Back Conversion from Product to Parent: Methyl Triclosan to Triclosan

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Back Conversion from Product to Parent: Methyl Triclosan to Triclosan in Plants QIUGUO FU, Chunyang Liao, Xinyu Du, Daniel Schlenk, and Jay J. Gan Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00071 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Environmental Science & Technology Letters

Back Conversion from Product to Parent: Methyl Triclosan to Triclosan in Plants

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Qiuguo Fu,†,‡, Chunyang Liao,† Xinyu Du,† Daniel Schlenk,† Jay Gan†,*

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†

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United States

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‡

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Switzerland

Department of Environmental Sciences, University of California, Riverside, California 92521,

Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf,

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*Corresponding author: [email protected]

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ACS Paragon Plus Environment


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Abstract Numerous man-made chemicals pass through wastewater treatment plants (WWTPs), where

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biological and chemical treatments often result in the formation of a wide range of

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transformation products via reactions such as conjugation and alkylation. Such transformation

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products may come into contact with plants when treated wastewater and biosolids are used in

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agricultural production or when plants are used for mitigation purposes (e.g., treatment wetlands).

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Using the high-volume antimicrobial triclosan as a model compound, we showed that its primary

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transformation product in the WWTPs, methyl triclosan, was readily converted back to the parent

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in plants. When exposed to environmentally relevant concentrations of methyl triclosan, triclosan

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was detected in Arabidopsis thaliana cells within 12 h, and the level of triclosan increased over

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time. The molar fraction of triclosan to methyl triclosan in the cells was estimated to be 0.17 at

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144 h. When grown in nutrient solution containing methyl triclosan, lettuce and carrot seedlings

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were also capable of transforming methyl triclosan back to triclosan after 4 d, with triclosan

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levels reaching >10 µg/g in lettuce tissues and >3 µg/g in carrot tissues. The back and forth

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conversions of triclosan in various environmental compartments effectively prolong its

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environmental persistence and exposure. Future assessment of this and other emerging

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contaminants should consider such inter-conversions to obtain a better understanding of their fate

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and risks.

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Key words: methyl triclosan, triclosan, emerging contaminants, transformation products, back

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conversion

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Introduction

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Numerous pharmaceuticals and person care products (PPCPs) and their human metabolites

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are deposited into wastewater treatment plants (WWTPs), where they are subjected to multiple

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treatment processes including chemical and microbial transformations. These processes often

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lead to the formation of additional transformation intermediates, such as conjugates and

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alkylated products. Many PPCPs and their transformation products have been found in WWTP

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effluents and biosolids. For example, triclosan, a high-production-volume antibacterial agent, is

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widely used in various household and healthcare products including soaps, toothpaste, cosmetics,

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shampoo, and textiles.1 Methyl triclosan has been found as the primary microbial transformation

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product of triclosan during wastewater treatement.2 Both triclosan and methyl triclosan have

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been shown to exhibit cytotoxic, genotoxic, and endocrine disrupting activity towards non-target

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organisms.3,4 Triclosan and methyl triclosan tend to be enriched in biosolids due to their

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hydrophobicity, and thus have been found at mg/kg levels in active sludge and biosolids,2,5–7

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while trace levels also remained in the effluent.4

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When treated wastewater is used for irrigation or biosolids are applied as soil amendments,

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PPCPs and their metabolites can come into contact with plants.8–12 Moreover, native plants are

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often used to remove nutrients and trace contaminants and improve water quality through

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semi-engineered systems such as wetlands, grassed waterways, and vegetative buffers.13,14 Most

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studies to date have focused on PPCPs in their parent form, while few studies have considered

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the likelihood of back conversion from products to the parent. Qu. et al.15 reported that

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trenbolone acetate, the photolytic product of the steroidal promoter trenbolone, was converted to

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its parent form in surface water under ambient conditions. However, to date the potential of

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product-to-parent conversion in higher plants has not been examined for PPCPs. In this study we tested the hypothesis that methyl triclosan may be back converted to

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triclosan through demethylation in plants. Back conversion to triclosan was targeted for

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characterization in Arabidopsis thaliana cells as well as lettuce and carrot whole plants.

