Progress 09/01/12 to 09/30/15
Outputs
Target Audience:Targeted audiences of the proposed work include environmental research community, environmental consultants, regulatory agencies, and coordinators of biosolids land application program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project is being primarily executed by Ph.D.-level graduate students - Ms. Sara Nason and Ms. Elizabeth Miller. How have the results been disseminated to communities of interest?The graduate students presented their research results (to date) at the Society for Environmental Toxicology and Chemistry North America meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the past year (Oct. 2014-Sep. 2015), we have published a critical review of plant root uptake of pharmaceutical and personal care product ingredients (PPCPs) in the journal Environmental Science and Technology (Miller et al. 20151). We adapted a new extraction method for PPCPs in vegetable tissues and continued to work on plant-induced rhizosphere pH changes, sorption of PPCPs to clay, and partitioning of PPCPs to lipid membranes. We also began testing PPCP toxicity and how exposure to one PPCP may affect how plants respond to other PPCPs. We presented our work at the Society for Environmental Toxicology and Chemistry (SETAC) Young Environmental Scientists meeting in Petnica, Serbia and at the SETAC North America meeting in Salt Lake City, Utah. Our critical review covers pertinent soil chemistry, plant biology, models for plant uptake of organic contaminants, and published literature of uptake of PPCPs by specific crops, with an eye towards developing better predictive models for plant uptake of current and future PPCPs. We reviewed the processes expected to influence root uptake of PPCPs, evaluated the current literature on uptake of PPCPs, assessed models for predicting uptake of these compounds, and provided recommendations for future research that will improve mechanistic understanding and model development. In the lab, we adapted the methods of Goldstein et al. 20142 and Malchi et al. 20143 to work for our compound list. This method uses only one ASE cycle (instead of multiple ASE cycles with different solvents) and yields improved recoveries compared to our previous extraction method. We plan to continue our work with Arabidopsis mutants using this new extraction method. We are also determining liposome-water partitioning coefficients (Klipw) for the pharmaceuticals lamotrigine and sulfamethoxazole partitioning into different types of phospholipid liposomes as a potential surrogate measure for partitioning to plant membranes. Currently, n-octanol-water partitioning coefficients are used to model uptake and bioaccumulation of organic contaminants, but this metric is a poor surrogate for ionizable compounds partitioning into biological membranes (which contain polar moieties). We believe Klipw may be a better surrogate, but little information about Klipw for PPCPs is available in the literature. We are testing different ionization states and different liposome compositions to determine the extent that partitioning is affected by pharmaceutical/membrane charge and phospholipid properties. We also continued our work on how plant-induced changes to rhizosphere pH may affect uptake and bioaccumulation of PPCPs. To test whether plants change rhizosphere pH and how this affects uptake of PPCPs, we developed a method of growing wheat (based off of the method of Bravin et al. 20094) that separates roots grown in a mat from a thin layer of sand or clay to simulate the rhizosphere. When the wheat plants are grown on different nitrate: ammonia nutrient ratios, they excrete compounds that alter the pH of the sand/clay. We are testing whether this pH change also affects wheat shoot bioaccumulation of ionizable PPCPs, starting with lamotrigine. In conjunction with these experiments, we are performing clay sorption experiments to account for changes in sorption to the clay as a function of pH. PPCPs and their metabolites may induce effects in plants that impact uptake processes operative in the root or elsewhere in the plant. We are using root length assays,5 in which wild-type Arabidopsis thaliana seeds are sprouted and grown for seven days on agar-filled petri dishes containing one or more PPCP to determine PPCP toxicity to plants. At the end of the exposure period, the length