Source: UNIVERSITY OF KENTUCKY submitted to NRP
REDUCTION OF TOBACCO-SPECIFIC N-NITROSAMINES (TSNA) IN DARK TOBACCOS
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0232042
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Plant and Soil Sciences
Non Technical Summary
TSNAs are known carcinogens in tobacco, and dark tobacco, particularly dark-fired tobacco has the potential to produce higher TSNA than other types. TSNAs are produced primarily during the curing process but agronomic practices occuring in the field, as well as after curing, may also influence TSNA levels. Reducing TSNA in raw tobacco leaf will have a direct impact on reduced TSNA in tobacco products. The tobacco industry has had a major interest in reduced TSNA for several years and interest has increased with FDA regulation of tobacco products. If research finds practical methods that could be used to reduce TSNA in raw leaf, these methods and management practices will be included in University extension recommendations, which should result in lower overall TSNA levels in raw leaf and potentially reduced health risk to tobacco consumers.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20419991060100%
Goals / Objectives
Objectives: 1) Determine the effect of nitrogen application timing on TSNA development in dark fire-cured tobacco. 2) Determine impact of use of pale yellow dark tobacco lines on potential changes in fire-curing schedules and resulting TSNA levels. 3) Determine the effect of pre-harvest antioxidant and biofungicide application on TSNA development in dark fire-cured tobacco. 4) Determine the effect of forced air during early stages (yellowing) of curing on TSNA formation in dark fire-cured tobacco. 5) Determine typical spatial variability in curing conditions within dark air-curing barns and correlation to resulting TSNA levels. 6) Determine the effect of excessive heat during fire-curing on TSNA levels in dark fire-cured tobacco. 7) Determine the effect of ordering method on TSNA in dark air-cured tobacco.
Project Methods
For objective 1, trials will be conducted comparing a single pretransplant application of 300 lbs N/A to multiple applications made pretransplant and after transplanting. Tobacco will be fire-cured and cured leaf samples will be collected for TSNA analysis. Trials will be randomized complete block with 4 replications and will be conducted for 2 years. For objective 2, pale yellow lines and their normal green counterparts of the same lines will be tested in a randomized complete block design with 4 replications and 2 plots of each line within each replication. Within each line and replication, one plot will be cured using normal fire-curing procedures (firing started 7 days after housing) while the other plot will be cured using aggressive fire-curing procedures with firing started 1 day after housing). Following curing, cured leaf samples of from each plot (each line and firing procedure) will be collected for TSNA analysis. This experiment will be conducted for at least 2 years. For objective 3, preharvest applications of ferulic acid and bacilus pumilus will be made 7 days and 1 day prior to harvest. Tobacco will be fire-cured and cured leaf samples will be collected for TSNA analysis. Treatments will be arranged in a randomized complete block with 3 replications and trials will be conducted for at least 2 years. For objective 4, two identical barns will be used to compare the standard practice of natural ventilation for 7 days prior to first firing to an extended period of 14 days with forced air prior to first firing. Once firing begins, normal procedures will be used for curing and cured leaf samples will be collected for TSNA analysis. This experiment will be conducted for at least 2 years. For objective 5, two locations will be used to evaluate the spatial variability of curing conditions and corresponding TSNA production during air-curing of dark tobacco. Each barn will be monitored at 27 locations and cured leaf samples will be collected for low and high-converter lines at each of the 27 locations (54 samples per location). These experiments will be conducted for at least 2 years. For objective 6, two identical fire-curing barns will be used with one barn intentionally cured with excessive temperature (>160 F) at least during the drying phase, with the other barn fire-cured at normal temperatures (approx. 130 F) during the drying phase. Cured leaf samples will be collected from designated locations in each barn for TSNA analysis. These experiments will be conducted for at least 2 years. For objective 7, low and high-converter lines of dark tobacco will be harvested and cured on 3 scaffold wagons. Following curing, one wagon will be brought into order with natural moisture, while the other 2 wagons are ordered by either mist or steam application. Cured leaf samples will be collected from each line on each wagon/ordering method for TSNA analysis. This experiment will be conducted for at least 2 years