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Materials and Methods

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Chemicals and Arabidopsis Cell Line

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Standards of methyl triclosan (CAS# 4640-01-1) and methyl triclosan-d3 were obtained from

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Toronto Research Chemicals (Ontario, Canada). Triclosan (CAS# 3380-34-5) and triclosan-d3

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were purchased from Alfa Aesar (Ward Hill, MA) and C/D/N Isotopes (Pointe-Claire, Quebec,

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Canada), respectively. N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA)

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(Sigma-Aldrich, St. Louis, MO) was used as the derivatization reagent. The stock solutions of all

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target substances were prepared by dissolving the standards in hexane/acetone (v/v, 9:1) or

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methanol and stored in amber glass vials at -20 °C before use. The working solutions were

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prepared from the stock solutions through serial dilution with hexane/acetone or methanol before

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use. Ultrapure water was prepared by a Milli-Q water purification system. All organic solvents

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including dichloromethane, ethyl acetate, acetone, hexane, methanol and acetonitrile were of

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HPLC or GC grade and obtained from Fisher Scientific (Fair Lawn, NJ).

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The Arabidopsis thaliana cell line PSB-D was provided by Arabidopsis Biological Resource

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Center at Ohio State University (Columbus, OH). Cell line suspensions were grown under sterile

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conditions in the incubator (25 °C, 130 rpm, dark). Further details about the cells, medium

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composition and preparation are given in Text S1 in the Supporting Information.

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A. thaliana Cell Cultivation Experiment

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The A. thaliana cell line was inoculated in 30 mL of Murashige and Skoog medium in

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Erlenmeyer flasks.16,17 After 4 d pre-cultivation, 20 µL of methanol (0.07% v/v) containing

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methyl triclosan (450 mg/L) was introduced to the medium to yield an initial concentration of

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300 µg/L. Additionally, two control treatments were simultaneously prepared, including solvent

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control (medium spiked with 20 µL pure methanol), and medium control (medium pre-cultured

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without cells, but spiked with 20 µL methanol containing methyl triclosan). The incubation was

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terminated immediately (0 h) or after 12, 24, 48, 120, and 144 h for analysis. Cell material was

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separated from the culture medium by centrifugation at 3000 rpm for 30 min and washed twice

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using ultrapure water. Detailed description of extraction, cleanup, and analysis of triclosan and

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methyl triclosan in the medium and cell matters are given in SI. The final extracts dissolved in

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hexane/acetone (v/v, 9:1) were mixed with MTBSTFA and kept at 70 °C for 60 min for

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derivatization prior to analysis by gas chromatography-mass spectrometry (GC-MS/MS).

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Whole Plant Cultivation Experiment

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Conversion of methyl triclosan to triclosan was further evaluated using whole plants of

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lettuce (Lactuca sativa) and carrot (Daucus carota, Scarlet Nantes) grown hydroponically in

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nutrient solution (Oasis® 16-4-17 hydroponic fertilizer 3.16 g/L) in the growth chamber (22 °C,

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16 h day/20 °C 8 h night cycle, relative humidity of 75-80%). In brief, lettuce and carrot

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seedlings (35 d old) were transplanted from potting mix to the glass jar containing 1 L nutrient

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solution with methyl triclosan at 1.0 mg/L, a level likely within the range found in active sludge

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and biosolids.2,5,6 During the hydroponic incubation, only plant root was in contact with the

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nutrient solution. After 4 d of cultivation, the whole plant and nutrient medium were collected

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separately. Plant roots were washed with distilled water and separated from the leaves.

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A second trial using intact lettuce plants was performed to determine if triclosan formed in

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the plant was released back into the growth medium. Lettuce seedlings were purchased from a

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local retail store and were transplanted into the nutrient solution after carefully washing off loose

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solids (potting mix) from the roots. The plants were cultivated for 4 d after methyl triclosan

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treatment (550 µg/L), after which they were then transferred to a clean nutrient solution (i.e.,

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without methyl triclosan). After cultivation for another 4 d, the nutrient solution was sampled for

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analysis of triclosan. Additionally, treatment with the lettuce plant but without methyl triclosan,

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and treatment with methyl triclosan but without the lettuce plant, were included as the blank

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controls. To evaluate the potential contribution of plant rhizosphere microbial or abiotic

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transformations to the conversion of methyl triclosan to triclosan, lettuce plants were cultivated

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in 1 L glass jar containing nutrient solution in the growth chamber for 4 d and then removed. The

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used nutrient solution was immediately spiked with methyl triclosan (nominal concentration 550

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µg/L) and incubated for an additional 4 d. In a parallel treatment, NaN3 (200 mg/L) was added to

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inhibit the microbial activity in the growth medium to detect any abiotic transformation.

