1 Uptake and Accumulation of Pharmaceuticals in Overhead- and

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Uptake and Accumulation of Pharmaceuticals in Overhead- and Surface-Irrigated Greenhouse Lettuce Gemini D Bhalsod, Ya-Hui Chuang, Sangho Jeon, Wenjun Gui, Hui Li, Elliot T Ryser, Andrey K Guber, and Wei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04355 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Journal of Agricultural and Food Chemistry

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Uptake and Accumulation of Pharmaceuticals in Overhead- and Surface-Irrigated

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Greenhouse Lettuce

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Gemini D. Bhalsod,†,¶ Ya-Hui Chuang,† Sangho Jeon,†,▲ Wenjun Gui,†,ǁ Hui Li,† Elliot T. Ryser,‡

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Andrey K. Guber,† and Wei Zhang*,†,§

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†

Department of Plant, Soil and Microbial Sciences, ‡Department of Food Science and Human

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Nutrition, and §Environmental Science and Policy Program, Michigan State University, East

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Lansing, MI 48824, United States

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ǁ

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China

Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058,

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¶

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▲

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Republic of Korea

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*Corresponding author. Dr. Wei Zhang, Address: 1066 Bogue ST RM A516, East Lansing, MI

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48824, United States; Tel: 517-353-0471; Fax: 517-355-0270; Email: [email protected].

Cook County Unit, University of Illinois Extension, Arlington Heights, IL 60004, United States National Institute of Agricultural Sciences, Rural Development Administration, Wanju 54875,

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1 ACS Paragon Plus Environment


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ABSTRACT: Understanding the uptake and accumulation of pharmaceuticals in vegetables

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under typical irrigation practices is critical to risk assessment of crop irrigation with reclaimed

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water. This study investigated the pharmaceutical residues in greenhouse lettuce under overhead

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and soil-surface irrigations using pharmaceutical-contaminated water. Compared to soil-surface

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irrigation, overhead irrigation substantially increased the pharmaceutical residues in lettuce

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shoots. The increased residue levels persisted even after washing for trimethoprim, monensin

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sodium, and tylosin, indicating their strong sorption to the shoots. The post-washing

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concentrations in fresh shoots varied from 0.05 ± 0.04 µg/kg for sulfadiazine to 345 ± 139 µg/kg

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for carbamazepine. Root concentration factors ranged from 0.04 ± 0.14 for tylosin to 19.2 ± 15.7

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for sulfamethoxazole. Translocation factors in surface-irrigated lettuce were low for

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sulfamethoxalzole, trimethoprim, monensin sodium and tylosin (0.07–0.15), but high for caffeine

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(4.28 ± 3.01) and carbamazepine (8.15 ± 2.87). Carbamazepine was persistent in soil and

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hyperaccumulated in shoots.

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KEYWORDS: pharmaceuticals, uptake, lettuce, Lactuca sativa, irrigation

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INTRODUCTION

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Pharmaceuticals are considered contaminants of emerging concern, because they are widely

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detected in the environment, are not routinely monitored or regulated, and could pose potential

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risks to human and ecosystem health.1, 2 The ubiquitous presence of pharmaceuticals in the

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environment results from their large use in healthcare and animal agriculture.3 For instance, as an

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important group of pharmaceuticals, antibiotics are commonly used in livestock production for

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growth promotion, and disease prevention and treatment.3, 4 Like many other pharmaceuticals, a

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significant portion of the administered antibiotics is released to agroecosystems via sewage

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sludge, wastewater effluents, animal manure, and agricultural wastewater, due to incomplete

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drug metabolism or insufficient removal during wastewater treatment.3, 5-7 The anthropogenic

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loading of antibiotics to agroecosystems has been linked to the proliferation of antibiotic

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resistance in bacteria populations.4, 8, 9 Antibiotic resistance is an eminent global health threat,10

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thus demanding more research in both clinical and agricultural settings. When contaminated soil

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and water are used for agricultural production, potential risks of pharmaceuticals to food safety

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and human health need to be examined in terms of chronic low-level exposure and the

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proliferation of antibiotic resistant bacteria and genes.9, 11, 12

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Currently over 70% of the world’s freshwater is used for crop irrigation.13, 14 Due to

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increasing water shortage, alternative irrigation water sources (often of lower water quality) must

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be considered.15 Treated municipal or agricultural wastewaters are increasingly being reclaimed

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for crop irrigation,13, 15-17 particularly in water-stressed regions. However, many pharmaceuticals

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have been detected in wastewater effluents at ng/L to µg/L levels.7 For instance, acetaminophen,

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caffeine, carbamazepine, sulfamethoxazole and trimethoprim was found up to 11.7, 15.2, 3.1,

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22.0, and 2.5 µ/L in the wastewater effluents, respectively.6, 7, 18 Thus, concerns have been raised



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regarding potential risks of crop irrigation with reclaimed water to food safety and human

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health.11, 16, 18, 19 In the US, 52 million acres of cropland are irrigated,20 and about 51% of

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irrigation is performed by overhead sprinkler systems, 42% by surface flood irrigation, and 7%

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by micro-irrigation.21 Irrigation practices with reclaimed water (e.g., overhead spray irrigation

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and soil surface irrigation) vary substantially across the world, depending on source and quality