and curvature of the roots is measured using ImageJ or RootNav software and compared to that of unexposed plants. We have performed root length assays for individual compounds at 0.1 - 1000 mg/L to determine baseline individual toxicity of PPCPs. Existing terrestrial phytotoxicity studies have focused mainly on antibiotics, and there is minimal information available about other classes of compounds. The existing literature also focuses on testing the toxicity of individual compounds, but effluent-irrigated crops are exposed to PPCPs as mixtures. To test the hypothesis that plants exposed to mixtures of PPCP would exhibit increased toxicity than those exposed to individual compounds, we are performing root length assays with 2-20 PPCPs in combination. We have found that sulfamethoxazole and trimethoprim have a synergistic toxic effect on A. thaliana that is greater than the sum of their individual activity and that carbamazepine decreases the toxicity of lamotrigine. We are planning more root length assays using different plant species to determine whether these affects are also applicable to crop species. References: (1)Miller, E. L.; Nason, S. L.; Karthikeyan, K.; Pedersen, J. A. Root Uptake of Pharmaceutical and Personal Care Product Ingredients. Environ. Sci. Technol. 2015, acs.est.5b01546. (2)Goldstein, M.; Shenker, M.; Chefetz, B. Insights into the uptake processes of wastewater-borne pharmaceuticals by vegetables. Environ. Sci. Technol. 2014, 48, 5593-5600. (3)Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B. Irrigation of root vegetables with treated wastewater: Evaluating uptake of pharmaceuticals and the associated human health risks. Environ. Sci. Technol. 2014, 48, 9325-9333. (4)Bravin, M. N.; Martí, A. L.; Clairotte, M.; Hinsinger, P. Rhizosphere alkalisation - A major driver of copper bioavailability over a broad pH range in an acidic, copper-contaminated soil. Plant Soil 2009, 318, 257-268. (5)Hillis, D. G.; Fletcher, J.; Solomon, K. R.; Sibley, P. K. Effects of ten antibiotics on seed germination and root elongation in three plant species. Arch. Environ. Contam. Toxicol. 2011, 60, 220-232.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Miller, E. L.; Nason, S. L.; Karthikeyan, K.; Pedersen, J. A. Root Uptake of Pharmaceutical and Personal Care Product Ingredients. Environ. Sci. Technol. 2015, acs.est.5b01546.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
The Society of Environmental Toxicology and Chemistry (SETAC) 4th Young Environmental Scientists Meeting, 14-19 March, 2015, Petnica, Serbia
Title: "Influence of Casparian strip thickness and energy production on root uptake of pharmaceutical and personal care product ingredients" Authors: Elizabeth Lianne Miller, Sara L. Nason, K.G. Karthikeyan, and Joel Pedersen.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
The Society of Environmental Toxicology and Chemistry (SETAC) 4th Young Environmental Scientists Meeting, 14-19 March, 2015, Petnica, Serbia.
Title: "Assessment of pharmaceutical phytotoxicity in Arabidopsis thaliana"
Authors: Sara L. Nason, Elizabeth Lianne Miller, K.G. Karthikeyan, and Joel A. Pedersen.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
The Society of Environmental Toxicology and Chemistry (SETAC) North America 36th Annual Meeting, 1-5 November, 2015, Salt Lake City, Utah. Title: "Mixture effects on uptake of pharmaceuticals and personal care products by Arabidopsis thaliana"
Authors: Sara L. Nason, Elizabeth Lianne Miller, K.G. Karthikeyan, and Joel A. Pedersen.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Targeted audiences of the proposed work include environmental research community, environmental consultants, regulatory agencies, and coordinators of biosolids land application program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project is being primarily executed by two Ph.D.-level graduate students - Ms. Sara Nason and Ms. Elizabeth Miller. How have the results been disseminated to communities of interest? Both the graduate students presented their research results (to date) at the the Society for Environmental Toxicology and Chemistry North America meeting in Vancouver, BC (Nov. 2014). What do you plan to do during the next reporting period to accomplish the goals? We will continue all activities as originally proposed and approved.
Impacts
What was accomplished under these goals?
