AGRONOMIC AMENDMENTS FOR MITIGATION OF ORGANIC ARSENIC UPTAKE BY RICE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1007964
• Grant No.: 2016-67012-24673
• Project No.: DEL00750
• Proposal No.: 2015-03495
• Program Code: A7201
• Project Start Date: Jan 1, 2016
• Project End Date: Dec 31, 2017
• Grant Year: 2016

Project Director
Limmer, M. A.

Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717

Performing Department
Plant and Soil Sciences

Non Technical Summary
Worldwide, arsenic frequently contaminates rice, presenting a risk to food safety. Additions of silicon have shown promise in reducing uptake of arsenite, but reducing uptake of organic arsenic is less studied. Uptake of organic arsenic compounds may also cause straight-head disease in rice, thereby lowering yields. The overall goal of this research is to develop agronomic techniques for reducing rice uptake of organic arsenic compounds, particularly monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Towards this goal, the project will investigate the following three factors' ability to limit rice uptake of organic arsenic: silicon amendments, organic acid amendments, and nitrogen source. Amendments successful in reducing uptake of DMA and MMA in hydroponic studies will be tested in field trials to improve the nation's food safety via reduced risk to consumers. This project also includes several career development and mentorship aspects, including the co-development of a plant-contaminant interactions course, mentorship of students, attending career development workshops, and learning of new analytical skills. My mentor, Dr. Seyfferth, will be an invaluable asset due to her experience with uptake of inorganic contaminants by agronomic crops. This project will aid my personal goal of becoming a leading teacher-scholar at an academic institution through the unique skills and experiences of my mentor and the facilities at the University of Delaware.

Animal Health Component

0%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	1530	2000	50%
711	1530	2020	50%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
1530 - Rice;

Field Of Science
2020 - Engineering; 2000 - Chemistry;

Keywords

arsenic

Goals / Objectives
The long-term goal of this research is understand how As, particularly organic species, enter rice and to develop techniques for reducing the risk As presents to human health via exposure to contaminated rice. To accomplish this goal, I have set the following objectives:1. Investigate the effect of Si amendments on organic As uptake in hydroponic rice.Hypothesis: Si amendments will have minimal effects on organic As uptake.2. Test additional amendments for their ability to outcompete DMA and MMA uptake by hydroponic rice.Hypothesis: A compound with similar physicochemical properties to DMA and MMA will compete with the transport of these organic As compounds.3. Assess the effect of ammonium addition on organic As uptake in hydroponic rice.Hypothesis: Ammonium addition will acidify the apoplastic space increasing uptake of MMA and DMA.4. Perform field trials with amendments identified in objectives 1, 2, and 3.Hypothesis: Hydroponic experimental results will be representative of field trial results.

Project Methods
Objective 1 Methods:Rice seedlings will be grown hydroponically in 1-L vessels and exposed to no DMA/MMA, 5 µM DMA, or 5 µM MMA and varying amounts of bioavailable Si in triplicate. Si will be added as silicic acid at 5 different levels: 50, 250, 500, 750 and 1500 µM. The lowest level of silicic acid is representative of field sites with minimally bioavailable Si, while the highest level is near the solubility of many environmentally relevant forms of Si. After 7 days of exposure, biomass will be recorded and the plants will be processed following published methodology. Briefly, shoots will rinsed with deionized water while roots will be rinsed in a buffer solution to remove apoplastic As. Tissues will then be frozen in liquid nitrogen and ground by mortar and pestle. The ground tissue will then be extracted via sonication with 20 mL of 2 mM NaH2PO4 and 0.2 mM Na2-EDTA for 1 hour. Extracts will be filtered through 0.45 µm syringe filters prior to analysis by HPLC-ICP-MS using published methodology. This method is able to quantify arsenite, arsenate, and both DMA and MMA (at oxidation states of +5), as well as their reduced equivalents [DMA(III) and MMA(III)]. For QA/QC, spike recovery will be tested in the extracts and certified reference materials will be analyzed. The As concentrations and biomass production can be analyzed by two-way ANOVA to test the effects of As and Si additions on plant uptake of organic As and plant growth.Objective 2:In this objective, rice plants will be grown hydroponically and exposed to 5 µM DMA and MMA along with different weak acids to explore other potential competing analytes (i.e., Malic acid, Lactic acid, Carbonic acid, Glutamic acid, Aspartic acid, Citric acid, Acetic acid, Formic acid, Succinic acid, Abscisic acid, Auxins (NAA)). These compounds must be present in plant roots, should be similar in size and shape to MMA and DMA, and possess a similar pKa to MMA and DMA (contrasted with silicic acid). Some of the compounds are found in rice root exudates, such as the acidic sugars and amino acids. Others, such as abscisic acid and auxin, are well known signaling molecules. Rice seedlings grown hydroponically will receive one of these potential competing analytes at 500 µM. After 24 hours of exposure, the shoots will be harvested as in Objective 1 and processed for analysis by HPLC-ICP-MS.The resulting concentrations of MMA and DMA in the rice shoot can be analyzed by 1-way ANOVA.Objective 3:Rice seedlings will be grown hydroponically using differing nitrogen sources while exposed to 5 µM of DMA and MMA. For the ammonium treatment, the only supplied nitrogen source will be 0.33 mM (NH4)2SO4, while for the nitrate treatment, 0.66 mM of KNO3 will be supplied. After one week of exposure, the rice shoots will be harvested and processed as in above objectives to assess shoot concentrations of DMA and MMA. Analysis will follow 1-way ANOVA.Objective 4:In this objective, I will leverage the UD RICE Facility, which is designed to perform field uptake studies of inorganic As by rice and is well-suited to study uptake of organic As species. This objective will remain less well defined until the completion of Objectives 1-3. Upon the completion of these hydroponic studies, the treatments which resulted in the largest decrease in MMA and/or DMA uptake will be selected for field study. In the field study, rice will be grown in triplicate with amendments added during establishment. The UD RICE Facility consists of 12 rice paddy mesocosms, with the capacity to conduct 4 treatments, with three plot replicates per treatment. Specific concentrations of amendments will depend on the results of Objectives 1-3. Rice plants will be harvested at 3 time points: once during vegetative growth, once during panicle emergence, and once after rice kernel maturation. The following plant parts will be analyzed for MMA and DMA concentrations as before: leaf, tiller, bran layer and husked grain. Biomass will be recorded to look for improvements in plant health resulting from lessened DMA uptake.