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The primary target audience for this project has been the approximately 1800 dark tobacco growers in Kentucky and Tennessee. An additional target audience would be those involved in the dark tobacco manufacturing industry who are not dark tobacco growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided content for dark tobacco agent trainings that were held each year for the duration of this project (5 years, 2013-2017). In total, 10 hours of training were provided to nearly 100 county agricultural extension agents during this 5 year period. Additionally, this project has provided content for annual good agricultural (GAP) trainings for dark tobacco growers. There are approximately 1,800 dark tobacco growers in KY and TN, and each grower is required by the industry to attend annual GAP trainings where a portion of the content has included information that was provided by this project. Over the past 5 years, these 1,800 dark tobacco growers have received approximately 5 hours of annual training where approximately 2 hours of this training was derived from information provided by this project. How have the results been disseminated to communities of interest?Much of these results have been disseminated to communities of interest through tobacco educational events such as annual GAP trainings, county-level tobacco production meetings for tobacco growers, and dissemination through production of the TSNA chapter in three editions of the "Burley and Dark Tobacco Production Guide", which is now distributed throughout tobacco production regions of KY, TN, VA, and NC. In addition, some of the results of this research are published in preceedings and presentations at the Tobacco Workers Conference national tobacco research conference, and at the CORESTA international tobacco research conferences. Some of these results are also published on our dark tobacco website (http://darktobacco.ca.uky.edu). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Relative to Objective 1, we determined that nitrogen application rate may have a much greater impact on TSNA than nitrogen application timing. As long as all nitrogen applications are complete by 6 weeks after tobacco transplanting, nitrogen application timing should not have a significant impact on TSNA levels in cured dark tobacco leaf. Total nitrogen application rates should be no more than 300 lbs N/A. Relative to Objective 2, we determined that the use of pale yellow dark tobacco lines do not have a significant impact on TSNA levels in cured dark tobacco leaf. Final TSNA levels in pale yellow and conventional dark tobacco lines are smilar when cured under the same conditions. However, leaf quality may be greater in pale yellow lines when firing practices begin earlier in the cure. Relative to Objective 3, we have found no consistent reduction in TSNA development in dark fire-cured tobacco as a result of making pre-harvest applications of anti-odxidants or biofungicides. Relative to Objective 4, we have found benefits of the judicial use of forced air during early (yellowing) stages of curing in reducing TSNA formation. Relative to Objective 5, our paper that we published in Tobacco Science journal in 2017 showed that spatial variability in curing conditions within air-curing barns was very difficult to fully correlate with resulting TSNA levels. Relative to Objective 6, we found a very strong correlation between the use of excessive heat during fire-curing and increased TSNA levels in the cured leaf. Curing temperatures within dark fire-curing barns that exceeded 150 F, even for short periods of time, resulting in increases in TSNA compared to fire-curing barns that did not exceed 130 F. As a result, our recommendation to dark-fired tobacco growers is to not allow barn temperatures to exceed 130 F during fire-curing. Relative to Objective 7, we found no consistent effect of ordering method on TSNA formation in dark air-cured, or burley, tobacco. Any ordering method may be used (misting, steaming, or natural ordering), provided that the moisture levels in the tobacco are not excessive as a result of over ordering.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Richmond, M. D., Pearce, R. C., Goff, B. M., *Bailey, W. A. (2017). Analysis of Variability in Curing Conditions and Tobacco-Specific Nitrosamines Within Barns of Dark Air-Cured Tobacco, (vol. 54, pp. 6-14) Tobacco Science. http://www.tobaccoscienceonline.org/doi/pdf/10.3381/17-060
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Jack, A. M., Bush, L. P., Bailey, W. A. (2017). TSNAs in Burley and Dark Tobacco, ID-160: Burley and Dark Tobacco Production Guide (2017-2018 ed., pp. 62-66) Lexington KY: University of Kentucky Agricultural Communications Service. http://www2.ca.uky.edu/agc/pubs/ID/ID160/ID160.pdf
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Keeney, A. B., Bailey, W. A. 2017. CORESTA Agronomy/Phytopathology Group Meeting, "Dark air-cured and dark fire-cured tobacco TSNA levels in response to potassium source and rate," CORESTA - Cooperation Center for Scientific Research Relative to Tobacco, Accepted, National, Santa Cruz do Sul, Brazil.