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Additionally, a treatment without methyl triclosan was included as the blank control. Sample

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preparation of nutrient solution and plant tissues are given in the SI.

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Data Analysis

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Data were calculated as mean ± standard deviation (SD). One-way analysis of variance

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(ANOVA) and Student’s t-test were carried out with GraphPad 6 to evaluate systematic

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differences between multiple groups and two groups, respectively (α = 0.05).

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Results and Discussion

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Conversion of Methyl triclosan to Triclosan in Arabidopsis Cells

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To test if A. thaliana cells were able to take up and transform methyl triclosan, we exposed A.

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thaliana cells to methyl triclosan and monitored the disappearance of methyl triclosan from the

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growth medium and appearance of methyl triclosan in the cells. Rapid dissipation of methyl

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triclosan from the medium containing viable A. thaliana cells was observed throughout the

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incubation, with its concentration decreasing from 296 ± 56 µg/L initially to 21 ± 10 µg/L after

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12 h of incubation, or a loss of about 93% at the end of incubation (Figure 1A). Concurrent to

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the decline in the medium, methyl triclosan appeared in the cells and the level quickly increased

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within the first 12 h to a maximum of 25 ± 7 µg/g dry weight, suggesting uptake of methyl

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triclosan into A. thaliana cells (Figure 1B). After 12 h, the level of methyl triclosan in the cells

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linearly decreased to 16 ± 2 µg/g at the end of 144-h incubation (slope -0.07 ± 0.007, p = 0.01,

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R2 = 0.98) (Figure 1B). For the solvent control, methyl triclosan was not found in the medium or

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cells. In addition, approximately 22% of methyl triclosan was unaccounted for when it was

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incubated in the cell-free medium, likely due to sorption to the container surfaces. Taken together,

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these observations suggested that methyl triclosan was accumulated into and subsequently

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transformed in A. thaliana cells.

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Triclosan appeared in A. thaliana cells shortly after exposure to methyl triclosan, and the

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level of triclosan continued to increase over time, reaching 2.8 ± 0.3 µg/g at the end of 144-h

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incubation (Figure 1). The molar fraction of triclosan to methyl triclosan in the cells was

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estimated to be 0.17 at 144 h, suggesting that at least 15% of methyl triclosan was back

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converted to triclosan. As triclosan likely underwent further transformations,18,19 it is possible

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that the actual back conversion fraction was greater. Figure 2 is an example of chromatograms

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from the extract of A. thaliana cells after 144 h of incubation. Both triclosan and methyl triclosan

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were structurally confirmed using authentic standards after derivatization. The chromatograms

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show that triclosan was present in the cells after exposure to methyl triclosan at levels

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significantly above the detection limit (Figure 2A), while it was absent in the control (Figure

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2B).

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Formation of Triclosan in Vegetable Plants Exposed to Methyl Triclosan To test if the demethylation of methyl triclosan to triclosan would also occur in whole plants,

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lettuce and carrot seedlings were exposed to methyl triclosan under hydroponic conditions.

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Triclosan was found in leaves and roots of both lettuce and carrot. In addition, triclosan was

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detected at higher levels in lettuce than in carrot, with the concentrations up to 11.5 ± 5.5 µg/g in

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lettuce and 3.7 ± 2.2 µg/g in carrot tissues (Table 1). Within each plant species, the levels of

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triclosan were similar (p > 0.05) between the root and leaf tissues (Table 1).

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The second trial using whole plants of lettuce with additional control treatments confirmed

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the formation of triclosan in the plant. Methyl triclosan and triclosan were detected in lettuce

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tissues at 6.3-7.0 µg/g and 0.2-1.0 µg/g, respectively (Figure S1). Trace amounts (0.15 ± 0.04

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µg/L, Table S2) of triclosan were found in the nutrient medium after 4 d of plant cultivation

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without methyl triclosan. This may be attributed to background contamination in the lettuce

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seedlings as they were grown in potting mix before use. In the depuration experiment, the level

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of triclosan in the growth medium after 4 d was similar to that in the control without methyl

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triclosan (Figure S1), suggesting that once formed, triclosan was not released appreciably from

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the root. This differed from a previous study on benzotriazole metabolites,20 and may be

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attributed to the high hydrophobicity of triclosan. Additionally, microorganisms in the growth

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medium did not appear to convert methyl triclosan to triclosan, as the level of triclosan (0.04 ±

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0.06 µg/L) in the nutrient solution was not statistically different from the control (Table S2).

