Grantee Research Project Results
2019 Progress Report: Reclaimed Water Irrigation: Plant Accumulation and Risks of Contaminants of Emerging Concern (CECs)
EPA Grant Number: R835829Title: Reclaimed Water Irrigation: Plant Accumulation and Risks of Contaminants of Emerging Concern (CECs)
Investigators: Gan, Jay , Trumble, John T. , Dudley, Stacia
Current Investigators: Gan, Jay , Trumble, John T. , Dudley, Stacia , Pennington, Marcus , sun, Chengliang
Institution: University of California - Riverside
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 2015 through August 31, 2018 (Extended to February 28, 2020)
Project Period Covered by this Report: September 1, 2018 through August 31,2019
Project Amount: $749,631
RFA: Human and Ecological Health Impacts Associated with Water Reuse and Conservation Practices (2014) RFA Text | Recipients Lists
Research Category: Water , Human Health
Objective:
In arid and semi-arid regions such as the southwestern U.S., municipally treated wastewater (or reclaimed water) is potentially a valuable water resource that may be used for augmenting agricultural irrigation, thus alleviating water scarcity caused by urbanization and droughts. A hurdle to this beneficial reuse, however, is the potential uptake of and risks from contaminants of emerging concern (CECs) from the treated wastewater via food crops. In this project, we aim to identify CECs with the highest potential for plant accumulation, obtain first-hand values of occurrence of CECs in edible products of common vegetables and other food crops, understand processes governing uptake, translocation, metabolism and accumulation, and characterize acute and sublethal effects of selected CECs on a range of important insects. This project will provide the much-needed first-hand information on the occurrence and potential risks of CECs in food plants when treated wastewater is used for irrigation. The study findings will be of great value to the scientific community and the society-at-large for developing knowledge that promotes the safe reuse of treated wastewater.
Progress Summary:
Novel Analytical Methods:
Plants are complex matrices for analysis and identification of trace contaminants and their metabolites in plant tissues is a great challenge. In particular, it is often formidable to identify unknown biotransformation products in the absence of reference standards, and this analytical challenge is particularly true for contaminants of emerging concern (CECs) that are mostly polar molecules without characteristic structures (e.g., Cl, Br) and in complex matrices such as plants. Using the fibrate drug gemfibrozil as a model CEC and Arabidopsis thaliana as a model plant, we developed and demonstrated a novel analytical framework coupling deuterium stable isotope labeling with high-resolution mass spectrometry (SILAMS) in identifying plant biotransformation products. When exposed in A. thaliana cells, gemfibrozil was quickly taken up into the cells and extensively metabolized. The use of non-labeled and deuterated gemfibrozil at 3:1 ratio created unique diagnostic patterns in mass spectra, enabling the identification of 11 novel Phase II amino acid/peptide conjugates. Similarity in mass fragmentation patterns and chromatographic behaviors was then employed to establish the probable structures. Two major metabolites were further confirmed as glutamate and glutamine conjugates using authentic standards. Most of the identified conjugates were also detected in the whole A. thaliana plant. Therefore, SILAMS offers unique advantages by excluding false matrix positives and helping discern unknown metabolites, including polar conjugates with endogenous biomolecules, with a high degree of confidence. This novel framework may be readily applied to other CECs for high-throughput metabolite screening in plants to improve our understanding of their food safety and human health risks and potential deleterious effects on other species living on plants.
Plant Uptake and Metabolism of CECs:
We have carried out experiments to evaluate uptake potentials of different emerging contaminants by vegetable plants, and also explore metabolism pathways of some common CECs using Arabidopsis thaliana cells and commonly consumed vegetables (i.e., radish, cucumber, lettuce, and carrots) as model plants. These studies have provided novel information on what processes and factors regulate plant uptake of trace contaminants such as CECs, and highlighted that plants can extensively metabolize CECs, but their metabolites, such as conjugates, may maintain partial biological activity and should be considered in comprehensive risk assessment.
In one study, we tested the hypothesis that addition of biosolids could increase the sorption of certain CECs in soil, decreasing their bioavailability. Accumulation of triclosan and triclocarban was measured in roots of radish and carrot grown in soils with or without biosolids. Addition of biosolids significantly prolonged the persistence of triclosan in soil. When expressed in bioaccumulation factor (BCF), accumulation of triclosan drastically decreased in biosolids-amended soils, while the effect was limited for triclocarban. Compared to the unamended soil, amending biosolids at 2% (w/w) decreased BCF of triclosan in the edible tissues of radish and carrot by 85.4 and 89.3%, respectively. Measurement using a thin-film passive sampler provided direct evidence showing that the availability of triclosan greatly decreased in biosolids-amended soils. Partial correlation analysis using data from this and published studies validated that biosolids decreased plant uptake primarily by increasing soil organic carbon content and subsequently sorption. Therefore, contamination of food crops by biosolids-borne contaminants does not linearly depend on biosolids use rates. This finding bears significant implications in the overall risk evaluation of biosolids-borne contaminants.