  • Type: Other Status: Published Year Published: 2017 Citation: Bailey, W. A. 2017. Dark Fired Tobacco, University of Kentucky Agricultural Communications Service. https://www.youtube.com/watch?time_continue=123&v=f6e05gUKWDE
  • Type: Websites Status: Published Year Published: 2017 Citation: CORESTA TSNA Sub-Group website, W. A. Bailey, website manager. continual updates. http://darktobacco.ca.uky.edu/coresta


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audience is primarily dark tobacco growers, some of which are in racial minorities and some could be considered socially, economically, or educationally disadvantaged. Target audience would also include dark tobacco manufacturers, dealers, and processors, and others in associated agribusinesses. Efforts include science-based educational programs presented at annual county production meeting and field days for growers, short courses for more intensive classroom training for growers, and annual training of county extension agents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Results from all of the research relative to this project have been presented at annual county agent trainings in KY and TN. Many of these results have also been presented at annual winter educational meetings and trainings for tobacco growers. How have the results been disseminated to communities of interest?Although results from this project has been published at scientific meetings/conferences as well as journal articles, the major dissemination to our main communities of interest will be through publication of the chapter "TSNAs in Burley and Dark Tobacco" by A. Jack, L. Bush, and A. Bailey In (. Pearce, A. Bailey, and E. Walker (eds.) Burley and Dark Tobacco Production Guide, published in every two years by the Cooperative Extension Services of the University of Kentucky, the University of Tennessee, Virginia Tech, and North Carolina State University. What do you plan to do during the next reporting period to accomplish the goals?During the final year of this project (2017), we will be repeating several experiments to confirm results as well as publishing these results. A manuscript will be submitted to Tobacco Science journal in the 2017 relative to objective 5 (determine typical spatial variability in curing conditions within dark air-curing barns and correlation to resulting TSNA levels). Additional presentations and abstracts will be published at the CORESTA Agro-Phyto conference in Fall 2017, and will be presented at production meetings and trainings for county extension agents and tobacco growers in Winter 2017.

Impacts
What was accomplished under these goals? During the past year, major accomplishments of this project were related to objectives 4, 5, 6, and 7. Relative to objective 4 (determine the effect of forced air during yellowing stage of curing on TSNA formation in dark fire-cured tobacco), an experiment that was ran in 2016 determined that TSNA from tobacco subjected to forced air for 12 days during yellowing and prior to firing was reduced 49% compared to TSNA from tobacco that was subjected to only natural ventilation for 12 days during yellowing and prior to firing. This experiment will be repeated in 2017 and published in upcoming conference proceedings. Relative to objective 5 (determine typical spatial variability in curing conditions within dark air-curing barns and correlation to resulting TSNA levels), this project has now been completed and a graduate student has written a journal manuscript for publication in Tobacco Science journal. The manuscript is now in the final stages of internal review before it is submitted to the journal for review. Relative to objective 6 (determine effect of excessive heat during fire-curing on TSNA levels in dark fire-cured tobacco), dramatic increased of approximately 2- to 10-fold have been observed when fire-curing temperatures exceed 160 F as compared to the target maximum temperature of 130 F. These results have been published in two conference presentations and abstracts published in 2014 and will also be published in the upcoming 2017-2018 edition of the Burley and Dark Tobacco Production Guide (ID-160, published by the University of Kentucky, Cooperative Extension Service). Relative to objective 7 (determine the effect of ordering method on TSNA in dark air-cured tobacco), an experiment was conducted in 2016 that determined there was no statistically significant difference between tobacco subjected to ordering by misting compared to tobacco subjected to ordering by steaming. This experiment will be repeated in 2017 and published in upcoming conference proceedings.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Richmond, M. D., W. A. Bailey, and R. C. Pearce. May 2015. Analysis of Variability in Curing Conditions and TSNA within Barns of Dark Air-Cured Tobacco. CORESTA Study Grant Final Report. https://www.coresta.org/sites/default/files/technical_documents/main/TSNA-CRR_2012-2014-TSNA-Hobo-Study-Final-Report.pdf


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audience is primarily dark tobacco growers, some of which are in racial minorities and some could be considered socially, economically, or educationally disadvantaged. Target audience would also include dark tobacco manufacturers, dealers, and processors, and others in associated agribusinesses. Efforts include science-based educational programs presented at annual county production meeting and field days for growers, short courses for more intensive classroom training for growers, and annual training of county extension agents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is providing data to answer basic questions regarding TSNA development in dark tobacco. These results are resulting in the development of best management practices and various recommendations for reducing TSNA in dark tobacco. These data also lead to various opportunities to train growers on these best management practices, and train county agents that can also extend this information to more growers. This project has also provided valuable information that is used in the CORESTA Sub-group TSNA in air-cured and fire-cured tobacco, for which I have been chairman. One of the goals of this subgroup is to develop a manual of best management practices to minimize TSNA development in tobacco that can be used by tobacco growers all over the world. How have the results been disseminated to communities of interest?Conclusive findings thus far have been included in industry and grower educational meetings where the focus has been on TSNA. The development of the chapter on TSNA in the last two editions of the KY/TN Tobacco Production Guide has also contributed to grower knowledge as this production guide is disseminated to tobacco growers and others interested througout KY and TN. The 2015-2016 version of this guide was also distributed in NC and VA. What do you plan to do during the next reporting period to accomplish the goals?We have now completed the projects related to effects of spacial variability in air-curing conditions (Objective 5), excessive heat during fire-curing (Objective 6), and ordering methods (Objective 7). During the next reporting period we plan to complete manuscripts of these projects and submit to scientific journals (Tobacco Science journal or other appropriate journal outlets) for publication.