Progress 01/01/16 to 12/31/17

Outputs
Target Audience:Several audiences were reached by efforts performed this year. Several scientific presentation were given to disseminate the research. Those in attendance included soil scientists, plant scientists, agronomists at the CSA, SSSA and ASA meeting. A presentation given to the American Chemical Society included a wide variety of scientists, engineers and students interested in contamination of food and water. The research was also presented at the Rice Technical Working Group Conference to an audience focused on rice research and production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this grant, I attended the X-ray summer school at the Stanford Synchrotron Radiation Lightsource (SSRL), where I learned the details of x-ray fluorescence imaging. I have also travelled to SSRL several times to image rice grains from my experiments. By learning this advanced imaging technique, I can explore where the arsenic is localized in rice grains as well as the chemistry associated with the arsenic. I have also had the opportunity to attend a vareity ofconferences where I presented my research and gave 3 seminars at local universities. I have also improved my teaching skills as a co-instructor for a new class I developed with my advisor. I have also improved my teaching pedagogy through a semester-long class taught at the University of Delaware. Finally, I have had the opportunity to mentor several undergraduate researchers and have worked closely with 4 master's and PhD students. How have the results been disseminated to communities of interest?Results have been disseminated through a number of avenues. Results were presented at a variety of scientific conferences as well as departmental seminars. Conference attendees ranged from international researchers, to state university extension specialists, to rice processors, enabling wide dissemination of the results to interested communities. The results of the research were also integrated into a class developed by the PD. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research is investigating sustainable approaches to minimizerice uptake of arsenic species. Arsenic is readily accumulated by rice, resulting in global concerns about consumption of rice. Arsenic is a known carcinogen, and rice is widely consumed throughout the world. To date, no single solution has been effective in reducing arsenic concentrations in rice without negatively affecting farmers or consumers, making implementation unlikely. The most promising sustainable solution involves the addition of silicon fertilizers. Rice requires large amounts of silicon and has a well-developed uptake system to acquire silicon from the soil. Arsenic uses this same transport pathway to enter the rice plant, so the addition of silicon fertilizer can be used to out-compete arsenic for uptake. This solution has the potential to reduce toxic arsenic concentrations in rice while also closing the global silicon cycle. Silicon is beneficial for rice, but is often depleted in rice-growing areas due to years of rice cropping. This research will not only improve food quality for millions of people, but can also improve food security, as arsenic can negatively affect the amount of rice produced by each plant. Objectives: 1. The results of this work are currently under revision (submitted to ES&T). In this objective, rice plants were grown hydroponically in a 3 factor experimental design. The factors were arsenic supplied (DMA, MMA or no arsenic), silicon supplied (50, 250, 500, 750, 1500 uM Si) and maturity (harvested at maturity or vegetative state). The plants were harvested and divided into various parts. Each part was analyzed by ICP-MS and ICP-OES for metal content. MMA was found to be phytotoxic at all concentrations of Si, resulting into stunted plants that did not reach maturity. Conversely, DMA plants exhibited no phytotoxicity and did not differ in appearance from control plants. Interestingly, increasing silicon concentrations in the hydroponic media significantly