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of reclaimed water, crop types (nonfood and food crops), and adequacy of reuse infrastructure

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and management.13, 16, 22 For wheat crop spray-irrigated with wastewater effluent,

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sulfamethoxazole, trimethoprim, olfoxacin and carbamazepine were detected at 0.1–5.2 µg/kg on

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wheat grain surface and 0.6–2.3 µg/kg in wheat grains, respectively.18 Therefore, understanding

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the transfer of pharmaceuticals from contaminated soil and water to and their residue levels in

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crops under typical irrigation practices is critical to informed assessment of exposure level and

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health risks. It is particularly important to evaluate the effect of irrigation methods on

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pharmaceutical uptake by vegetables such as lettuce (Lactuca sativa), because vegetables are

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often consumed with minimal processing.

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A number of recent studies have examined the uptake and accumulation of pharmaceuticals

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in plants including vegetable crops.23-26 These previous studies often examined the

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pharmaceutical uptake through plant roots,27-30 whereas little work has been directed to foliar

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uptake of pharmaceuticals. Two recent studies reported that foliar uptake most likely occurs for

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lipophilic compounds.31, 32 Lu et al.31 observed greater accumulation of relatively lipophilic

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bisphenol A (log Kow = 3.40) and nonylphenol (log Kow = 4.48) in lettuce and tomato through the

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foliar exposure rather than the subsurface root exposure. Similarly, Calderón-Preciado et al.32

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found greater retention of lipophilic contaminants in leaves with closed stomata in the dark.

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Therefore, foliar uptake of pharmaceuticals in vegetables under overhead sprinkler irrigation


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deserves further study.

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This study aimed to compare the uptake and accumulation of pharmaceuticals in greenhouse

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lettuce irrigated with pharmaceutical-contaminated water via overhead or soil-surface irrigation.

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We selected eleven commonly used pharmaceuticals, including a fever reducer and pain reliever

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(acetaminophen), a stimulant (caffeine), an anticonvulsant (carbamazepine), and 8 antibiotics

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(sulfadiazine, sulfamethoxazole, carbadox, trimethoprim, lincomycin, oxytetracycline, monensin

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sodium, and tylosin), based on their large use in humans and animals, and their varying

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physicochemical properties such as molecular weight, water solubility, charge behaviors (pKa),

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and hydrophobicity (log Dow, the pH-adjusted log Kow by accounting for neutral species) (Table

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S1). This study was conducted in a controlled greenhouse setting so that the pharmaceutical

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residues in lettuce shoots, roots, and soils under two irrigation treatments could be compared to

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infer major uptake pathways.

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MATERIALS AND METHODS

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Chemicals and Materials. Eleven pharmaceuticals (acetaminophen, caffeine,

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carbamazepine, sulfadiazine, sulfamethoxazole, carbadox, trimethoprim, lincomycin,

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oxytetracycline, monensin sodium, and tylosin) were purchased from Sigma-Aldrich (St. Louis,

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MO, USA). Their detailed physicochemical properties are provided in Table S1 in Supporting

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Information. These pharmaceuticals were dissolved in HPLC-grade methanol to prepare stock

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solutions at concentrations ranging from 10 to 1000 mg/L. Acetonitrile and anhydrous sodium

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sulfate (Na2SO4) were purchased from EMD Chemicals (Gibbstown, NJ, USA), ceramic

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homogenizers, C18 and primary-secondary amine (PSA) from Agilent Technologies (Santa

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Clara, CA, USA), and disodium ethylenediaminetetraacetate (Na2EDTA), formic acid, and

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sodium chloride (NaCl) from J.T. Baker (Phillipsburg, NJ, USA). All chemicals used were of 5 ACS Paragon Plus Environment


®

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analytical grade or better. Oasis hydrophilic-lipophilic balance (HLB) extraction cartridges

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(6cc) were purchased from Waters Corporation (Milford, MA, USA).

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A soil sample was collected at Charlotte, MI, and was air-dried, passed through a 2-mm

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sieve, and stored in a covered plastic container before use. The soil was tested at the Soil and

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Plant Nutrient Laboratory of Michigan State University (East Lansing, MI) according to the

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standard methods. The soil had 81.3% of sand, 10.5% of silt, and 8.2% of clay, and was

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classified as a loamy sand as per the USDA classification. Soil pH was 7.4 measured in a 1:1

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soil:water mixture. Organic matter content was 2.5% measured by loss-on-ignition at 360 °C.

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Bray P1 extractable phosphorus concentration was 71 mg/kg. Potassium, magnesium, and

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calcium concentrations in 1-mol/L ammonium acetate extractant were 71, 50, 126, and 1298

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mg/kg, respectively. Cation exchange capacity was 7.0 meq/100 g as measured by the

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ammonium saturation method. The soil was found free of the selected pharmaceuticals. The

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loamy sand was used because it is a common soil type in many areas where lettuce is produced

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(particularly in California). Also, coarse-textured soils typically demand higher amount of

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irrigation water, which may necessitate the use of reclaimed water and represent a “worse-case”

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scenario. Similar loamy sand soils were also used in previous studies on the uptake of

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pharmaceuticals by lettuce and other vegetables.23, 24, 33, 34

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Greenhouse Growth Experiments. This study was conducted in a greenhouse under

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controlled condition with a 16-h photoperiod, air temperature of 24 ± 10 °C, and relative

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humidity of 43 ± 17%. To prepare free-draining nursery pots for growing lettuce, approximately

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1455 g of soil sample were uniformly packed into each nursery pot (14.6 cm in diameter at top

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and 10.8 cm in height) to a depth of 9 cm, resulting in a bulk density of 1.35 g/cm3.