The potential for food crops to accumulate CECs depends largely on their metabolism in plants, which is poorly understood. We evaluated the metabolism of naproxen and ibuprofen, two of the most used human drugs from the profen family, in Arabidopsis thaliana cells and Arabidopsis plant. The complementary use of high-resolution mass spectrometry and 14C labeling allowed characterization of both free and conjugated metabolites, as well as non-extractable residues. Naproxen and ibuprofen, in their parent form, were conjugated quickly and directly with glutamic acid and glutamine, and further with peptides, in A. thaliana cells. For example, after 120 h, the metabolites of naproxen accounted for >90% of the extractable chemical mass, while the intact parent itself was negligible. The structures of glutamate and glutamine conjugates were confirmed using synthesized standards, and further verified in whole plants. Amino acid conjugates may easily deconjugate, releasing the parent molecule. This finding highlights the possibility that the bioactivity of such CECs may be effectively preserved through direct conjugation - a previously overlooked risk. Many other CECs are also carboxylic acids like the profens. Therefore, direct conjugation may be a common route for plant metabolism of these CECs, making it imperative to consider conjugates when assessing their risks.
We further investigated the metabolism of diclofenac by employing 14C tracing, and time-of-flight (TOF) and triple quadruple (QqQ) mass spectrometers to unravel its metabolism pathways in Arabidopsis thaliana cells. Diclofenac was found to be quickly taken up into A. thaliana cells. Phase I metabolism involved hydroxylation and successive oxidation and cyclization reactions. However, Phase I metabolites did not accumulate appreciably; they were instead rapidly conjugated with sulfate, glucose, and glutamic acid through Phase II metabolism. In particular, diclofenac parent was directly conjugated with glutamic acid, with acyl-glutamatyl-diclofenac accounting for >70% of the extractable metabolites after 120-h incubation. In addition, at the end of incubation, >40% of the spiked diclofenac was in the non-extractable form, suggesting extensive sequestration into cell matter. The rapid formation of non-extractable residue and dominance of diclofenac-glutamate conjugate uncover previously unknown metabolism pathways for diclofenac. In particular, the rapid conjugation of parent highlights the need to consider conjugates of emerging contaminants in higher plants, and their biological activity and human health implications.
Metabolism was further studied for the antibiotic sulfamethoxazole in Arabidopsis thaliana cells as well as cucumber seedlings grown under hydroponic conditions. Using high-resolution mass spectrometry and 14C tracing allowed for sulfamethoxazole metabolism to be comprehensively characterized through all metabolic phases. Seven phase I and II metabolites were identified in A. thaliana cell cultures and cucumber seedlings. Sulfamethoxazole metabolism followed hydroxylation and then rapid conjugation with glutathione and amino acids. Direct conjugation with the parent compound was also observed via acetylation and glycosylation. At the end of 96 and 168 h incubation, >50% of the radiolabeled sulfamethoxazole became non-extractable in both A. thaliana cells and cucumber seedlings, suggesting extensive phase III metabolism and detoxification. The study findings provided information for a better understanding of the uptake and metabolism of sulfamethoxazole in higher plants, highlighting the need to consider metabolic intermediates and terminal fate when assessing the risk of CECs in the soil-plant continuum.
In yet another study, the metabolism of diazepam was investigated in Arabidopsis thaliana cells and cucumber (Cucumis sativus) and radish (Raphanus sativus) seedlings grown in hydroponic solution following acute (7 d)/high concentration (1 mg L-1), and chronic (28 d)/low concentration (1 μg L-1) exposures. Liquid chromatography paired with mass spectrometry, 14C tracing, and enzyme extractions, were used to characterize the metabolic phases. The three major metabolites of diazepam - nordiazepam, temazepam and oxazepam - were detected as Phase I metabolites, with the longevity corresponding to that of human metabolism. Nordiazepam was the most prevalent metabolite at the end of the 5d, 7d and 28d cultivation in A. thaliana cells. At the end of 7d cultivation, non-extractable residues (Phase III) in radish and cucumber seedlings accounted for 14% and 33% of the added 14C-diazepam, respectively. By the end of 28d incubation, the non-extractable radioactivity fraction further increased to 47% and 61%, indicating Phase III metabolism as an important destination for diazepam. Significant changes to glycosyltranferase activity were detected in both cucumber and radish seedlings exposed to diazepam at 1 mg L-1 and 1 μg L.-1 Findings of this study also highlighted the need to consider the formation of bioactive transformation intermediates and different phases of metabolism to achieve a comprehensive understanding of risks of CECs in agroecosystems.
