Article pubs.acs.org/est
Rapid Phytotransformation of Benzotriazole Generates Synthetic Tryptophan and Auxin Analogs in Arabidopsis Gregory H. LeFevre,†,‡ Claudia E. Müller,†,‡ Russell Jingxian Li,§ Richard G. Luthy,†,‡ and Elizabeth S. Sattely*,†,∥ †
ReNUWIt Engineering Research Center, ‡Department of Civil & Environmental Engineering, §Department of Chemistry, Department of Chemical Engineering, Stanford University, Stanford, California 94305, United States
∥
S Supporting Information *
ABSTRACT: Benzotriazoles (BTs) are xenobiotic contaminants widely distributed in aquatic environments and of emerging concern due to their polarity, recalcitrance, and common use. During some water reclamation activities, such as stormwater bioretention or crop irrigation with recycled water, BTs come in contact with vegetation, presenting a potential exposure route to consumers. We discovered that BT in hydroponic systems was rapidly (approximately 1-log per day) assimilated by Arabidopsis plants and metabolized to novel BT metabolites structurally resembling tryptophan and auxin plant hormones; 60% of BT. Glycosylated BT was excreted by the plants into the hydroponic medium, a phenomenon not observed previously. The observed amino acid metabolites are likely formed when tryptophan biosynthetic enzymes substitute synthetic BT for native indolic molecules, generating potential phytohormone mimics. These results suggest that BT metabolism by plants could mask the presence of BT contamination in the environment. Furthermore, BT-derived metabolites are structurally related to plant auxin hormones and should be evaluated for undesirable biological effects.
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INTRODUCTION Climate change and continued population growth necessitate the use of nontraditional water supplies, particularly in arid regions.1 “Recycled” or “reclaimed” water (i.e., highly treated wastewater) and captured stormwater runoff are increasingly used for landscape and agricultural irrigation or for groundwater recharge.2 Discoveries of contaminants of emerging concern (CECs) or micropollutants incompletely removed during wastewater treatment3,4 have raised concerns about the potential health impacts of recycled water. 5 For example, some pharmaceuticals6−8 and antimicrobials9 are taken up into food crops following plant exposure to recycled water. The biological effects of CECs in the environment are often unknown or poorly predicted.10 Benzotriazoles (BTs) are high-volume production, xenobiotic anticorrosive agents used in a variety of consumer and industrial products, such as plastic films, engine coolant, brake fluid, deicing fluids, and dishwashing detergents.11,12 BTs are present in stormwater runoff as a component of antifreeze coolant, especially near airports where deicing operations occur.13,14 Common BTs in aquatic environments are 1-H benzotriazole (BT) and tolyltriazole (a mixture of 4- and 5-1-H-methylbenzo© 2015 American Chemical Society
triazole; 4- or 5-MeBT). BTs are considered recalcitrant CECs due to their biological and chemical stability in the environment.11,12,17,18 The relatively polar nature (BT log KOW = 1.3) and high microbial recalcitrance11,12,19 results in incomplete BT removal via conventional wastewater treatment practices and concomitant release to receiving waters. Reported microbial transformation half-lives for BT range from ∼1 to 114 days;18,20 5-MeBT half-lives range from ∼1 to 31 days.20,21 BT in the environment may undergo some photodegradation, but reaction rates are considered relatively slow.22 Ultraviolet (UV) disinfection processes in wastewater plants are considered insufficient15,23 to remove BT, requiring ozone or other advanced oxidation treatment.23,24 BTs have been measured at several hundred nanograms per liter (ng/L) in recycled water in Australia following reverse osmosis and UV treatment.15,16 Indeed, BT has been proposed as a proxy for polar wastewater Received: Revised: Accepted: Published: 10959
June 4, 2015 August 14, 2015 August 24, 2015 August 24, 2015 DOI: 10.1021/acs.est.5b02749 Environ. Sci. Technol. 2015, 49, 10959−10968
Article