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Concurrently, 4–6 seeds of Burpee Black Seeded Simpson Lettuce (Burpee, Warminster, PA,

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USA) were planted in a sterile potting mix for approximately three weeks to produce transplant

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seedlings for the subsequent irrigation experiments. The selected lettuce is a commonly grown

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lettuce type. The potting mix was watered with deionized (DI) water and applied with a fertilizer

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solution with a nitrogen concentration of 125 mg/L (Peters Professional water soluble 20-20-20

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general purpose fertilizer, Scotts, Marysville, OH, USA) as needed. Seedlings were thinned and

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transplanted into individual soil pots. Before transplanting, excess potting mix was removed

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from the transplants, and the soil pots were watered with DI water and then free-drained for 10–

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15 mins. After the transplanting, the pots were watered again, and the lettuce plants were left to

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acclimate for about 2 days before beginning the irrigation experiments.

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®

Thirty-six lettuce plants were planted (i.e., 36 soil pots), and placed randomly in a custom-

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built automatic irrigation system, as described in Supporting Information S1. The automatic

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irrigation system was able to accurately control water volume and timing during the overhead

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and soil-surface irrigations. As illustrated in Figure S1, fifteen lettuce plants were placed under

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either the overhead or surface irrigation line fed with an opaque pharmaceutical water tank,

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whereas three lettuce plants were irrigated with the pharmaceutical-free water for overhead or

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surface irrigation, respectively. The pharmaceutical water tank was filled with prepared irrigation

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water containing the eleven pharmaceuticals and covered to avoid exposure to light. Detailed

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procedure on irrigation water preparation is described in Supporting Information S1. Two trials

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were performed at two varying pharmaceutical concentrations of 50 (Trial 1) and 30 µg/L (Trial

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2). These concentrations were on the high end of pharmaceutical concentrations observed in

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wastewater effluents and other environmental waters,6, 7 but were needed to allow for the

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detection of pharmaceutical residues in lettuce so that the effect of irrigation methods could be

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compared. Because stunt and major necrosis were observed on the lettuce plants in Trial 1, the 7 ACS Paragon Plus Environment


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irrigation water in Trial 2 was fertilized with the 20-20-20 general purpose fertilizer to a final

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nitrogen concentration of 125 mg/L. A lower pharmaceutical level of 30 µg/L was also selected.

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No fertilizer was added in the irrigation water in Trial 1, and the fertilizer solution (125 mg N/L)

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was applied to the pots periodically. In Trial 1 each lettuce plant received about 0.27 L of the

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fertilizer solution (or 34 mg nitrogen), whereas in Trial 2 each lettuce plant received

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approximately 3.78 L of the fertilizer solution (i.e., 472 mg nitrogen). The pharmaceutical-free

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controls were included to examine if there was any phytotoxicity of pharmaceuticals to the

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lettuce. It was also used to obtain background matching matrices for the water, lettuce, and soil

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samples in the LC/MS-MS analysis. The irrigation water in both water tanks was used to irrigate

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lettuce plants until the water level reached the tank outlet, after which the tanks were cleaned out

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and refilled twice during the trials.

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The lettuce plants in Trial 1 and Trial 2 were irrigated with about 25–125 mL of irrigation

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water daily (equivalent to an irrigation depth of 1.5–7.5 mm), depending on the water demand of

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lettuce at various growth stages. The irrigation amounts were in the typical range of average

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daily irrigation for lettuce in the field.35 The irrigation water amounts were recorded for

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calculating total amount of each pharmaceutical applied to lettuce plants, totaling 2.58 L in Trial

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1 and 3.78 L in Trial 2. In the overhead-irrigated pots, a perforated transparent screen was placed

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around each lettuce plant to minimize water loss from the overhead spray while allowing for air

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exchange. In the Trial 2, surface-irrigated pots also had a screen placed around the lettuce plant.

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Volumetric soil water content in the soil pots was measured in situ using 5TE sensors and EM 50

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data loggers (Decagon® Devices, Pullman, WA). Average volumetric soil water contents in the

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overhead- and surface-irrigated pots were 0.20 ± 0.05 and 0.16 ± 0.04 in Trial 1, and 0.15 ± 0.04


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and 0.13 ± 0.05 in Trial 2, respectively, which were much lower than the saturation level. No

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free drainage occurred at the bottom of the soil pots.

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Sampling and Extraction of Lettuce and Soil Samples. The lettuce plants irrigated with

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the pharmaceutical-containing water were randomly harvested weekly in triplicates, whereas the

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pharmaceutical-free control plants were harvested at the end of Week 2, 4 and 5. The lettuce

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shoots were washed in 200 mL DI water, and the wash water in Trial 1 was saved for later

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analysis. The washing was to simulate the possible wash-off of pharmaceutical residues by post-

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harvest washing typically performed by vegetable producers or consumers prior to consumption.