Effect of CECs on Plant Development and Hormone Homeostasis and Enzymatic Response
We have carried out a series of laboratory experiments to understand if and how exposure to low levels of CECs affects plant development, as well as the role of various enzymes in mediating or mitigating the toxicity. These studies provide novel information on the interactions of CECs with plant hormones and enzymes.
Contamination of agricultural soils by pharmaceutical and personal care products resulting from the application of treated wastewater, biosolids and animal wastes may confer phytotoxicity to plants. We investigated the uptake and transport, physiological responses and detoxification of a mixture of 17 CECs in cucumber seedlings. All CECs were detected at higher concentrations in roots compared to leaves, with root activity inhibited in a dose-dependent manner. At 5-50 µg/L, the mature leaves exhibited burnt edges as well as a reduction in photosynthesis pigments. Reactive oxygen species (ROS) production and lipid peroxidation increased with increasing CEC concentrations; and their contents were greater in roots than in leaves for all CEC treatments. Enzymes involved in various functions, including oxidative stress (superoxide dismutase and ascorbate aperoxidase) and xenobiotic metabolism (peroxidase and glutathione S-transferase), were elevated to different levels depending on the CEC concentration. Glutathione content gradually increased in leaves, while a maxima occurred at 0.5 μg L-1 CECs in roots, followed by a decrease thereafter. The findings illustrated the complexity of phytotoxicity after exposure to CEC mixtures, and provided insights into the molecular mechanisms likely responsible for the detoxification of CECs in higher plants.
In a follow up study, we used acetaminophen, one of the most-used pharmaceuticals, to explore roles of glutathione (GSH) conjugation in its biotransformation in crop plants. Acetaminophen was taken up by plants, and conjugated quickly with GSH. After exposure to 5 mg L-1 acetaminophen for 144 h, GSH-acetaminophen conjugates were 15.2±1.3 nmol g-1 and 1.2±0.1 nmol g-1 in cucumber roots and leaves, respectively. Glutathione-acetaminophen was also observed in common bean, alfalfa, tomato, and wheat. Inhibition of cytochrome P450 decreased GSH conjugation. Moreover, the GSH conjugate was found to further convert to cysteine and N-acetylcysteine conjugates. Glutathione S-transferase activity was significantly elevated after exposure to acetaminophen, while levels of GSH decreased by 55.4% in roots after 48 h, followed by a gradual recovery thereafter. Enzymes involved in GSH synthesis, regeneration and transport were consistently induced to maintain the GSH homeostasis. Therefore, GST-mediated conjugation likely played a crucial role in minimizing phytotoxicity of acetaminophen and other CECs in plants.
In a related study, we investigated the mechanism by which plant development is affected by triclosan, the most commonly used antimicrobial agent. Microscopic, pharmacological and biochemical analyses, and histochemical dye staining were used to explore the effects of triclosan on root growth in wheat plants. Exposure to triclosan inhibited root elongation, and significantly triggered hydrogen peroxide (H2O2) production and lipid peroxidation in wheat roots. The inhibition of root growth by triclosan was reversed by dimethylthiourea, a H2O2 scavenger, indicating that alterations of endogenous H2O2 concentrations in root cells were likely linked to triclosan-induced root growth inhibition. The addition of butylated hydroxyanisole, a lipophilic antioxidant, during triclosan treatment completely prevented the increase of lipid peroxidation, but did not alleviate triclosan-induced reduction of root growth. In triclosan-treated wheat roots, the level of indole-3-acetic acid decreased by 68.3%, while the contents of two indole-3-acetic acid oxidative metabolites, indole-3-aldehyde and indole-3-carboxylic acid, increased by 71.3% and 314.4%, respectively. Moreover, the oxidation of auxin induced by triclosan in wheat roots was prevented by dimethylthiourea. These results together suggested that the triclosan-enhanced production of H2O2 induced auxin oxidation, thus leading to the suppression of root growth. Findings of this study improve our mechanistic understanding on how antimicrobial agents such as triclosan affect plant root growth.
