a  3 33.438 40.156 0.017 0.030 n/a n/a 0.006 0.005  4 21.728 34.273 0.011 0.019 13.089 11.427 n/a n/a  5 2.359 1.763 0.325 0.036 6.646 3.661 0.026 0.045 Carbadox 1 2.472 1.787 0.584 0.332 1.753 1.589 n/a n/a  2 3.538 1.976 0.464 0.149 5.584 7.313 0.310 0.175  3 2.490 1.950 0.221 0.034 0.648 0.107 0.488 0.155  4 1.086 0.518 0.439 0.099 1.281 0.464 0.398 0.188  5 2.957 2.376 0.412 0.277 3.008 2.192 0.465 0.268 Trimethoprim 1 n/a n/a 0.840 0.463 n/a n/a 0.058 0.064  2 n/a n/a 0.213 0.110 n/a n/a 0.002 0.002  3 n/a n/a 0.048 0.013 n/a n/a 0.003 0.001  4 16.444 27.824 0.040 0.041 1.533 0.524 0.003 0.001  5 1.031 0.272 0.016 0.007 n/a n/a 0.004 0.003 Lincomycin 1 1.354 1.377 1.479 0.382 1.496 1.458 1.980 0.292  2 1.778 0.181 1.253 0.288 0.885 0.820 0.383 0.104  3 0.893 0.760 0.748 0.052 0.625 0.184 0.552 0.113  4 0.891 0.543 1.340 0.382 0.718 0.145 0.968 0.103  5 n/a n/a 1.074 0.254 0.910 0.340 1.000 0.188 Oxytetracycline 1 n/a n/a 0.464 0.196 n/a n/a 0.540 0.166  57  Table 10  2 n/a n/a 1.629 1.684 n/a n/a 0.777 0.657  3 n/a n/a 0.681 0.066 n/a n/a 0.354 0.053  4 n/a n/a 1.193 0.490 n/a n/a 0.597 0.162  5 n/a n/a 1.136 0.275 n/a n/a 1.236 0.597 Monensin Sodium 1 0.108 0.012 9.297 2.669 0.081 0.057 n/a n/a  2 0.258 0.239 6.942 5.218 0.118 0.059 n/a n/a  3 0.193 0.086 7.573 3.922 0.371 0.157 n/a n/a  4 0.039 0.043 n/a n/a 0.144 0.086 n/a n/a  5 0.251 0.060 8.463 0.370 0.266 0.237 0.561 0.928 Tylosin 1 0.184 0.070 4.776 3.526 0.360 0.264 0.165 0.169  2 0.260 0.098 3.359 1.343 0.189 0.035 0.107 0.150  3 0.121 0.021 2.181 0.486 0.235 0.128 0.118 0.125  4 0.086 0.077 3.158 2.404 0.068 0.011 0.086 0.037  5 0.107 0.038 1.878 0.493 0.121 0.024 0.201 0.183    58  Table 11. Mass balance for pharmaceuticals in the 50-µg/L trial.     Overhead  Surface  Pharmaceutical Har-vest Week Cumulative amount of Pharmaceu-tical Applied in Irrigation Water (µg) Total Shoot  Accumulation (µg) Total Root Accumulation (µg) Total Soil Accumulation (µg) %  Captured  Total Shoot  Accumulation (µg) Total Root Accumulation (µg) Total Soil Accumulation (µg) %  Captured  Acetaminophen 1 26.936 0.289 0.002 3.438 13.8 0.262 0.000 2.065 8.6  2 56.647 0.116 0.123 2.295 4.5 0.246 0.014 3.110 5.9  3 87.850 0.233 0.115 3.943 4.9 0.185 0.101 2.780 3.5  4 121.434 0.355 0.146 5.371 4.8 0.211 0.061 2.585 2.4  5 161.468 0.588 0.141 3.655 2.7 0.313 0.000 3.678 2.5 Caffeine 1 22.615 0.303 0.709 22.063 102.0 0.135 0.386 48.143 215.2  2 50.203 0.929 0.070 38.805 79.3 0.065 0.190 30.671 61.6  3 80.385 0.870 0.250 58.625 74.3 1.243 0.529 43.256 56.0  4 112.182 1.228 0.662 46.802 43.4 0.862 0.188 46.487 42.4  5 147.840 1.394 0.058 28.999 20.6 0.878 1.222 67.364 47.0 Carbamazepine 1 27.050 0.207 1.134 9.887 41.5 0.067 0.044 51.113 189.4  2 57.066 1.782 0.355 36.282 67.3 0.236 0.019 24.964 44.2  3 88.138 1.949 0.200 39.089 46.8 1.684 0.030 41.288 48.8  4 122.485 1.652 0.627 58.485 49.6 2.130 0.190 65.246 55.2  5 163.449 2.940 0.071 83.263 52.8 2.590 0.090 87.449 55.1 Sulfadiazine 1 24.860 0.000 0.000 2.727 11.0 0.000 0.007 1.446 5.8  2 53.530 0.000 0.001 0.911 1.7 0.000 0.000 0.190 0.4  3 83.307 0.001 0.004 0.842 1.0 0.003 0.002 2.726 3.3  4 115.445 0.008 0.006 3.303 2.9 0.000 0.003 6.939 6.0  5 151.642 0.000 0.002 0.813 0.5 0.023 0.003 1.886 1.3 Sulfamethoxazole 1 26.654 0.100 0.064 5.467 21.1 0.008 0.099 0.000 0.4  2 