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Soil samples at the top (0-3 cm), middle (3-6 cm), and bottom (6-9 cm) layers of the pots were

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also collected. Detailed procedures on sampling of the lettuce shoots, roots, and soils could be

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found in Supporting Information S1. The lettuce shoot, root and soil samples were freeze-dried

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and ground before extraction and analyses for the pharmaceutical residues.

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The pharmaceutical residues in the lettuce shoots and roots were extracted following a quick,

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easy, cheap, effective, rugged, and safe (QuEChERS) method.36 The pharmaceutical recovery in

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vegetable samples by this method was about 72–96%.36 Detailed extraction and clean-up

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procedure is provided in Supporting Information S1. This QuEChERS method was also adapted

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for extracting the pharmaceutical residues from the soil samples. In general, the extraction with

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the Na2EDTA concentration of 150 mg/L produced better results than the Na2EDTA level of 300

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mg/L (Table S2).

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Sampling and Extraction of Water Samples. Water samples of 20 mL were collected from

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both water tanks daily in Trial 1 and 2–3 times a week in Trial 2. The collected water samples

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were stored in amber glass vials with polyurethane caps. Using the HLB cartridges, the

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extraction and clean-up of water samples was adopted from Chuang et al.36 and described in



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detail in Supporting Information S1 (Table S3). The extracts were stored in the −20 °C freezer

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for later analysis.

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LC-MS/MS Analysis. The extracts were analyzed for the pharmaceutical concentrations

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using a Shimadzu Prominence high performance liquid chromatograph (Colombia, MD, USA)

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coupled with an Applied Biosystems Sciex 4500 QTrap mass spectrometer (Foster City, CA,

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USA). An Agilent Eclipse Plus C18 Column (2.1 mm × 50 mm, particle size of 5 µm) was used

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for separation. The mobile phase consisted of phase A (0.3% formic acid in DI water) and phase

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B, i.e., acetonitrile/methanol mixture (1/1 by volume) with 0.3% formic acid. The flow rate was

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0.35 mL/min, and the sample injection volume was 10 µL. Pharmaceutical concentrations were

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quantified using a matrix-matched calibration curve. Precursor ions and product ions for

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qualification and quantification, along with mass spectrometer parameters, can be found in Table

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S4.

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Calculations and Statistical Analyses. The pharmaceutical concentrations measured in the

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freeze-dried shoot and root samples were converted to their concentrations based on the fresh

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weight of lettuce shoots and roots (i.e., Cshoot and Croot), according to the measured fresh and dry

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weights of the shoots and roots. We selected to calculate the pharmaceutical concentrations in

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lettuce by fresh weight27 because lettuce is often consumed in fresh. The gravimetric water

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content was 0.92 ± 0.03 in the fresh shoots and 0.92 ± 0.02 in the fresh roots, respectively. Thus,

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the pharmaceutical concentrations by dry weight in the lettuce was on average 12.5 times of the

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concentrations by fresh weight.

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To elucidate the uptake pathways of pharmaceuticals in lettuce, bioconcentration factors including root concentration factors (RCF) and translocation factors (TF) were calculated via:

RCF =

Croot Csoil

(1) 10 ACS Paragon Plus Environment

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TF =

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Cshoot Croot

(2)

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where Croot and Cshoot are the pharmaceutical concentrations by fresh weight of lettuce. Thus, the

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calculated RCF would be on average 12.5 times less than the RCF by dry weight. The TF by

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fresh weight would be similar to its values by dry weight due to similar water content in the

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lettuce shoots and roots. RCF and TF were calculated weekly, allowing for assessing their

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changes throughout the growth stages of the lettuce.

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Total mass balance of applied pharmaceuticals in the soil-lettuce system was calculated as

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described in Supporting Information S1. The unrecovered fraction might be dissipated through

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transformation and degradation in soils and within plants.3, 19, 24, 37 Or a fraction of certain

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pharmaceuticals may be irreversibly bound with soil matrices, and thus could not be extracted,38,

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39

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which is often considered not bioavailable to plants.5 All statistical analyses were conducted using GraphPad PRISM 7. Lettuce biomass

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comparisons by trials were analyzed as grouped unpaired t-tests. Statistical significance was

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determined using the Holm-Sidak method. Lettuce biomass at each week was analyzed

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individually, without assuming a consistent variance. Lettuce biomass comparisons between

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irrigation methods within trials were analyzed in the same manner.

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RESULTS AND DISCUSSION

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Lettuce Shoot Biomass. In the pharmaceutical-free control, the overhead-irrigated lettuce

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shoots harvested on Week 2, 4, and 5 consistently had greater fresh and dry weight than the

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surface-irrigated shoots in both trials (Figure 1a and b). Thus, the overhead-irrigated lettuce grew

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better than the surface-irrigated lettuce. Upon exposure to the pharmaceuticals, there was no

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significant difference in the shoot biomass between two irrigation methods, and the fresh weight

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in Week 2 and dry weight in Week 3 were even lower under overhead irrigation than under 11 ACS Paragon Plus Environment


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surface irrigation. Thus, the pharmaceuticals in the irrigation water negated the positive effect of

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the overhead irrigation on the lettuce shoot growth. The lettuce shoot weights in Trial 2 were

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significantly greater than Trial 1, probably due to increased fertilizer input and reduced

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phytotoxicity with the lower pharmaceutical concentrations in Trial 2. The difference in fertilizer

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application cannot be solely responsible for the observed difference in the lettuce growth,

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because the difference of shoot weight between Trial 1 and Trial 2 was in general much greater

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with the pharmaceutical exposure than that without the pharmaceutical exposure. For example,

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on Week 4 the fresh weight of shoots between Trial 1 and Trial 2 differed by a factor of 6.2–10.6

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in the presence of pharmaceuticals, and only by a factor of 3.0–4.5 in the absence of

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pharmaceuticals. The similar trends were observed for the dry weight of the shoots.