We further explored nitric oxide (NO) production and its roles in regulating triclosan tolerance in plants using wheat as an example. The NO content were 1.4-fold higher than that of the control after treatment with 5 µg L-1 triclosan, and increased further at higher triclosan treatment levels. Simultaneously, a significant increase in reduced glutathione, GSH, and a decrease of oxidized glutathione, GSSG, occurred in wheat roots. The GSH:GSSG ratio was 2.2-fold higher than that of the control in the triclosan-treated roots. The elevated GSH level and GSH:GSSG ratio were significantly suppressed by cPTIO, a specific NO scavenger. Enzymes involved in GSH regeneration were also induced by triclosan, with γ-glutamylcysteine synthetase, S-nitrosoglutathione reductase and glutathione reductase enhanced by 51.2, 50.1 and 81.7%, respectively. The elevated activity of these enzymes, however, was similarly abolished by cPTIO. These results clearly suggested that triclosan triggered NO formation to maintain cellular redox by targeting GSH metabolism, and that NO plays a central role in facilitating plant performance under the stress of bioactive xenobiotics analogous to triclosan.
In a separate study, we evaluated the effect of low-dose, chronic exposure to a mixture of 10 CECs, including 4 antibiotics, 3 anti-inflammatory drugs, 1 antiepileptic, 1 beta-blocker, and 1 antimicrobial, on lettuce (Lactuca sativa) and cucumber (Cucumis sativa L.) plants. The CEC mixture was added in nutrient media at 1 to 20X of their typical levels in treated wastewater effluents. Biological endpoints including germination, growth, phytohormone homeostasis, and CEC bioaccumulation were determined. Exposure to the CEC mixture did not affect the germination rate of lettuce seeds, but stimulated root elongation and increased the root-to-shoot biomass ratio during a 7 d cultivation. A dose-dependent decrease in biomass was observed in cucumber seedling after a 30 d exposure, with the highest rate CEC treatment resulting in decreases of 51.2 ± 20.9, 26.3 ± 34.1, and 33.2 ± 41.7% in the below-ground, above-ground, and total biomass, respectively. Levels of abscisic acid were significantly elevated (p < 0.05) in the leaves, but decreased (p < 0.05) in the roots. The dose-response of auxin was characterized by a hormesis effect. A significant 6-fold increase in the stem auxin level was observed at the 1X CEC rate, followed by a decrease to 2-fold the control at the 20X rate. Leaf auxin concentrations also significantly increased at the 1X CEC rate to 16-fold, followed by a decrease at the highest CEC rate. The results of this study suggested that chronic exposure to low levels of CEC mixtures may compromise the fitness of plants, and the impairments are underlined by alterations in hormone balances.
Effects of CECs on Terrestrial Invertebrates:
In a parallel line of research, we evaluated potential biological effects of CECs on agriculturally and economically important insects, including Megaselia scalaris, Trichoplusia ni, and Myzus persicae.
In a study published in the prestigious scientific journal PNAS, we assessed the effects of common pharmaceuticals on an agricultural pest, Trichoplusia ni (Lepidoptera: Noctuidae). Larvae were reared on artificial diets spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations. Trichoplusia ni showed increased developmental time and mortality when reared on artificial diets containing antibiotics, hormones, or a mixture of contaminants. Mortality was also increased when T. ni were reared on tomatoes grown hydroponically with the same concentrations of antibiotics. The antibiotic-treated plants translocated ciprofloxacin through their tissues to roots, shoots, and leaves. Microbial communities of T. ni changed substantially between developmental stages and when exposed to CECs in their diets. The results suggest that use of reclaimed wastewater for irrigation of crops can affect the developmental biology and microbial communities of an insect of agricultural importance.
In a separate study, we assessed the effects of common pharmaceuticals on a cosmopolitan saprophagous insect, Megaselia scalaris (Diptera: Phoridae). Larvae were reared on artificial diets spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations. Female flies showed no oviposition preference for treated or untreated diets. Larvae exposed to caffeine in diets showed increased mortality, and larvae fed antibiotics and hormones showed signs of slowed development, especially in females. The normal sex ratio observed in M. scalaris from control diets was affected by exposure to caffeine and pharmaceutical mixture treatments. There was an overall effect of treatment on the flies’ microbial communities; notably, caffeine fed insects displayed higher microbial variability. Eight bacterial families accounted for approximately 95% of the total microbes in diet and insects. The results suggest that CECs at environmentally relevant concentrations can affect the biology and microbial communities of an insect of ecological and medical importance.