55.812 0.012 0.030 0.000 0.1 0.000 0.012 36.130 64.8  3 87.825 1.810 0.016 27.282 33.1 1.042 0.015 6.332 8.4  4 121.498 0.060 0.136 22.848 19.0 0.022 0.006 9.245 7.6  5 161.967 0.366 0.010 35.734 22.3 0.000 0.029 19.395 12.0 Carbadox 1 21.250 0.033 0.044 3.200 15.4 0.018 0.165 1.402 7.5  2 47.500 0.016 0.009 1.282 2.8 0.000 0.009 0.000 0.0  3 71.250 0.031 0.015 0.000 0.1 0.155 0.023 5.709 8.3  4 97.500 0.000 0.019 42.999 44.1 0.000 0.000 11.456 11.8  5 128.750 0.000 0.056 2.796 2.2 0.000 0.026 4.446 3.5 Trimethoprim 1 25.661 0.255 0.017 2.885 12.3 0.003 0.049 7.047 27.7  2 57.036 0.217 0.047 9.572 17.2 0.006 0.013 12.391 21.8  3 90.674 0.816 0.061 10.057 12.1 0.115 0.023 24.416 27.1  4 126.924 0.604 0.090 20.214 16.5 0.033 0.025 48.845 38.5  5 168.703 0.571 0.080 57.033 34.2 0.022 0.047 54.571 32.4 Lincomycin 1 23.305 0.072 0.045 16.183 69.9 0.144 1.772 40.573 182.3  2 50.148 0.229 1.330 26.536 56.0 0.028 2.067 26.950 57.9  3 77.402 2.415 0.058 11.987 18.7 3.314 0.451 17.895 28.0  59  Table 11  4 107.921 0.358 0.673 36.067 34.4 0.365 0.072 91.934 85.6  5 143.343 0.250 0.051 6.252 4.6 0.086 0.046 12.500 8.8 Oxytetracycline 1 21.250 0.004 0.000 36.184 170.3 0.002 0.000 27.032 127.2  2 47.500 0.006 0.000 34.028 71.6 0.004 0.000 36.571 77.0  3 71.250 0.040 0.000 38.872 54.6 0.027 0.000 42.747 60.0  4 97.500 0.020 0.000 49.474 50.8 0.135 0.000 49.551 51.0  5 128.750 0.030 0.012 39.460 30.7 0.122 0.000 38.491 30.0 Monensin  Sodium 1 19.819 0.064 0.005 16.270 82.4 0.046 0.049 12.173 61.9  2 38.931 0.152 0.009 0.000 0.4 0.000 0.018 4.733 12.2  3 59.871 0.253 0.028 0.189 0.8 0.059 0.017 8.506 14.3  4 79.014 0.479 0.088 13.926 18.3 0.011 0.001 8.640 10.9  5 105.547 0.514 0.008 3.958 4.2 0.013 0.005 5.108 4.9 Tylosin 1 23.653 0.029 0.006 0.000 0.1 0.015 0.003 3.135 13.3  2 51.255 0.079 0.010 4.527 9.0 0.000 0.002 5.137 10.0  3 80.510 0.288 0.021 3.926 5.3 0.002 0.002 8.815 11.0  4 112.134 0.274 0.054 12.875 11.8 0.003 0.017 15.475 13.8  5 148.649 0.307 0.026 16.225 11.1 0.008 0.015 24.511 16.5     60  Table 12. Mass balance for pharmaceuticals in the 30-µg/L trial.     Overhead  Surface  Pharmaceutical Har-vest Week Cumulative amount of Pharmace-utical Applied in Irrigation Water (µg) Total Shoot Accumulation (µg) Total Root Accumulation (µg) Total Soil Accumulation (µg) % Captured  Total Shoot Accumulation (µg) Total Root Accumulation (µg) Total Soil Accumulation (µg) % Captured    Acetaminophen 1 9.076 0.298 0.918 0.000 13.4 0.214 0.838 0.000 11.6  2 22.965 0.090 0.571 0.000 2.9 0.106 0.278 0.000 1.7  3 49.541 0.294 0.476 0.000 1.6 2.071 0.345 0.000 4.9  4 81.136 0.191 0.200 0.000 0.5 0.093 0.146 0.000 0.3  5 113.991 0.658 0.211 0.000 0.8 0.371 0.109 0.000 0.4 Caffeine 1 14.093 0.243 0.018 15.902 114.7 0.032 0.001 2.564 18.4  2 36.929 0.208 0.021 7.193 20.1 0.093 0.013 63.095 171.1  3 66.343 0.267 0.023 11.616 17.9 0.327 0.012 13.428 20.8  4 100.660 0.405 0.006 22.113 22.4 0.037 0.002 8.100 8.1  5 137.041 1.039 0.007 16.826 13.0 0.204 0.016 5.212 4.0 Carbamazepine 1 16.408 0.822 0.036 11.047 72.6 0.252 0.012 10.533 65.8  2 41.413 3.441 0.066 17.073 49.7 3.219 0.081 23.231 64.1  3 73.383 7.121 0.128 23.179 41.5 10.487 0.151 26.738 50.9  4 109.501 13.607 0.136 37.967 47.2 16.714 0.170 36.034 48.3  5 148.076 29.290 0.387 46.724 51.6 23.339 0.289 34.049 39.0 Sulfadiazine 1 