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The lettuce plants also showed stunt and major necrosis under a higher pharmaceutical

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exposure in Trial 1, but appeared much healthier in Trial 2 (Figure S3). Boxall et al.23 reported

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that lettuce growth was significantly reduced upon exposure to oxytetracycline, but not to

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sulfadiazine, trimethoprim, and tylosin at the pharmaceutical concentration of 1 mg/kg in a

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loamy sand. The phytotoxicity of pharmaceuticals to plants varies with pharmaceutical type,

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plant species, and exposure level.5 For example, the reported median effective concentrations

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(EC50) of the pharmaceuticals ranged between 0.1 to 5 mg L−1 for duckweed.2 Liu et al.40 found

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that the lowest concentrations of chlortetracycline, tetracycline, tylosin, sulfamethoxazole,

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sulfamethazine, and trimethoprim to negatively affect the seeding height and root length of rice

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and cucumber were all above 10 mg/kg in a silt loam soil. In our study, the cumulative

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concentrations of pharmaceuticals applied to the soil were less than 0.12 mg/kg individually.

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Nonetheless, a significant negative impact on the lettuce growth was observed. Thus, the lettuce

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might access the pharmaceuticals in irrigation water more easily than the soil-sorbed


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pharmaceuticals in the previous studies.23, 40 Also, the bioavailability of the pharmaceuticals in

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the loamy sand soil might be higher than other fine-textured soils.23 Finally, there might be an

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additive and/or synergistic toxicity of a pharmaceutical mixture as already demonstrated for

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algae and duckweed,41, 42 which should be explored in future studies.

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Pharmaceutical Residues in Lettuce Shoots. During harvest in both trials, the lettuce

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shoots were washed in DI water to remove any weakly sorbed pharmaceuticals on the shoots.

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The concentrations of washable pharmaceutical residues were higher in the overhead-irrigated

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shoots than in the surface-irrigated shoots (Figure S4), with the exception of carbadox and

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oxytetracycline (Figures S4f and i). In fact, the washable pharmaceutical residues was essentially

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nonexistent in the surface-irrigated shoots (Figure S4). The negligible levels of washable

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carbadox and oxytetracycline residues in the shoots likely resulted from photodegradation43 or

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strong foliar sorption. Clearly, the direct exposure of pharmaceuticals to the lettuce with

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overhead irrigation could substantially increase their residues in the shoots. This effect was

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particularly pronounced during the early stage of lettuce growth when the shoot biomass was

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lower, except for lincomycin and tylosin. As lettuce is often washed either by producers or

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consumers prior to consumption, the washable fraction of pharmaceutical residues in the lettuce

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shoots might be effectively removed during washing, and are of less concern. Thus, they were

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only measured in Trial 1. Rather, the remaining pharmaceutical residues after washing would

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represent a higher exposure risk to consumers, and were thus focused in this study.

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The post-washing concentrations of each pharmaceutical in the lettuce shoots in both trials

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are shown in Figures 2 and S5, respectively. In contrast to the concentrations of washable

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pharmaceutical residues, only trimethoprim, monensin sodium and tylosin consistently showed

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greater post-washing concentrations in the shoots under overhead irrigation than under surface



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irrigations (Figure 2g, 2j and 2k; Figure S5g, 5j and 5k). For other pharmaceuticals such as

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acetaminophen, caffeine, carbamazepine, sulfadiazine, sulfamethoxazole, carbadox, and

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oxytetracycline, no conclusive difference in their concentrations in the shoots between overhead

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and surface irrigations was observed over time (Figure 2 and S5). The post-washing

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pharmaceutical residues in the lettuce shoots could originate directly from the irrigation water

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under overhead irrigation, or be translocated from the roots. Thus, they could be collectively

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controlled by exposure route (i.e., foliar vs root exposure), in-plant metabolism, and root-to-

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shoot translocation, which would vary with individual pharmaceuticals. Thus, a large variation in

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the post-washing pharmaceutical concentrations in the shoots was expected, e.g., ranging from

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1.4 ± 0.1 µg/kg of tylosin to 327 ± 99 of carbamazepine in the overhead-irrigated mature lettuce

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shoot on Week 5 (Figure 2). While the post-washing concentrations of most pharmaceuticals in

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the shoots did not increase with time, the concentrations of carbamazepine, trimethoprim, and

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lincomycin in Trial 2 clearly demonstrated an increasing trend over time (Figure 2). The increase

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of trimethoprim concentration was only observed for the overhead-irrigated shoot (Figure 2g and