Two graduate students worked on the project for the entire duration of 2014. In the past year, we have compiled and analyzed the pharmaceuticals and personal care products (PPCP) crop uptake literature and submitted a critical review of this literature to the journal Environmental Science and Technology. We finalized the development of our extraction and quantification method for PPCPs in vegetable tissues and used the method to test for PPCPs in a variety of commercially-available produce. We also analyzed uptake by Arabidopsis thaliana wild-type and esb1 mutant plants to putatively identify PPCP root uptake pathways. Finally, we presented our method and Arabidopsis work at the Society for Environmental Toxicology and Chemistry North America meeting in Vancouver, BC. Our critical review covers pertinent plant biology, models for plant uptake of organic contaminants, and published literature of uptake of PPCPs by specific crops, with an eye towards how these three often disparate directions of study should influence one another to develop and validate better predictive models for plant uptake of current and future PPCPs. We review the many descriptive PPCP uptake studies that have been conducted and what has been learned from them, focusing on trends within compound classes and exposure risk from consumption of contaminated crops, and suggest areas of study to improve current uptake models and ways in which descriptive studies could be performed/reported to better inform future model development. In the laboratory, we finished developing a novel, streamlined accelerated solvent extraction and liquid chromatography-tandem mass spectrometry method for detecting PPCPs spanning a broad range of physicochemical properties in plant matrices. Samples are sequentially extracted using three solvents: neutral, acidic, and basic. Minimal clean-up is required prior to sample analysis. Compounds are separated by single-injection high performance liquid chromatography and detected via triple quadrupole mass spectrometry with electrospray ionization and polarity switching. Most previously published methods require solid phase extraction clean up and multiple injections to analyze a full range of basic, neutral, and acidic PPCPs. Our method is simpler and more easily automated, while achieving similar recoveries and reporting limits. We validated this method by measuring accumulation of PPCPs in Arabidopsis thaliana grown hydroponically in spiked nutrient solution and investigated PPCP occurrence in opportunistically sampled commercially available strawberries, iceberg lettuce, spinach, celery, kale, swiss chard, and onions. The method allowed detection of all target analytes in A. thaliana. None of the targeted analytes were detected in the commercial produce samples tested. We also used A. thaliana esb1 mutants to investigate the influence of Casparian strip thickness on uptake route of PPCPs with varying physicochemical properties. We compared uptake into roots and above-ground tissues of wild-type and mutant A. thaliana to test the hypotheses that the degree of PPCP uptake via the apoplastic route decreases with increasing thickness and integrity of the Casparian strip. We further hypothesized that bioaccumulation increases with compound lipophilicity and with the fraction of ionizable PPCPs in the neutral form by influencing their ability to pass the Casparian strip (the waxy structure made of suberin and lignin that lines the endodermis and acts as a barrier to uptake into the transpiration stream by blocking the space between cells surrounding the vasculature). Esb1 mutants produce twice the amount of suberin as wild-type plants. A. thaliana were grown on liquid nutrient solution spiked with 100 µg/L of the PPCPs for 2 weeks prior to harvest and analysis of tissues. PPCPs were extracted from plant tissue and quantified using our novel method described above. We found that the PPCP concentrations in nutrient solution were stable, and all PPCPs were taken up into roots and/or above-ground tissues at detectable levels. Trimethoprim, valsartan, and fluoxetine may be transported via the apoplastic route (through the intercellular space), as they show significantly reduced uptake to above-ground tissues with increased suberin content. The other PPCPs in this study are likely transported via the symplastic pathway (through cells), as their uptake to above-ground tissues was the same or increased with increased root suberin content. In addition to the above projects, we have also begun work investigating the effects on PPCP uptake of blocking plant energy production, plant-induced rhizosphere pH changes, sorption of PPCPs to clay, and partitioning of PPCPs to lipid membranes.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
1) Miller, E.L., S.L. Nason, C.J. Hedman, K.G. Karthikeyan, J.A. Pedersen. 2014.Influence of Casparian Strip Thickness on Root Uptake of Pharmaceuticals and Personal Care Products (Poster Presentation at the Society for Environmental Toxicology and Chemistry North America meeting in Vancouver, BC. - Nov. 2014).
2) Nason, S.L., E.L. Miller, C.J. Hedman, K.G. Karthikeyan, J.A. Pedersen. 2014. Pharmaceuticals and Personal Care Products in Commercial Produce and Hydroponically Grown Arabidopsis thaliana (Poster Presentation at the Society for Environmental Toxicology and Chemistry North America meeting in Vancouver, BC. - Nov. 2014).
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Targeted audiences of the proposed work include agricultural producers, environmental research community, environmental consultants, regulatory agencies, and coordinators of biosolids land application program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project is being primarily executed by two Ph.D.-level graduate students - Ms. Sara Nason and Ms. Elizabeth Miller. How have the results been disseminated to communities of interest? We are only in Year 1 of the project and are currently working on two manuscripts for publication in peer-reviewed journals. In addition, the graduate students will start presenting project results at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue all activities as originally proposed and approved.
Impacts
What was accomplished under these goals?