decreased arsenic concentrations in the roots, shoots, husk and grain for the mature plants. For the vegetative plants, silicon did not affect arsenic concentration in any plant part. This suggests that a majority of arsenic uptake occurs during reproduction, which coincides with the period of greatest silicon uptake. It also suggests that experiments conducted with rice seedling may not accurately represent the plant later in life. Importantly, this experiment demonstrates that silicon additions can reduce the uptake of DMA by rice. 2. In this objective, plants were grown hydroponically in the presence of DMA and a potential competing analyte. Competitors included silicic acid, boric acid, glycine, lactic acid, glycerol, urea, phosphoric acid, serine, calcium, and a negative control. Plants were harvested and digested, and analyzedvia ICP-MS and ICP-OES. Of all the elements, only silicon was able to rescue yield and prevent straighthead disorder. Additionally, only Si was able to substantially decrease DMA accumulation in the grain. This suggests that of the competitors investigated, DMA primarily enters the plant through silicon transporters. A manuscript is in preparation. 3. In this objective, plants were grown hydroponically in the presence of DMA and differing nitrogen sources. Nitrogen was supplied as 100% nitrate, 50% nitrate 50% ammonium, or 100% ammonium. Plants were harvested and digested, and analyzed via ICP-MS and ICP-OES. Growth of plants with ammonium resulted in a decreased solution pH, which was correlated to an increased accumulation of DMA. A manuscript is in preparation. 4. Based upon data collected, silicon was determined to be the most promising amendment forfield trials. Silicon was added to rice paddies as either calcium silicate, rice husk, or charred rice husk. The last two are sustainable sources of silicon that would be readily available to farmers globally. The field study is ongoing, but the first year's results were recently published in Science of the Total Environment. Overall, the silicon amendments were able to increase plant concentrations of silicon while decreasing plant concentrations of As. Importantly, grain concentrations of inorganic arsenic were slightly lowerd (~15%). Grain concentrations of organic arsenic were slightly increased, although this is of less concern because organic arsenic is much less toxic than inorganic arsenic.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Teasley, W. A., Limmer, M. A., & Seyfferth, A. L. How rice (Oryza sativa L.) responds to elevated As under different Si-rich soil amendments. Environ. Sci. Technol., 2017, 51(18), 10335-10343.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Limmer, M. A., Mann, J., Amaral, D. C., Vargas, R., & Seyfferth, A. L. (2018). Silicon-rich amendments in rice paddies: Effects on arsenic uptake and biogeochemistry. Sci. Tot. Environ., 2018, 624, 1360-1368.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Seyfferth, A.L., Limmer, M.A. & Dykes, G.E. On the Use of Silicon as an Agronomic Mitigation Strategy to Decrease Arsenic Uptake by Rice. Adv. Agron. In press.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Limmer, M.A.*, Wise, P., Dykes, G., Mann, J., Amaral, D., Vargas, R., Seyfferth, A. Arsenic Uptake by Rice: Interactions with Silicon. SETAC. Brussels, Belgium. May 8-11, 2017.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Limmer, M.A.*, Seyfferth, A. Decreasing Arsenic Uptake by Rice Through Silicon Additions. AEESP, Ann Arbor, MI. June 20-22, 2017.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Limmer, M.A.*, Wise, P., Dykes, G., Mann, J., Amaral, D., Vargas, R., Seyfferth, A. Silicon-Rich Amendments in Rice Paddies: Effects on Arsenic Uptake and Biogeochemistry. Soil Science Society of America. Tampa, FL. Oct 22-23, 2017.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Limmer, M.A.*, Wise, P., Dykes, G., Mann, J., Amaral, D., Vargas, R., Seyfferth, A. Silicon amendments for managing arsenic accumulation in rice. Rice Technical Working Group. Long Beach, CA. Feb 19-22, 2018.

Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Several audiences were reached by efforts performed this year. Several scientific presentation were given to disseminate the research. Those in attendance included soil scientists, plant scientists, agronomists at the CSA, SSSA and ASA meeting. A presentation given to the American Chemical Society included a wide variety of scientists, engineers and students interested in contamination of food and water. The research was also presented to a largely Chinese audience at the 13th International Phytotechnologies Conference held in Hangzhou, China. The research was also incorporated into a class developed by the PD entitled "Plant-Contaminant Interactions". This class was attended by graduate students and upper-class undergraduates from a variety of cultures. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, I attended the X-ray summer school at the Stanford Synchrotron Radiation Lightsource (SSRL), where I learned the details of x-ray fluorescence imaging. I have also travelled to SSRL several times to image rice grains from my experiments. By learning this advanced imaging technique, I can explore where the arsenic is localized in rice grains as well as the chemistry associated with the arsenic. I have also had the opportunity to attend 3 conferences where I presented my research and gave 3 seminars at local universities. I have also improved my teaching skills as a co-instructor for a new class I developed with my advisor. How have the results been disseminated to communities of interest?Results have been disseminated through a number of avenues. Results were presented at a variety of scientific conferences as well as departmental seminars. The results of the research were also integrated into a class developed by the PD. What do you plan to do during the next reporting period to accomplish the goals?To accomplish objective 1, the manuscript is under preparation and will be submitted this year. Objectives 2 and 3 are awaiting data analysis, which will occur this year followed by manuscript preparation and submission. Details regarding objective 4 will be finalized upon completion of Objectives 2 and 3.

Impacts
What was accomplished under these goals? This research is investigating sustainable approaches to minimized rice uptake of arsenic species. Arsenic is readily accumulated by rice, resulting in global concerns about consumption of rice. Arsenic is a known carcinogen, and rice is widely consumed throughout the world. To date, no single solution has been effective in reducing arsenic concentrations in rice without negatively affecting farmers or consumers, making implementation unlikely. The most promising sustainable solution involves the addition of silicon fertilizers. Rice requires large amounts of silicon and has a well-developed uptake system to acquire silicon from the soil. Arsenic uses this same transport pathway to enter the rice plant, so the addition of silicon fertilizer can be used to out-compete arsenic for uptake. This solution has the potential to reduce toxic arsenic concentrations in rice while also closing the global silicon cycle. Silicon is beneficial for rice, but is often depleted in rice-growing areas due to years of rice cropping. This research will not only improve food quality for millions of people, but can also improve food security, as arsenic can negatively affect the amount of rice produced by each plant. Objectives: 1. In this objective, rice plants were grown hydroponically in a 3 factor experimental design. The factors were arsenic supplied (DMA, MMA or no arsenic), silicon supplied (50, 250, 500, 750, 1500 uM Si) and maturity (harvested at maturity or vegetative state). The plants were harvested and divided into various parts. Each part was analyzed by ICP-MS and ICP-OES for metal content. MMA was found to be phytotoxic at all concentrations of Si, resulting into stunted plants that did not reach maturity. Conversely, DMA plants exhibited no phytotoxicity and did not differ in appearance from control plants. Interestingly, increasing silicon concentrations in the hydroponic media significantly decreased arsenic concentrations in the roots, shoots, husk and grain for the mature plants. For the vegetative plants, silicon did not affect arsenic concentration in any plant part. This suggests that a majority of arsenic uptake occurs during reproduction, which coincides with the period of greatest silicon uptake. It also suggests that experiments conducted with rice seedling may not accurately represent the plant later in life. Importantly, this experiment demonstrates that silicon additions can reduce the uptake of DMA by rice. 2. In this objective, plants were grown hydroponically in the presence of DMA and a potential competing analyte. Competitors included silicic acid, boric acid, glycine, lactic acid, glycerol, urea, phosphoric acid, serine, calcium, and a negative control. Plants were harvested and digested, but analysis via ICP-MS and ICP-OES have not yet occurred. 3. In this objective, plants were grown hydroponically in the presence of DMA and differing nitrogen sources. Nitrogen was supplied as 100% nitrate, 50% nitrate 50% ammonium, or 100% ammonium.Plants were harvested and digested, but analysis via ICP-MS and ICP-OES have not yet occurred. 4. Based upon data collected thus far, silicon appears to be the most promising amendment to consider in field trials. However, a final decision will be made after all the data have been analyzed.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.*, Seyfferth, A. Rice & Organic Arsenic Species: Interactions with Silicon. Soil Science Society of America. Phoenix, AZ. Nov. 6-9, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.*, Seyfferth, A. Arsenic Uptake by Rice: Competition with Silicon. 13th Phytotechnologies Conference. Hangzhou, China. Sept. 26-29, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.*, Seyfferth, A. Rice Uptake of Organic Arsenic Species: Competition with Silicon. 252nd ACS Conference. Philadelphia, PA. Aug. 21-25, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.* Phytoforensics: Plants as Biomonitors of Subsurface Contamination. University of Maryland, Baltimore County Center for Urban Environmental Research and Education Seminar. Oct. 7, 2016. Invited Seminar.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.* Plant Interactions with Environmental Contaminants: Applications in Phytoforensics & Food Safety. University of Delaware Department of Plant & Soil Science Seminar. March 18, 2016.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Limmer, M.A.* Plant Uptake of Organic Contaminants: Applications in Phytoforensics & Food Safety. Towson University Chemistry Department Seminar. Feb. 18, 2016. Invited Seminar.