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Figure S5g), and thus most likely resulted from the accumulation of trimethoprim directly

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received from the irrigation water. However, for carbamazepine and lincomycin, the surface-

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irrigated shoots also showed an increasing concentration over time, suggesting the translocation

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from the roots. In particular, the concentration of carbamazepine was very high (Figure 2c and

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Figure S5c), and was hyperaccumulated in the lettuce shoot, similar to its hyperaccumulation in

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radish leaf, ryegrass,33 cucumber leaf, and tomato leaf.24

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Pharmaceutical Residues in Lettuce Root. The concentrations of each pharmaceutical in

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the lettuce roots in Trail 1 and Trial 2 are shown in Figures S6 and S7, respectively. In Trial 2,

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there was no statistically significant difference in the pharmaceutical concentrations in the roots


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with regard to the irrigation method, indicating that irrigation methods do not play a large role in

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the root accumulation of pharmaceuticals in lettuce (Figure S7). This observation was expected,

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because all the applied pharmaceuticals in both overhead and surface irrigations eventually

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drained to the soils, and became accessible by the roots. In Trial 1, due to the low root biomass

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(Figure 1), all of the roots collected from the three pots at each harvest were combined, and

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therefore no statistical significance could be inferred. Closer examination of the results from

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Trial 2 revealed that the concentration in the roots increased over time for carbamazepine,

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carbadox, trimethoprim and lincomycin (Figure S7c, f, g and h), suggesting the effective root

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accumulation of these pharmaceuticals, despite their disparate log Dow values (−1.22–2.45, Table

315

S1). Interestingly, there was a decrease in the concentration of acetaminophen in the root over

316

time (Figure S7a). This observation is supported by a hydroponic study,37 in which the

317

concentrations of acetaminophen in the Indian mustard shoots and roots sharply decreased in

318

only one week. The concentrations of caffeine, sulfadiazine, sulfamethoxazole, monensin

319

sodium, and tylosin were relatively stable with time, implying either ineffective root uptake or

320

equilibrium between root uptake, translocation and dissipation.

321

Pharmaceutical Residues in Soils. The pharmaceutical residue levels in the soils were

322

similar regardless of irrigation methods (Figure S8 and S9). This was expected because the soils

323

in both trials received about similar amount of pharmaceuticals as the soil surface was not

324

covered, despite minor interception of pharmaceuticals by lettuce shoots under overhead

325

irrigation. Most pharmaceuticals were accumulated in the soils to a varying degree, except for

326

acetaminophen and sulfadiazine that were quickly dissipated in both trials (Figure S8 and S9).

327

Compared to Trial 1, oxytetracycline also diminished in the soils in Trial 2 (Figure S9i). The

328

dissipation of pharmaceuticals in the soils could result from either degradation/transformation or



329

irreversible sorption to soil matrix.38, 39 For example, about 73–93% of acetaminophen and 64%

330

of sulfadiazine initially applied to soils became non-extractable fraction.38, 39 Sulfadiazine has

331

been reported to degrade in soils with half-lives ranging from 12–18 days in aerobic soils to 57–

332

237 days in anoxic soils.44 In loamy sand soils, the dissipation of sulfadiazine in our study

333

appeared to be much faster (a half-life < 45 days) than the reported half-life < 103 days,23 likely

334

because a greater organic content of the soil in our study promoting greater microbial activities

335

or stronger sorption of sulfadiazine to the soil.

336

The concentrations of acetaminophen, caffeine, sulfamethoxazole, lincomycin,

337

oxytetracycline and monensin sodium showed no patterns with the soil depth, likely resulted

338

from either their high mobility or quick dissipation.45-48 For instance, caffeine has been reported

339

to have a high desorption capacity (>15%), especially in sandy loam soils,48 which likely resulted

340

in its leaching downwards. Sulfonamides such as sulfamethoxazole often has lower sorption and

341

higher mobility in soils at neutral and basic pH.46, 49 The trend for sulfadiazine in Trial 2 could

342

not be assured due to its low concentrations. Monensin has a half-life of less than 4 days in

343

soils.47 Conversely, the concentrations of carbamazepine, carbadox, trimethoprim, and tylosin in

344

the top soil layer was greater than the lower layers and increased with time, probably due to their

345

lower mobility or dissipation in soils.46, 50-54 For instance, carbamazepine has low leaching

346

potential and high persistence in soils, with the half-lives greater than 40 days.33, 52, 54

347

Bioconcentration Factors of Pharmaceuticals in Lettuce. Bioconcentration factors

348

including RCF and TF were calculated weekly for 5 weeks in Trial 2 (Table S5), but not for Trial

349

1 due to unhealthy lettuce plants and low plant growth (Figure 1 and S3). RCFs and TFs were

350

not significantly different throughout the lettuce growth stages, except for the TFs of

351

carbamazepine in the surface-irrigated lettuce that increased with mature plants. Thus, the RCFs


Page 16 of 29

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352

and TFs for the 5 weeks were averaged and reported in Table 1. RCFs were similar for both

353

overhead- and surface-irrigated lettuce, which was again expected due to similar pharmaceutical

354

concentrations in the soils. Comparing with other pharmaceuticals, sulfamethoxazole, carbadox,

355

trimethoprim, and oxytetracycline had greater RCF values, suggesting that they tend to

356

accumulate in the roots.