15.941 0.002 0.003 1.341 8.4 0.001 0.003 1.264 8.0  2 38.187 0.007 0.008 1.165 3.1 0.009 0.005 1.140 3.0  3 66.831 0.006 0.009 1.687 2.5 0.002 0.009 1.181 1.8  4 99.406 0.005 0.006 1.981 2.0 0.106 0.006 4.980 5.1  5 134.345 0.027 0.013 2.140 1.6 0.003 0.009 1.434 1.1 Sulfamethoxazole 1 7.800 0.000 0.009 19.822 254.2 0.013 0.004 0.000 0.2  2 17.618 0.000 0.009 0.013 0.1 0.000 0.006 5.540 31.5  3 42.586 0.008 0.049 0.219 0.6 0.001 0.013 0.118 0.3  4 71.088 0.005 0.021 3.537 5.0 0.000 0.015 0.262 0.4  5 102.708 0.051 0.021 1.674 1.7 0.013 0.016 0.447 0.5 Carbadox 1 15.000 0.017 0.012 1.352 9.2 0.016 0.005 0.395 2.8  2 38.250 0.053 0.028 1.570 4.3 0.036 0.016 1.607 4.3  3 63.000 0.083 0.062 4.483 7.3 0.111 0.032 6.476 10.5  4 87.000 0.185 0.043 9.209 10.8 0.180 0.051 21.980 25.5  5 113.250 0.200 0.065 5.190 4.8 0.228 0.071 6.116 5.7 Trimethoprim 1 7.800 0.016 0.009 0.000 0.3 0.001 0.002 0.000 0.0  2 19.482 0.017 0.020 0.000 0.2 0.000 0.019 0.000 0.1  3 47.116 0.012 0.038 0.119 0.4 0.002 0.065 1.557 3.4  4 80.194 0.012 0.049 15.109 18.9 0.001 0.034 4.152 5.2  5 115.562 0.011 0.087 11.709 10.2 0.002 0.067 2.205 2.0 Lincomycin 1 14.928 0.046 0.011 6.872 46.4 0.020 0.003 30.965 207.6  2 38.814 0.151 0.029 2.406 6.7 0.058 0.020 9.072 23.6  3 66.185 0.228 0.048 55.603 84.4 0.135 0.032 7.033 10.9  61    4 97.628 0.462 0.036 7.620 8.3 0.236 0.026 6.600 7.0  5 131.872 0.521 0.061 4.096 3.5 0.268 0.036 6.730 5.3 Oxytetracycline 1 15.000 0.021 0.017 0.000 0.3 0.017 0.008 0.145 1.1  2 38.250 0.107 0.015 0.000 0.3 0.073 0.015 0.271 0.9  3 63.000 0.092 0.022 0.015 0.2 0.079 0.029 0.000 0.2  4 87.000 0.256 0.022 0.125 0.5 0.126 0.023 0.118 0.3  5 113.250 0.300 0.031 0.660 0.9 0.503 0.039 0.000 0.5 Monensin Sodium 1 14.622 0.126 0.005 9.123 63.3 0.000 0.003 6.998 47.9  2 36.487 0.185 0.013 7.017 19.8 0.000 0.006 8.557 23.5  3 63.614 0.379 0.010 5.734 9.6 0.000 0.020 6.885 10.9  4 94.879 0.546 0.003 8.685 9.7 0.000 0.004 16.127 17.0  5 127.258 0.820 0.011 6.238 5.6 0.058 0.013 8.689 6.9 Tylosin 1 14.625 0.027 0.002 2.736 18.9 0.001 0.001 0.946 6.5  2 36.184 0.059 0.004 2.655 7.5 0.006 0.006 5.299 14.7  3 61.474 0.096 0.007 6.990 11.5 0.009 0.014 8.633 14.1  4 91.157 0.117 0.005 13.509 15.0 0.003 0.004 10.692 11.7  5 122.260 0.129 0.009 10.818 9.0 0.008 0.006 8.223 6.7  62     Figure 1. Schematic of automatic irrigation system (Pump = P, Pressure Gauge = G, and Valve = V).    63    Figure 2. Pharmaceutical concentrations in irrigation water over time in 50 µg/L Trial. 64    Figure 3. Pharmaceutical concentrations in irrigation water over time in 30 µg/L Trial.  65    Figure 4. Images of lettuce in the 50-µg/L and 30-µg/L trials at week 3   50-µg/L Trial 30-µg/L Trial  66    Figure 5. Fresh and dry shoot biomass for Trial 1 and Trial 2 (nominal pharmaceutical concentrations of 50 and 30 µg/L in irrigation water, respectively).  67    Figure 6. Holm-Sidak two-tailed unpaired t-test showing significant difference (p < 0.05) between plant biomass between Trial 1 and Tria 2 (Trial 1 = nominal pharmaceutical concentration of 50 µg/L in irrigation water, Trial 2 = nominal pharmaceutical concentration of 30 µg/L in irrigation water).    68     Figure 7. Pharmaceutical concentrations in shoot wash waters for overhead and surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water).     