In another study, we further assessed the effects of common pharmaceuticals on an agricultural pest, the aphid Myzus persicae (Sulzer, Hemiptera: Aphididae). Second instar nymphs were transferred to bell peppers (Capsicum annuum) that were grown hydroponically. Treatment plants were spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations found in reclaimed wastewater. M. persicae displayed no differences in population growth or microbial community differences due to chemical treatments. Plants, however, displayed significant growth reduction in antibiotic and mixture treatments, specifically in wet root masses. Antibiotic treatment masses were significantly reduced in the total and root wet masses. Mixture treatments displayed an overall reduction in plant root wet mass. The results suggest that the use of reclaimed wastewater for crop irrigation would not affect aphid populations, but could hinder or delay crop production.
Journal Articles on this Report : 13 Displayed | Download in RIS Format
Other project views: | All 23 publications | 16 publications in selected types | All 16 journal articles |
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Dudley S, Sun C, Jiang J, Gan J. Metabolism of sulfamethoxazole in Arabidopsis thaliana cells and cucumber seedlings. Environmental Pollution 2018;242(Pt B):1748-1757. |
R835829 (2018) R835829 (2019) |
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Dudley S, Sun C, McGinnis M, Trumble J, Gan J. Formation of biologically active benzodiazepine metabolites in Arabidopsis thaliana cell cultures and vegetable plants under hydroponic conditions. The Science of the Total Environment 2019;662:622-630. |
R835829 (2019) |
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Fu Q, Wu X, Ye Q, Ernst F, Gan J. Biosolids inhibit bioavailability and plant uptake of triclosan and triclocarban. Water Research 2016;102:117-124. |
R835829 (2016) R835829 (2017) R835829 (2019) |
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Fu Q, Zhang J, Borchardt D, Schlenk D, Gan J. Direct conjugation of emerging contaminants in Arabidopsis: indication for an overlooked risk in plants? Environmental Science & Technology 2017;51(11):6071-6081. |
R835829 (2017) R835829 (2019) |
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Fu Q, Ye Q, Zhang J, Richards J, Borchardt D, Gan J. Diclofenac in Arabidopsis cells: rapid formation of conjugates. Environmental Pollution 2017;222:383-392. |
R835829 (2017) R835829 (2019) |
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Fu Q, Dudley S, Sun C, Schlenk D, Gan J. Stable isotope labeling-assisted metabolite probing for emerging contaminants in plants. Analytical Chemistry 2018;90(18):11040-11047. |
R835829 (2018) R835829 (2019) |
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Pennington MJ, Rothman JA, Dudley SL, Jones MB, MdFredrick QS, Gan J, Trumble JT. Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant. Proceedings of the National Academy of Sciences of the United States of America 2017;114(46):E9923-E9931. |
R835829 (2017) R835829 (2019) |
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Sun C, Dudley S, McGinnis M, Gan J. Hydrogen peroxide mediates triclosan-induced inhibition of root growth in wheat seedlings. Environmental Pollution 2018;243(Pt A):472-479. |
R835829 (2018) R835829 (2019) |
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Sun C, Dudley S, Trumble J, Gan J. Pharmaceutical and personal care products-induced stress symptoms and detoxification mechanisms in cucumber plants. Environmental Pollution 2018;234:39-47. |
R835829 (2017) R835829 (2019) |
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Sun C, Dudley S, McGinnis M, Gan J. Acetaminophen detoxification in cucumber plants via induction of glutathione S-transferases. Science of the Total Environment 2018;646:431-439. |
R835829 (2018) R835829 (2019) |
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Pennington MJ, Rothman JA, Jones MB, McFrederick QS, Gan J, Trumble JT. Effects of contaminants of emerging concern on Megaselia scalaris (Lowe, Diptera:Phoridae) and its microbial community. Scientific Reports 2017;7(1):8165. |
R835829 (2019) |
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McGinnis M, Sun C, Dudley S, Gan J. Effect of low-dose, repeated exposure of contaminants of emerging concern on plant development and hormone homeostasis. Environmental Pollution 2019;252(Pt A):706-714. |
R835829 (2019) |
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Sun C, Dudley S, Wang J, Gan J. Nitric oxide regulates triclosan-induced redox disequilibrium by enhancing glutathione metabolism in wheat seedlings. Environmental Science & Technology Letters 2019; 6(5):313-317. |
R835829 (2019) |
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Supplemental Keywords:
Water reuse; emerging contaminants; environmental sustainability; PPCPs.Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.