357

As the overhead-irrigated lettuce shoots also received the pharmaceuticals directly from the

358

irrigation water, the calculated TF values do not truly represent the root-to-shoot translocation of

359

a pharmaceutical. Thus, only the TF values of the surface-irrigated lettuce was examined closely.

360

In general, a pharmaceutical with a TF below 1 is not readily transported from roots to shoots in

361

plants, whereas a pharmaceutical with a TF above 1 suggests a readily root-to-shoot translocation

362

and accumulation in the shoots.28 Caffeine and carbamazepine had a TF of 4.28 ± 3.01 and 8.15

363

± 2.87 in the surface-irrigated lettuce, respectively. Thus, caffeine and carbamazepine tend to be

364

translocated up and accumulated in the lettuce shoots. The uptake factors were calculated as the

365

product of RCF and TF, and were about 6.4 and 81.5 for caffeine and carbamazepine (dry

366

weight), respectively. These values, while on the high end, were consistent with the previously

367

reported values, e.g., 2 for caffeine and 47 for carbamazepine in tomato and cucumber leaves,

368

and 59 for carbamazepine in rye grass grown in sandy soils.24, 34 The TF of carbamazepine

369

observed in this study (8.15 ± 2.87) was very similar to the calculated TF of 7.88 in the radish

370

leaf grown in a loamy sand.33 Since carbamazepine has a log Dow of 2.45 and is neutral over a

371

wide pH range (Table S1), it tends to be taken up by the plant roots, but not bind with the

372

function groups in the root tissues. Thus, carbamazepine can be freely transported by the

373

transpiration stream.



374

Other pharmaceuticals all had a TF value less than 1. In particular, sulfamethoxazole,

375

trimethoprim, monensin sodium, and tylosin had a TF value less than 0.15, indicating a very

376

limited root-to-shoot translocation. Insignificant root-to-shoot translocation of these

377

pharmaceuticals have been reported previously for cabbage, corn, cucumber, and onion, possibly

378

due to either their quick degradation in soils, their strong accumulation in roots, or their large

379

molecular sizes.19, 24, 25, 30

380

Acetaminophen, sulfadiazine, carbadox, lincomycin, and oxytetracycline had moderate TF

381

values ranging from 0.47 to 0.98, suggesting that they could be moderately transported by the

382

transpiration stream. Additionally, the TF values of trimethoprim, monensin sodium and tylosin

383

for the overhead-irrigated lettuce were much greater than the ones for the surface-irrigated

384

lettuce (Table 1). This obviously resulted from the foliar exposure of these pharmaceuticals from

385

the irrigation water, despite the shoots were washed in DI water. Trimethoprim is predominantly

386

cationic and neutral with a low log Dow (0.54) (Table S1). Therefore, it could either bind with

387

negatively charged surface function groups or diffuse into the waxy cuticle layer on the lettuce

388

leaves.32 Monensin sodium and tylosin had relatively large log Dow (Table S1), thus being sorbed

389

to the lettuce leaves through hydrophobic interaction. Additionally, tylosin is mainly positively

390

charged (Table S1), which further promotes its binding with the negatively charged surface

391

function groups in the shoots. Due to the stronger interactions of trimethoprim, monensin sodium

392

and tylosin with the lettuce shoots, their increased foliar accumulation, as a result of overhead

393

irrigation, persisted even after the washing in DI water (Figure 2).

394

Mass Balance of Pharmaceuticals. The distribution of recovered pharmaceutical residues in

395

the shoots, roots, and soils on Week 5 is shown in Figure 3 and S10. Detailed mass distribution

396

and total recovered percentage over the initially applied amount of each pharmaceutical are


Page 18 of 29

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397

provided in Table S6 and S7. The pharmaceutical residues were predominantly distributed in the

398

soils, and the uptake and accumulation of the pharmaceuticals (except for carbamazepine in Trial

399

2) in the lettuce might be minor (Figure 2). In the case of carbamazepine, it was likely that in

400

Trial 2 the better lettuce growth allowed the hyperaccumulation of carbamazepine in the shoots

401

to the level comparable to its mass in the soil. The mass recoveries of the pharmaceuticals were

402

much lower in Trial 2 than in Trial 1 (Figure 3 and S10), likely because the biodegradation and

403

metabolism of pharmaceuticals were enhanced by greater microbial activities and lettuce growth

404

in Trial 2 due to higher fertilizer application. The biodegradation in soils and in-plant

405

metabolism played an important role in the fate of pharmaceuticals in the soil-lettuce systems, in

406

addition to abiotic sorption and degradation.3, 19, 24, 29, 37 For example, Bartha et al.37 proposed

407

acetaminophen could be degraded through both a plant-dependent pathway and a plant-

408

independent pathway. About 50% of total carbamazepine mass in tomato and cucumber leaves

409

were metabolized to two main metabolites (10,11-epoxide-carbamazepine and 10,11-dihydro-

410

10,11-dihydroxy-carbamazepine).24 Some pharmaceuticals, such as caffeine and acetaminophen,

411

have been shown to degrade through photolysis.55, 56

412

Overall, our findings may have interesting implications on utilizing reclaimed water to