Impacts
What was accomplished under these goals? Relative to Objective 1, a new project was initiated in 2015 to compare TSNA accumulation in dark fire-cured and dark air-cured tobacco that received nitrogen rates of 100, 200, or 300 lbs N/A. Relative to Objective 3, a new project was initiated in 2015 to evaluate the effect of preharvest application of the yeast species Debaryomyces hansenii on TSNA accumulation. Relativet o Objective 4, an on-going research project continues to evaluate the effect of forced air during the yellowing phase prior to first firing on TSNA accumulation in dark fire-cured tobacco.

Publications

  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Jack, A., L. Bush, and A. Bailey. 2015. TSNAs in Burley and Dark Tobacco. Pp. 49-53 In 2015-2016 Burley and Dark Tobacco Production Guide. Coop. Ext. Service. Univ. of KY.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Richmond, M. D. 2014. Evaluation of correlation between within-barn curing environment and TSNA accumulation in dark air-cured tobacco. M.S. Thesis, University of Kentucky
  • Type: Other Status: Published Year Published: 2015 Citation: Richmond, M. D., W. A. Bailey, and R. C. Pearce. 2015. Analysis of variability in curing conditions and TSNA within barns of dark air-cured tobacco. Final Study Grant Report submitted to CORESTA Scientific Commission.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audience is primarily dark tobacco growers, some of which are in racial minorities and some could be considered socially, economically, or educationally disadvantaged. Target audience would also include dark tobacco manufacturers, dealers, and processors, and others in associated agribusinesses. Efforts include science-based educational programs presented at annual county production meeting and field days for growers, short courses for more intensive classroom training for growers, and annual training of county extension agents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project is providing data to answer basic questions regarding TSNA development in dark tobacco. These results are resulting in the development of best management practices and various recommendations for reducing TSNA in dark tobacco. These data also lead to various opportunities to train growers on these best management practices, and train county agents that can also extend this information to more growers. This project has also provided valuable information that is used in the CORESTA Sub-group TSNA in air-cured and fire-cured tobacco, for which I am chairman. One of the goals of this subgroup is to develop a manual of best management practices to minimize TSNA development in tobacco that can be used by tobacco growers all over the world. How have the results been disseminated to communities of interest? Conclusive findings thus far have been included in industry and grower educational meetings where the focus has been on TSNA. The development of the new chapter on TSNA in the KY/TN Tobacco Production Guide has also contributed to grower knowledge as this production guide is disseminated to tobacco growers and others interested througout KY and TN What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Relative to objective 2, work in this project continues to evaluate if the use of pale yellow dark tobacco lines may lead to potential changes in fire-curing methods that may potentially reduce TSNA. Relative to objective 4, work in this project suggest that forced air applied continuously during yellowing and even into early color setting prior to firing may play a role in reducing TSNA in cured leaf. Relative to objective 5, work in this project is showing that slight changes in relative humidity levels during air-curing due to physical differences in specific areas of air-curing barns may result in significant differences in TSNA at the end of air-curing. However, there is tremendous variability in TSNA and in the effects that relative humidity and temperature have on TSNA accumulation. Relative to objective 6, work in this project is conclusively showing that excessive heat (>160 F compared to normal <135 F) during fire-curing results in substantial increases in TSNA. Relative to objective 7, work in this project is suggesting that when comparing air-cured dark and burley tobacco ordered naturally, with misting, or with steaming; resulting TSNAs will be steaming > misting > natural ordering.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Bailey, W. A. 2014. Effect of nitrogen rate on growth, yield, quality, and leaf chemistry of dark tobacco. Tobacco Science. 51:13-22.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richmond, M. D., W. A. Bailey, and R. C. Pearce. 2014. Evaluation of correlation between curing environment and TSNA in dark air-cured tobacco. CORESTA Congress. Paper AP03.