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Similarly, no detectable abiotic conversion was observed under the experimental conditions, as

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the level of triclosan (0.14 ± 0.02 µg/L) also did not differ from the control (Table S2). Taken

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together, these results demonstrated that triclosan was formed via in-plant transformations after

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uptake of methyl triclosan by the root.

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Environmental Implications

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To the best of our knowledge, this was the first observation on the back conversion of

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methyl triclosan to triclosan in higher plants. Methyl triclosan is more persistent than triclosan5

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and has been frequently detected in effluent-impacted streams and biosolids.21 In

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WWTP-effluent impacted streams, methyl triclosan was found to bioaccumulate in aquatic

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organisms such as fish and algae.22,23 Furthermore, slow conversion of methyl triclosan to

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triclosan was previously observed in the liver and intestine of channel catfish, likely through

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O-demethylation.23 Cytochrome P450 enzymes (CYPs) were further proposed to catalyze the

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demethylation of methyl triclosan in catfish.23 In other studies, CYPs in human liver microsomes

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were reported to be involved in the dealkylation of naproxen and other drugs.24–26 Given that

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plants have isozymes of CYPs similar to fish and mammals 27 and that dealkylation is a common

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pathway in plant cell homeostasis,16,28 it is highly probable that CYPs were responsible for the

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catalytic conversion of methyl triclosan to triclosan in higher plants. Further mechanistic

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investigation is needed to identify and characterize the enzymes participating in the

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demethylation of methyl triclosan in higher plants.

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In a few previous studies, triclosan was detected in a range of food plants, including lettuce,

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cabbage, radish, carrot, barley, pinto bean, and soybean plant.29–34 In addition, in constructed

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wetlands receiving treated wastewater, various wetland plants, including cattail (Typha latifolia),

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pickerelweed (Pontederia cordata), and grassy arrowhead (Sagittaria graminea) accumulated

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triclosan and triclocarban, contributing to their removal from WWTP effluent.11 However, to date,

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in studies involving the soil-plant continuum, back conversion of methyl triclosan to triclosan

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has not been considered. Findings from this study suggested an additional pathway in the

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environmental cycle of triclosan, and also pointed to a potential cause for prolonged persistence

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of triclosan in ecosystems due to the back and forth transformations. Moreover, as observed for

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methyl triclosan in this study and other pharmaceuticals in humans in previous studies,24–26 it is

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likely that many other PPCPs and emerging contaminants may undergo alkylation and

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dealkylation in the soil-plant system. Therefore, this process should be taken into consideration

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to obtain a more complete understanding of their fate and risks.

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Supporting Information

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Additional details, including cell cultivation procedures, sample preparation procedures,

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instrument analytical conditions, and results from validation experiments, are available free of

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charge via the Internet at http://pubs.acs.org/.

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Acknowledgment

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This research was supported by the U.S. Environmental Protection Agency (Grant No. 835829).

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Table 1. Occurrence of triclosan in common vegetables lettuce and carrot after exposure to

321

methyl triclosan under hydroponic conditions

322 Triclosan (µg/g d.w.) Plant

Tissue Mean*

SD

leaf

10.7Aa

2.2

root

11.5Aab

5.5

leaf

3.1Bb

0.2

root

3.7Bab

1.1

Lettuce

Carrot 323 324

* Multiple comparisons were performed using two-way ANOVA with Tukey test. Letters in

325

uppercase indicate the difference between tissues of lettuce or carrot, and letters in lower case

326

indicate the difference between lettuce and carrot tissues. Groups sharing the same letter suggest

327

no significant differences.

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Figure Captions

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Figure 1. Kinetics of methyl triclosan and triclosan in the cell-medium system. (A)

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Concentration (µg/L) of methyl triclosan in cell culture medium; (B) Concentration (µg/g dry

332

weight) of methyl triclosan (filled circle) and triclosan (open circle) in Arabidopsis cells. Error

333

bars are standard deviation of triplicates.

334

Figure 2. Gas chromatograms (GC) of methyl triclosan and triclosan in (A) viable A. thaliana

335

cells; (B) dead A. thaliana cells at 144 h into incubation.

336

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Figure 1

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Figure 2

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Table of Content

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