69    Figure 8. Pharmaceutical concentrations in lettuce shoots for overhead and surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water).    70    Figure 9. Pharmaceutical concentrations in lettuce shoots for overhead and surface irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in irrigation water).  71     Figure 10. Pharmaceutical concentrations in lettuce roots for overhead and surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water).  72    Figure 11. Pharmaceutical concentrations in lettuce roots for overhead and surface irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in irrigation water).  73    Figure 12. Pharmaceutical concentrations in soil for overhead and surface irrigated plants in Trial 1 (nominal pharmaceutical concentrations of 50 µg/L in irrigation water).  74     Figure 13. Pharmaceutical concentrations in soil for overhead and surface irrigated plants in Trial 2 (nominal pharmaceutical concentrations of 30 µg/L in irrigation water).    75    Figure 14. Total percent of each pharmaceutical recovered for the 50 µg/L Trial.    76    Figure 15. Total percent of each pharmaceutical recovered for the 30 µg/L Trial .    77  CHAPTER THREE: CONCLUSIONS AND FUTURE RECOMMENDATIONS CONCLUSIONS Pharmaceuticals can enter the environment through their use in animal agriculture and human medicine. Because pharmaceuticals are not fully metabolized, they can be excreted through waste. Pharmaceuticals have been widely detected in wastewater treatment effluents, surface water and groundwater. Agricultural irrigation accounts for the majority of fresh water for human use. Due to rising worldwide water stress and scarcity, using reclaimed water for agricultural irrigation is an increasingly popular way to conserve freshwater. Unfortunately, conventional wastewater treatment practices are inefficient at removing pharmaceuticals from final effluent. Therefore, reclaimed water can be contaminated with common pharmaceuticals that can possibly accumulate in crops. Irrigation method may play a large role in final concentration and mass of pharmaceuticals in crops.  This study investigated the uptake and accumulation of pharmaceuticals in overhead and surface irrigated lettuce. When lettuce was grown in a greenhouse under simulated overhead and surface irrigation using water containing pharmaceuticals, those having low lipophilicity, low molecular weight, and high water solubility (acetaminophen, caffeine, carbamazepine, sulfadiazine, sulfamethoxazole, and carbadox) were similarly concentrated in les shoots over time. However, pharmaceuticals with high lipophilicity, high molecular weight, and lower water solubility (monensin sodium and tylosin) exhibited higher concentrations in overhead as opposed to surface-irrigated lettuce shoots. Exceptions to this were trimethoprim and lincomycin which have large molecular weights, high water solubility, but low log Kow. Both pharmaceuticals were more heavily concentrated in overhead-irrigated lettuce shoots likely because of diffusing into waxy leaf cuticles and binding with negatively charged leaf surface. Irrigation method  78  played no role in final concentration of pharmaceuticals in roots or soil. Carbamazepine, trimethoprim, carbadox, and tylosin showed increased concentrations in the top layer of soil