413

irrigate vegetable crops. Despite the wide use of overhead sprinkler systems, their use in

414

vegetable production needs to be carefully assessed when using reclaimed water for irrigation

415

due to greater concentration and mass of some pharmaceuticals (specifically trimethoprim,

416

monensin sodium and tylosin) in overhead- as opposed to surface-irrigated lettuce shoots. Most

417

applied pharmaceuticals from irrigation water were quickly dissipated in soils, thus suggesting

418

reduced exposure risks over time. However, carbamazepine was very persistent in soil, and was

419

hyperaccumulated in the lettuce shoots, thus posing a particular concern to food safety and



420

human health. This study used the pharmaceutical-containing irrigation waters that were

421

prepared in DI water, which differed from actual reclaimed waters in terms of water chemistry

422

such as dissolved organic matter (DOM), metal cations, and solution pH.22 As some

423

pharmaceuticals (e.g., tetracyclines) complex with DOM and metal cations, and solution pH

424

determines species distribution of ionizable pharmaceuticals,3 it is expected that water chemistry

425

of reclaimed water could influence the pharmaceutical residues in lettuce, specifically for the

426

overhead-irrigated shoots. Future study should examine the uptake and accumulation of

427

pharmaceuticals under diverse chemical conditions of reclaimed water. Finally, our study

428

showed the ubiquitous accumulation of pharmaceuticals in the lettuce upon exposure from

429

irrigation water, demonstrating the need for further assessing the environmental and food safety

430

risks associated with using pharmaceutical-contaminated water for irrigation.

431

ABBREVIATIONS USED: RCF, root concentration factors; TF, translocation factors.

432

ASSOCIATED CONTENT

433

Supporting Information

434

Supplemental Materials and Methods, and Supplemental Results. The Supporting Information is

435

available free of charge on the ACS Publications website at DOI:

436

ACKNOWLEDGMENT

437

This research was supported by Agriculture and Food Research Initiative Competitive Grant No.

438

2016-67017-24514 from the USDA National Institute of Food and Agriculture and the Project

439

GREEEN from Michigan State University AgBioResearch.

440

REFERENCES

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Table 1. Root concentration factors and translocation factors of pharmaceuticals in Trial 2. a Overhead Irrigation Surface Irrigation Pharmaceutical RCF TF RCF TF Acetaminophen na 0.17 (0.14) na 0.51 (0.71) Caffeine 0.13 (0.12) 28.1 (27.2) 0.12 (0.09) 4.28 (3.01) Carbamazepine 0.73 (0.22) 10.1 (1.84) 0.80 (0.37) 8.15 (2.87) Sulfadiazine 0.69 (0.19) 0.18 (0.08) 0.95 (0.18) 0.47 (0.84) Sulfamethoxazole 19.2 (15.7) 0.07 (0.14) 7.80 (5.72) 0.07 (0.13) Carbadox 2.51 (0.91) 0.41 (0.13) 2.45 (1.95) 0.51 (0.23) Trimethoprim 5.20 (5.90) 1.41 (0.31) 2.39 (1.30) 0.15 (0.13) Lincomycin 1.29 (0.40) 1.18 (0.28) 0.93 (0.34) 0.98 (0.62) Oxytetracycline 1.15 (0.27) 1.02 (0.46) 1.41 (0.69) 0.70 (0.34) Monensin Sodium 0.17 (0.09) 9.29 (2.88) 0.20 (0.12) 0.11 (0.25) Tylosin 0.09 (0.02) 3.07 (1.14) 0.04 (0.14) 0.14 (0.05) a RCF = root concentration factor, and TF = root-to-shoot translocation factor. The values are the mean with standard deviation in parentheses for the RCF and TF in 5 weeks as provided in Table S7. “na” means that RCF was not available due to zero concentrations of acetaminophen in the soil.

600



601

602 603 604 605 606 607

Figure 1. Fresh (a) and dry (b) weight of lettuce shoots in Trial 1 and Trial 2. Holm-Sidak twotailed unpaired t-test showed a significant difference in mean weight of lettuce shoots (p < 0.016) between Trial 1 and Trial 2. No significant difference in mean weight of lettuce shoots between overhead and surface irrigation unless noted the figure (p = 0.051–0.97). Error bar is the standard deviation of triplicates (n = 3).


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608

609

610

611 612 613 614 615 616 617 618

Figure 2. Post-washing pharmaceutical concentrations in overhead- or surface-irrigated lettuce shoots (fresh weight) in Trial 2 (nominal concentration of each pharmaceutical in irrigation water = 30 µg/L). (a) Acetaminophen, (b) Caffeine, (c) Carbamazepine, (d) Sulfadiazine, (e) Sulfamethoxazole, (f) Carbadox, (g) Trimethoprim, (h) Lincomycin, (i) Oxytetracycline, (j) Monensin Sodium, and (k) Tylosin. Error bar is the standard deviation of triplicates (n = 3).



619 620 621

Figure 3. Recovered percentages of applied pharmaceuticals in shoots, roots, and soils with overhead (a) or surface (b) irrigation at Week 5 in Trial 2.

622


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TOC Graphic

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1 Uptake and Accumulation of Pharmaceuticals in Overhead- and

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