Environmental Science & Technology micropollutants3 and treated as a “conservative pollutant” tracer in rivers,25 with little understanding of its ultimate fate. BTs are widely distributed in aquatic environments. In a recent survey4 of wastewater effluents (90 European treatment plants), BT was one of the most frequently detected compounds (97% occurrence) with concentrations of up to 221 μg/L (av 6.3 μg/ L). Concentrations of BT in rivers measured in Europe are in the nanograms to micrograms per liter range.4,11,13,17,26 The mean BT concentration in the Haihe River downstream from Beijing was 2 μg/L; 95% of this flow is subsequently used for agricultural irrigation.25 Concentrations of 4- and 5-MeBT have been measured >1 mg/L from an airport outfall in Wisconsin following deicing operations.14 5-MeBT was detected in >30% of streams measured throughout the United States27 at a median concentration of 0.39 μg/L, and biosolid-amended soils have been found to accumulate BTs.28 BTs have also been measured in human urine29 and indoor dust samples.30 BTs are toxic to aquatic life at high levels31,32 and may have endocrine disrupting properties.33,34 The Microtox EC50 values for BT and 5-MeBT are approximately 41 and 5 mg/L, respectively.32,35 Chronic toxicity tests indicated inhibition levels for 4,5-MeBT approximately between 5 and 24 mg/L,32 but little biological information exists on low-level or chronic exposure to BTs.11 Further toxicity results are summarized elsewhere.12 Benzotriazole-based UV stabilizers have also shown significant aryl hydrocarbon receptor ligand activity, suggesting the possibility of an immune modulatory response.36 The Health Council of The Netherlands has classified BT as a suspected human carcinogen,12 but BTs are unregulated by the U.S. Environmental Protection Agency (EPA) as a drinking water contaminant.37 Australia has established a value of 20 μg/L as a “guideline” for water recycling.15 There is growing interest but limited research in the uptake and transformation of CECs in plant tissues. For example, the antimicrobial triclosan is rapidly taken up and metabolized in carrot cell cultures,9 and the pharmaceutical carbamazepine and its metabolites have been measured in food crops.7 Plant metabolites are often assumed less harmful than the parent pollutant,9,38 but the biological effects of these metabolites are largely unknown. Some work suggests that BT can be taken up by plants39 but with no direct demonstration of phytotransformation or understanding of transformation products. Because humans and other animals could be exposed to BTs through recycled water applied to food crops or vegetated stormwater capture facilities, understanding plant processes is important for conducting exposure and risk assessments as well as understanding the fate of BT in the environment. This study investigated plant uptake of BT and identified multiple novel BT metabolites via untargeted metabolomics. Using a hydroponic system, we demonstrate that BT is rapidly taken up and metabolized at environmentally relevant concentrations by the model plant Arabidopsis thaliana. The results from this work suggest that the microbially recalcitrant micropollutant BT is transformed by plants, and that BT-derived metabolites that accumulate in plants may mask the presence of BT in the environment or have unknown biological effects such as phytohormone disruption.
0191, San Diego, CA). Synthesized chemicals are described in the SI. All solvents and chemicals used for LC-MS analysis were of LC-MS grade. Experimental Design. Plant Growth and BT Uptake. Wildtype Arabidopsis thaliana (Col-0 ecotype) seeds were sterilized using a bleach solution (see SI) and allowed to stratify overnight at 4 °C. Arabidopsis is commonly used as a model plant for many fundamental biological and metabolism experiments (e.g., refs 40−43). Seeds were added to autoclaved Magenta boxes (Magenta Corp., n = 30 seeds per box) with 25 mL of filtersterilized (0.22 μm PES, Corning) Murashige and Skoog (MS) basal medium. The MS medium contained (per 1L): Milli-Q water, 4.43g MS basal medium with vitamins (PhytoTechnology Laboratories; M519), 0.5 g of MES hydrate (PhytoTechnology Laboratories, CAS: 14522-94-8), 5.0 g of sucrose, and was adjusted to pH = 5.7 using 1N KOH. Magenta box edges were wrapped with breathable microporous tape (3M) and placed into a growth chamber (Percival) under fluorescent growth lights with a 16 h light/8 h dark period at 22 °C and a relative humidity of 50%. Plant seedlings were grown for 14 days prior to any BT exposure and were visually checked for any signs of microbial contamination. Observed germination rates were typically high (>90%) in the boxes. Any contaminated or poorly germinating plant boxes were discarded and not included for use in the experiments. All boxes were treated identically as biological replicates prior to BT exposure. Following the initial growth period in sterile hydroponic medium, plants were exposed to an environmentally relevant BT concentration (3 μg/L). Plant boxes were harvested immediately after spiking and then daily for 3 days. At each harvest point, n = 3 biological replicate boxes were harvested