overtime indicating stronger sorption to loamy sand soils. FUTURE RECOMMENDATIONS  Based on these findings, irrigation method could play a large role in final concentrations of pharmaceuticals in edible plants, depending on the chemical properties of pharmaceuticals and crop type. More research is necessary regarding different crops under both irrigation treatments, especially comparing root crops (i.e. carrots, radish, etc.) and fruit crops (i.e. tomatoes, cucumber, etc) pharmaceutical amounts. It is also necessary to understand how soil type plays a role in pharmaceutical uptake, so research should be done under both irrigation treatments with varying soil type and with possible soil amendments such as biochar to sorb contaminants. Degradation also plays a large role in final pharmaceutical amount, so more research should be done to help separate modes of degradation for specific pharmaceuticals.    Since using reclaimed water for agricultural irrigation is currently practiced in water scarce areas and its use is predicted to increase in the future, consideration on irrigation method and pharmaceutical type should be taken. Although it is difficult to predict final concentrations of pharmaceuticals in crops, the trends observed in this study can help inform growing practices and consumer washing practices to hopefully lower unintentional pharmaceutical exposure through food.     79             LITERATURE CITED      80  LITERATURE CITED   Bartha, B.; Huber, C.; Harpaintner, R.; Schröder, P. Effects of acetaminophen in Brassica juncea L. Czern.: investigation of uptake, translocation, detoxification, and the induced defense pathways. Environmental Science and Pollution Research 2010, 17 (9), 15531562.  2016.  http://www.beefusa.org/beefindustrystatistics.aspx (accessed Mar 04, 2016).  Boxall, A. B. A.; Johnson, P.; Smith, E. J.; Sinclair, C. J.; Stutt, E.; Levy, L. S. Uptake of  Veterinary Medicines from Soils into Plants. Journal of Agricultural and Food Chemistry 2006, 54 (6), 22882297.  Bruton, T.; Alboloushi, A.; de la Garza, B.; Kim, B.-O.; Halden, R. U. Fate of Caffeine in the Environment and Ecotoxicological Considerations. In Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations; Halden, R. U., Ed.; American Chemical Society: Washington, DC, 2010; Vol. 1048, pp 257273.  Cabello, F. C. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for  human and animal health and for the environment. Environmental Microbiology 2006, 8 (7), 11371144.  Calderón-Preciado, D.; Matamoros, V.; Bayona, J. M. Occurrence and potential crop uptake of  emerging contaminants and related compounds in an agricultural irrigation network. Science of The Total Environment 2011, 412-413, 1419.  Calderón-Preciado, D.; Matamoros, V.; Biel, C.; Save, R.; Bayona, J. M. Foliar sorption of emerging and priority contaminants under controlled conditions. Journal of Hazardous Materials 2013, 260, 176182.  Carter, L. J.; Harris, E.; Williams, M.; Ryan, J. J.; Kookana, R. S.; Boxall, A. B. A. Fate and  Uptake of Pharmaceuticals in SoilPlant Systems. Journal of Agricultural and Food Chemistry 2014, 62 (4), 816825.  Carvalho, P. N.; Basto, M. C. P.; Almeida, C. M. R.; Brix, H. A review of plantpharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands. Environmental Science and Pollution Research 2014, 21 (20), 1172911763.  