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bailey, W. A. and W. D. Pitt. 2014. Effect of excessive heat during curing on TSNA levels in dark fire-cured tobacco. CORESTA Congress. Paper AP02.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Target audienceis primarilydark tobacco growers, some of which are in racial minorities and some could be considered socially, economically, or educationally disadvantages. Target audience would also include dark tobacco manufacturers, dealers, and processors, and others inassociated agribusinesses. Efforts include science-based educational programs presented at annual county production meeting and field days for growers, short courses for more intensiveclassroom training for growers, and annual training ofcounty extension agents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project is providing data to answer basic questions regarding TSNA development in dark tobacco. These results are resulting in the development of best management practices and various recommendations for reducing TSNA in dark tobacco. These data also lead to various opportunities to traingrowers on these best management practices, and train county agents that can also extend this information to more growers. This project has also provided valuable information that is used in the CORESTA Sug-group TSNA in air-cured and fire-cured tobacco, which Iam chairman.One of the goals of thissubgroup is to develop a manual of best management practices tominimize TSNA development in tobacco that can be used by tobacco growers all over the world. How have the results been disseminated to communities of interest? Conclusive findings thus far have been included in industry and grower educational meetings where the focus has been on TSNA. The development of the new chapter on TSNA in the KY/TN Tobacco Production Guide has also contributed to grower knowledge as this production guide is disseminated to tobacco growers and others interested througout KY and TN. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Relative to objective 1, work in this project has determined that nitrogen application after 5 wks after tobacco transplanting may contribute to increased TSNA in cured leaf. Therefore, published recommendations are to not apply nitrogen later than 5 wks after transplanting. Relative to objective 2, workin this project continues to evaluate if the use of pale yellowdark tobacco lines may lead to potential changes in fire-curing methods that maypotentially reduce TSNA. Relative to objective 3, work in this projectsuggests that pre-harvest antioxidant and biofungicide applications tested have not resulted in consistent reduction in TSNA in cured leaf, and that this may notbe a practical method forreducingTSNA in dark tobacco. Relative to objective 4, work in this project suggest that forced air applied continuously during yellowing andeven into early color setting prior to firing may play a role in reducing TSNA in cured leaf. Relative toobjective 5, work in this project isbeginning to show that slight changes in relative humidity levels duringair-curingdue to physical differences in specific areas ofair-curing barns may result in significant differences inTSNA at the end ofair-curing. Relative toobjective6, work in this project is conclusively showing that excessive heat (>160 F compared to normal <135 F) during fire-curing results in substantial increases in TSNA. These results will be presented at scientific conferences and meetings in 2014 and also be included in the recommendations in the next KY/TN Tobacco Production Guide. Relative to objective 7, work in this project is suggesting that when comparing air-cured dark and burley tobacco ordered naturally, with misting, or with steaming; resulting TSNAs will be steaming > misting > natural ordering. If these results continue to be consistent, ordering method recommendations will be included in the next KY/TN Tobacco Production Guide.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Richmond, M. D., W. A. Bailey, and R. C. Pearce. 2013. Preliminary evaluation of correlation between within-barn curing environment and TSNA accumulation in dark air-cured tobacco. Proceedings of CORESTA Agro-Phyto Meeting, Brufa di Torgiano, Italy.
  • Type: Websites Status: Other Year Published: 2013 Citation: Continual updates and management of website for CORESTA Sub-group TSNA in air-cured and fire-cured tobacco: http://ces.ca.uky.edu/darktobacco/CORESTA TSNA SG
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Jack, A. M., L. P. Bush, and W. A. Bailey. 2013. TSNAs in burley and dark tobacco. In 2013-2014 Kentucky & Tennessee Tobacco Production Guide. ID-160. University of Kentucky Cooperative Extension Service.