Impact: Land application of biosolids and reclaimed water irrigation are quite popular practices worldwide, the former in both humid/dry regions and the latter in semi-arid/arid areas. Both practices offer numerous benefits (reliable water supply from reclaimed water; nutrients + organic matter inputs) that could enable agricultural production to be more sustainable, an important consideration given increasing global food demand and erratic rainfall patterns. However, the presence of certain constituents could impose restrictions and create serious environmental risks. Specifically, pharmaceuticals and personal care products (PPCPs), a subset of chemicals of emerging concern (CEC), deserve serious attention as wastewater treatment processes are not effective in their removal and they have been detected in wastewater effluents, biosolids and biosolid-applied soils, besides surface and ground waters. These CECs can be taken up by crop plants and there is a paucity of information on polar/ionogenic CECs (characteristics of PPCPs) and underlying mechanisms for CEC translocation to different plant components are poorly understood. To address these knowledge gaps, we are systematically evaluating the bioaccumulation of CECs with contrasting chemical characteristics by crop plants differing in their edible parts (tomato, carrot, and spinach). Specific project objectives are to: a) determine the role played by soils and biosolids on the bioaccumulation potential of CECs, and b) elucidate the influence of CEC chemical characteristics on plant uptake. Project results will increase our understanding of the fate of wastewater-derived chemicals (in biosolids and treated effluents) in agricultural systems. Accomplishments: Two graduate students have worked on this project during the 2013 project period. In 2013, we updated our test compounds list, developed and optimized methods for hydroponically growing three species of plants, tested several methods for extracting PPCPs from plant tissue, developed and optimized an LC-MS/MS method, and begun a review paper on PPCP uptake by plants. We developed methods for hydroponically growing Arabidopsis thaliana, spinach, and radishes to maximize plant survival and biomass production. Radishes were chosen over carrots because they grow more quickly and consistently in hydroponics. Our systems are entirely constructed of polypropylene to minimize PPCP sorption. In January 2014, we will begin uptake screening experiments using hydroponic Arabidopsis thaliana, as well as continuing work developing a method for growing tomatoes. While developing and testing our hydroponics methods, we developed and optimized our analytical procedures using grocery store-bought spinach spiked with known amounts of our chosen PPCPs. Our finalized list of PPCPs consists of: cimetidine, diphenhydramine, ranitidine, doxycycline, metformin, warfarin, sulfamethoxazole, triclocarban, triclosan, naproxen, metoprolol, gemfibrozil, carbamazepine, ciprofloxacin, caffeine, amitriptyline, and albuterol. Using published extraction procedures (sonication with acetonitrile and MTBE followed by clean up using SPE), we performed a time-course experiment and found that extracting plant material immediately after it is spiked yields higher recoveries than waiting between spiking and extracting. This is relevant for determining when to add the spike of internal standards during uptake experiments. For the remainder of our method experiments, we have performed extractions immediately after spiking. We also compared sample preparation methods and found that flash freezing spinach prior to extraction yields significantly higher compound recoveries than freeze drying for multiple compounds. Both methods are commonly used in the literature. Despite lower recoveries, we chose to use freeze dried spinach for the rest of our experiments because it is easier to work with and can be stored for longer duration without degradation occurring. Autoclaving the spinach prior to freeze-drying and extraction did not change yields, indicating that enzyme degradation did not affect our results. Other extraction methods we have tested include soxhlet extraction followed by cleanup with gel-permeation chromatography (GPC) and accelerated solvent extraction (ASE) with and without pH adjustments. The soxhlet and ASE (no pH adjustment) methods also had very low recoveries for the basic compounds. The pH adjusted ASE experiment is still in progress, but it is expected to be more successful for a wider variety of PPCPs. We plan to finalize this method and submit a manuscript in 2014. In addition to developing and optimizing our extraction procedure, we have also developed a detection/quantification method on an Agilent LC-MS/MS capable of polarity switching. While previous methods, such as EPA Method 1694, require four runs per sample to analyze all of our chosen compounds via LC-MS/MS, our new method requires only one run per sample. We plan to begin running extracted samples (not just standards) using this method in January 2014. Both graduate students on the project have been trained to use this instrument. In 2013, we also began a comprehensive review of the current plant PPCP uptake literature. This manuscript, an analysis and critique of all PPCP plant uptake studies and trends in their results, is currently in progress, and we plan to submit it to the journal Environmental Science and Technology in spring 2014.