Chiu, C.; Westerhoff, P. K. Trace Organics in Arizona Surface and Wastewaters. In  Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations; Halden, R. U., Ed.; American Chemical Society: Washington, DC, 2010; Vol. 1048, pp 81117.   81  Christian, T.; Schneider, R. J.; Färber, H. A.; Skutlarek, D.; Meyer, M. T.; Goldbach, H. E. Determination of antibiotic residues in manure, soil, and surface waters. Acta hydrochimica et hydrobiologica 2003, 31 (1), 3644.  Chuang, Y.-H.; Zhang, Y.; Zhang, W.; Boyd, S. A.; Li, H. Comparison of accelerated solvent extraction and quick, easy, cheap, effective, rugged and safe method for extraction and determination of pharmaceuticals in vegetables. Journal of Chromatography A 2015, 1404, 19.  Clarke, B. O.; Porter, N. A. Persistent Organic Pollutants in Sewage Sludge: Levels, Sources, and Trends. In Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations; Halden, R. U., Ed.; American Chemical Society: Washington, DC, 2010; Vol. 1048, pp 137171.  Daughton, C. G. Pharmaceutical Ingredients in Drinking Water: Overview of Occurrence and  Significance of Human Exposure. In Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations; Halden, R. U., Ed.; American Chemical Society: Washington, DC, 2010; Vol. 1048, pp 968.  Eggen, T.; Asp, T. N.; Grave, K.; Hormazabal, V. Uptake and translocation of metformin, ciprofloxacin and narasin in forage- and crop plants. Chemosphere 2011, 85 (1), 2633.  Eggen, T.; Lillo, C. Antidiabetic II Drug Metformin in Plants: Uptake and Translocation to Edible Parts of Cereals, Oily Seeds, Beans, Tomato, Squash, Carrots, and Potatoes. Journal of Agricultural and Food Chemistry 2012, 60 (28), 69296935.  EPA, Biosolids. January 28, 2016. https://www.epa.gov/biosolids (accessed Apr 2, 2016).   EPA, Environmental Protection Agency. Contaminants of Emerging Concern including  Pharmaceuticals and Personal Care Products. November 17, 2015. http://www.epa.gov/wqc/contaminants-emerging-concern-including-pharmaceuticals-and-personal-care-products (accessed Jan 20, 2016).  May 1998.  https://www3.epa.gov/npdes/pubs/bastre.pdf (accessed Mar 25, 2016)  Estévez, E.; Cabrera, M. del C.; Molina-Díaz, A.; Robles-Molina, J.; Palacios-Díaz, M. del P.  Screening of emerging contaminants and priority substances (2008/105/EC) in reclaimed water for irrigation and groundwater in a volcanic aquifer (Gran Canaria, Canary Islands, Spain). Science of The Total Environment 2012, 433, 538546.  Fatta-Kassinos, D.; Meric, S.; Nikolaou, A. Pharmaceutical residues in environmental waters and  wastewater: current state of knowledge and future research. Analytical and Bioanalytical Chemistry 2011, 399 (1), 251275.    82  FDA, Federal Drug Administration. 2013 Summary Report On Antimicrobials Sold or  Distributed for Use in Food-Producing Animals. April, 2015. http://www.fda.gov/downloads/ForIndustry/UserFees/AnimalDrugUserFeeActADUFA/UCM440584.pdf (accessed Mar 25, 2016).  Gao, P.; Ding, Y.; Li, H.; Xagoraraki, I. Occurrence of pharmaceuticals in a municipal  wastewater treatment plant: Mass balance and removal processes. Chemosphere 2012, 88 (1), 1724.  eló, D. Removal of pharmaceuticals during  wastewater treatment and environmental risk assessment using hazard indexes. Environment International 2010, 36 (1), 1526.  