in their entirety by removing all plants, yielding a total dry biomass of approximately 0.1 g. At t = 3 days, the hydroponic medium was drained from the plant boxes and replaced with new medium. Half of the systems were replaced with medium containing BT for re-exposure and half contained clean medium for depuration. These remaining sample boxes (n = 3 entire boxes harvested per day) were then harvested daily until t = 8 days. Concurrently, no-plant controls containing the sample BT spiked hydroponic medium were harvested in parallel to examine any abiotic (e.g., direct photolysis) loss processes. Similarly, no-plant BT negative control sample boxes spiked with filter-sterilized exudates harvested from Arabidopsis plants (see SI for details) were also conducted to detect any effects of exudate-catalyzed abiotic BT degradation (e.g., indirect photolysis) or other abiotic exudate effects.44 Plants with no BT exposure were also grown in parallel to act as positive controls (i.e., plant seeds were viable) and to compare overall biomass with the experimental treatments. The overall mass balance was determined by measuring BT in the hydroponic medium, plant tissue, and wall rinse. Samples were harvested at predetermined time intervals by removing the boxes from the growth chamber, separating the whole-plant tissue (i.e., both roots and shoots) from the liquid medium using forceps and immediately freezing the liquid medium in the closed Magenta box at −20 °C until liquid medium extraction and wall rinse. The plant tissue was lightly dabbed dry using a Kim-wipe, added to a “safe-lock” microcentrifuge tube (Eppendorf), and the fresh weight was determined. Plant samples were frozen at −20 °C until freezedrying. Freeze-drying was completed using a lyophilizer overnight. Dry biomass content was determined, then samples were frozen at −20 °C until extraction. Dry biomass and fresh
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METHODS AND MATERIALS Chemicals. Chemicals used in these experiments include the following: 1-H-benzotriazole (BT; Fluka, CAS 95-14-7), 1-Hbenzotriazole-d4 (BT-d4; CDN isotopes, CAS: 1185072-01-0), and hexopyranosyl-1H-benzotrizazole (GBT; ChemDiv 728710960
DOI: 10.1021/acs.est.5b02749 Environ. Sci. Technol. 2015, 49, 10959−10968
Article
Environmental Science & Technology
procedure except that no internal standard was added and only one extraction was conducted per sample. Quantification via LC-MS/MS. Benzotriazoles were quantified in positive mode using liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS; Applied Biosystems API 3000) with Shimadzu SCL-10A VP system controller and Analyst 1.5.2 software (AB SCIEX). The chromatography column was a Higgins Analytical Sprite Targa C18 (40 × 2.4 mm, 5 μm). The mobile phases were 0.4% formic acid in water (A) and in methanol (B). The mobile phase gradient (as percent B) was a follows: 10% for 0−1 min, 10−80% for 1−4 min, 80% for 4−5.5 min, and 80−20% for 4.4−6.5 min at a flow rate of 0.2 mL/min. Injection volume was 10 μL. A 6 min equilibration time was set between each sample run. The MS/ MS was set in multiple reaction monitoring mode (MRM, Table S.1). Two MRM transitions were used for each compound (Table S.1) for quality control. A six-point internal standard normalized external calibration curve was used to account for surrogate recovery and matrix effects during ionization. The instrument response was linear throughout the calibration range. The instrument detection limit45 for BT without enrichment was 16 ng/L. Hydroponic medium samples were both directly injected and enriched via SPE as described as a quality assurance step. Untargeted Metabolomics via LC-QTOF-MS. An Agilent 1260 LC coupled to a 6520 accurate mass quadrupole−time-offlight (QTOF) mass spectrometer with MassHunter Software (Agilent Technologies, version B.04.00) and Phenomonex Gemini-NX C18 (100 × 2.00 mm, 5 μm) column was used for untargeted metabolomics work. The mobile phases were 0.1% formic acid in water (A) and in acetonitrile (B). The mobile phase gradient (as percent B) was as follows: 5−50% for 25 min, 50−95% for 13 min, 95% for 5 min, 95−5% for 1 min, and hold at 5% 5 min at a flow rate of 0.4 mL/min for a total run time of 50 min. The injection volume was 10 μL. The first 60 s of each run was sent to waste. Fragmentor energy was 150 V. Mass range collected was over 50−1000 m/z at 1 spectra/s. Metabolomics runs were conducted both in positive and negative ionization mode (separately) because the chemical nature of the transformation products was unknown. The instrument was calibrated prior to each run, with maximum error residual during calibration of