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Land application of biosolids and reclaimed water irrigation are quite popular practices worldwide, the former in both humid/dry regions and the latter in semi-arid/arid areas. Both practices offer numerous benefits (reliable water supply from reclaimed water; nutrients + organic matter inputs) that could enable agricultural production to be more sustainable, an important consideration given increasing global food demand and erratic rainfall patterns. However, the presence of certain constituents could impose restrictions and create serious environmental risks. Specifically, pharmaceuticals and personal care products (PPCPs), a subset of chemicals of emerging concern (CEC), deserve serious attention as wastewater treatment processes are not effective in their removal and they have been detected in wastewater effluents, biosolids and biosolid-applied soils, besides surface and ground waters. These CECs can be taken up by crop plants and there is a paucity of information on polar/ionogenic CECs (characteristics of PPCPs) and underlying mechanisms for CEC translocation to different plant components are poorly understood. To address these knowledge gaps, we will systematically evaluate the bioaccumulation of CECs with contrasting chemical characteristics by crop plants differing in their edible parts (tomato, carrot, and spinach). Specific project objectives are to: a) determine the role played by soils and biosolids on the bioaccumulation potential of CECs, and b) elucidate the influence of CEC chemical characteristics on plant uptake. Project results will increase our understanding of the fate of wastewater-derived chemicals (in biosolids and treated effluents) in agricultural systems. Targeted audiences of the proposed work include agricultural producers, environmental research community, environmental consultants, regulatory agencies, and coordinators of biosolids land application program. The project will result in accelerated solvent extraction and LC-MS/MS methods optimized to recover and measure selected CECs in plant matrices, rigorous quantification of the extent of CEC uptake by food crops (in the presence and absence of soils and biosolids), and improved understanding of the influence of physico-chemical properties on CEC uptake by plants. Deliverables include annual reports and research articles. Results will be disseminated at the Tri-Societies (ASA-SSSA-CSA) meetings, ACS, SETAC, and published in peer-reviewed journals. PARTICIPANTS: 1) Ms. Sara Nason - Ph.D. student (Environmental Chemistry & Technology), University of Wisconsin-Madison. 2) Ms. Elizabeth Miller - Ph.D. student (Molecular and Environmental Toxicology), University of Wisconsin-Madison. 3) K.G. Karthikeyan - project PI. 4) Joel Pedersen - project PI. 5) Curtis Hedman - Wisconsin State Laboratory of Hygiene. TARGET AUDIENCES: Targeted audiences of the proposed work include agricultural producers, environmental research community, environmental consultants, regulatory agencies, and coordinators of biosolids land application program. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The proposed project will investigate the bioaccumulation of different (and chemically distinct) chemicals of emerging concern (CECs) by three types of agricultural crops under controlled-environmental conditions. Greenhouse hydroponic experiments will be conducted in the absence (soil-less) and presence of soils and with and without biosolids amendment. In addition, batch experiments will be performed to quantify sorption of CECs to soils and biosolids-derived dissolved organic matter. Thirteen CECs have been selected based on their occurrence in treated wastewater or biosolids. We will use Arabidopsis Thaliana for initial screening of plant uptake of CECs (all 13 CECs will be tested) in soil-less hydroponic systems. We will investigate uptake of all target CECs by Arabidopsis. From the CECs that are efficiently taken up by Arabidopsis, we anticipate choosing 4-5 compounds for further investigation of uptake by spinach, carrot and tomato in hydroponic (soil-less) systems. To determine the influence of soils and organic matter (from biosolids) on CEC bioaccumulation counterpart greenhouse studies will be conducted. These experiments represent flow-through irrigation systems with reclaimed wastewater in the presence and absence of biosolids. From the hydroponic studies, three CEC compounds with highest plant uptake potential with varying chemical characteristics will be chosen for subsequent pot experiments. Greenhouse pot experiments will involve three CEC compounds, two soil types (silt loam and loamy sand), three plants (+ one plant-free control). Experiments will be conducted with and without biosolids amendment. Results will be used to determine influence of CEC chemical characteristics on their accumulation in different plant components. We plan to extract CECs from water, soils, biosolids and plant tissues and analyze them by LC-MS. This project commenced only in Sep. 2012 and relevant activities conducted to date (4 months) include: (a) Recruitment of 2 Ph.D. level graduate students (Ms. Sara Nason and Ms. Elizabeth Miller) to work on this project, (b) Selection of Arabidopsis thaliana as the initial test plant including investigation of previous work and availability of mutants that can be used to isolate CEC transport pathways, (c) Final selection of test CEC compounds including compilation of essential physic-chemical properties, (d) Design of hydroponic system for growing Arabidopsis Thaliana, (e) Investigation of specialized methods to grow carrots hydroponically, (f) Optimization of LC-MS/MS methods for detection of selected CECs including initial training of graduate students to use this instrument, and (g) Initial testing of accelerated solvent extraction methods for plant samples.
Publications
- No publications reported this period
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