Goldstein, M.; Shenker, M.; Chefetz, B. Insights into the Uptake Processes of Wastewater-Borne Pharmaceuticals by Vegetables. Environmental Science & Technology 2014, 48 (10), 55935600.  Hirsch, R.; Ternes, T.; Haberer, K.; Kratz, K.-L. Occurrence of antibiotics in the aquatic  environment. Science of the Total Environment 1999, 225 (1), 109118.  Holm, J. V.; Ruegge, K.; Bjerg, P. L.; Christensen, T. H. Occurrence and distribution of pharmaceutical organic compounds in the groundwater downgradient of a landfill (Grindsted, Denmark). Environmental Science & Technology 1995, 29 (5), 14151420.  Jjemba, P. K. The potential impact of veterinary and human therapeutic agents in manure and  biosolids on plants grown on arable land: a review. Agriculture, Ecosystems & Environment 2002, 93 (1), 267278.  Kang, D. H.; Gupta, S.; Rosen, C.; Fritz, V.; Singh, A.; Chander, Y.; Murray, H.; Rohwer, C.  Antibiotic Uptake by Vegetable Crops from Manure-Applied Soils. Journal of Agricultural and Food Chemistry 2013, 61 (42), 999210001.  Kemper, N. Veterinary antibiotics in the aquatic and terrestrial environment. Ecological  Indicators 2008, 8 (1), 113.  Khan, S.; Cao, Q.; Zheng, Y. M.; Huang, Y. Z.; Zhu, Y. G. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environmental Pollution 2008, 152 (3), 686692.  Kim, K.-R.; Owens, G.; Kwon, S.-I.; So, K.-H.; Lee, D.-B.; Ok, Y. S. Occurrence and  Environmental Fate of Veterinary Antibiotics in the Terrestrial Environment. Water, Air, & Soil Pollution 2011, 214 (1-4), 163174.  Kong, W. D.; Zhu, Y. G.; Liang, Y. C.; Zhang, J.; Smith, F. A.; Yang, M. Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.). Environmental Pollution 2007, 147 (1), 187193.  83  Kümmerer, K. Antibiotics in the aquatic environment  A review  Part I. Chemosphere 2009,  75 (4), 417434.  Larsson, D. G. J.; de Pedro, C.; Paxeus, N. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. Journal of Hazardous Materials 2007, 148 (3), 751755.  Le, T. X.; Munekage, Y. Residues of selected antibiotics in water and mud from shrimp ponds in  mangrove areas in Viet Nam. Marine Pollution Bulletin 2004, 49 (11-12), 922929.  Li, D.; Han, Y.; Meng, X.; Sun, X.; Yu, Q.; Li, Y.; Wan, L.; Huo, Y.; Guo, C. Effect of Regular Organic Solvents on Cytochrome P450-Mediated Metabolic Activities in Rat Liver Microsomes. Drug Metabolism and Disposition 2010, 38 (11), 19221925.  Lu, J.; Wu, J.; Stoffella, P. J.; Wilson, P. C. Uptake and distribution of bisphenol A and nonylphenol in vegetable crops irrigated with reclaimed water. Journal of Hazardous Materials 2015, 283, 865870.  Macherius, A.; Eggen, T.; Lorenz, W. G.; Reemtsma, T.; Winkler, U.; Moeder, M. Uptake of Galaxolide, Tonalide, and Triclosan by Carrot, Barley, and Meadow Fescue Plants. Journal of Agricultural and Food Chemistry 2012, 60 (32), 77857791.  Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B. Irrigation of Root Vegetables with Treated Wastewater: Evaluating Uptake of Pharmaceuticals and the Associated Human Health Risks. Environmental Science & Technology 2014, 48 (16), 93259333.  Mathews, S.; Reinhold, D. Biosolid-borne tetracyclines and sulfonamides in plants. Environmental Science and Pollution Research 2013, 20 (7), 43274338.  Misra, A. K. Antibiotics as Crop Protectants. In Agricultural Uses of Antibiotics; Moats, W. A.,  Ed.; American Chemical Society: Washington, DC, 1986; Vol. 320, pp 4960.  Montforts, M. H.; Kalf, D. F.; van Vlaardingen, P. L.; Linders, J. B. The exposure assessment for  veterinary medicinal products. Science of the Total Environment 1999, 225 (1), 119133.  NOAA Fisheries, What is Aquaculture? January 31, 2012.   http://www.nmfs.noaa.gov/aquaculture/what_is_aquaculture.html (accessed Mar 26, 2016).   Pedrero, F.; Kalavrouziotis, I.; Alarcón, J. J.; Koukoulakis, P.; Asano, T. Use of treated municipal wastewater in irrigated agricultureReview of some practices in Spain and Greece. Agricultural Water Management 2010, 97 (9), 12331241.  Pereira, L. S.; Oweis, T.; Zairi, A. Irrigation management under water scarcity. Agricultural Water Management 2002, 57 (3), 175206.  84  Sallach, J. B.; Zhang, Y.; Hodges, L.; Snow, D.; Li, X.; Bartelt-Hunt, S. Concomitant uptake of antimicrobials and Salmonella in soil and into lettuce following wastewater irrigation. Environmental Pollution 2015, 197, 269277.  Sarmah, A. K.; Meyer, M. T.; Boxall, A. B. A. A global perspective on the use, sales, exposure  pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65 (5), 725759.  Stockwell, V. O.; Duffy, B. Use of antibiotics in plant agriculture. Rev. Sci. Tech. Off. Int. Epiz.  2012, 31 (1), 199210.  Tanoue, R.; Sato, Y.; Motoyama, M.; Nakagawa, S.; Shinohara, R.; Nomiyama, K. Plant Uptake  of Pharmaceutical Chemicals Detected in Recycled Organic Manure and Reclaimed Wastewater. Journal of Agricultural and Food Chemistry 2012, 60 (41), 1020310211.  Ternes, T. Occurrence of Drugs in German Sewage Treatment Plants and Rivers. Water Research 1998, 3 (11), 32453260.  Toze, S. Reuse of effluent waterbenefits and risks. Agricultural Water Management 2006, 80 (1-3), 147159.  USDA, Census of Agriculture News Release. USDA Reports 55.3 Million Acres of Irrigated U.S. Farmland. Nov 13, 2014. https://www.agcensus.usda.gov/Newsroom/2014/11_13_2014.php (accessed Jul 11, 2016).   USDA, National Agricultural Statistics Service. Quarterly Hogs and Pigs. June 2015a.  http://www.usda.gov/nass/PUBS/TODAYRPT/hgpg0615.pdf (accessed Mar 05, 2016).  USDA, National Agricultural Statistics Service. Poultry Production and Value 2014 Summary.  April 2015b. http://www.usda.gov/nass/PUBS/TODAYRPT/plva0415.pdf (accessed Mar 05, 2016).   USGS, The USGS Water Science School. Wastewater Treatment Water Use. December 29,  2015. http://water.usgs.gov/edu/wuww.html (accessed Mar 25, 2016).   USGS, Irrigation Water Use. May 2, 2016. http://water.usgs.gov/watuse/wuir.html (accessed Jul 11, 2016).  Vidaver, Anne K. Uses of Antimicrobials in Plant Agriculture. Clinical Infectious Diseases  2002, 34, S107S110.  Wang, C.; Teppen, B. J.; Boyd, S. A.; Li, H. Sorption of Lincomycin at Low Concentrations from Water by Soils. Soil Science Society of America Journal 2012, 76 (4), 1222.    85  Xiong, T.-T.; Leveque, T.; Austruy, A.; Goix, S.; Schreck, E.; Dappe, V.; Sobanska, S.;  Foucault, Y.; Dumat, C. Foliar uptake and metal(loid) bioaccessibility in vegetables exposed to particulate matter. Environmental Geochemistry and Health 2014, 36 (5), 897909.  Yi, L.; Jiao, W.; Chen, X.; Chen, W. An overview of reclaimed water reuse in China. Journal of Environmental Sciences 2011, 23 (10), 15851593.