QUALITATIVE ASSESSMENT OF NITRATE PRESENCE BY COUNTY EXTENSION AGENTS UTILIZING A DIPHENYLAMINE/SULFURIC ACID SOLUTION

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1 QUALITATIVE ASSESSMENT OF NITRATE PRESENCE BY COUNTY EXTENSION AGENTS UTILIZING A DIPHENYLAMINE/SULFURIC ACID SOLUTION K. Simon, M.S. Gadberry, and J. Jennings 1 Abstract Forty-seven county extension agents attending 1 of 4 different in-service trainings conducted in 2008 and 2009 were introduced to qualitative nitrate testing based on the diphenylamine in sulfuric acid reaction. Agents selected 1 of 23 available ground hay samples for subjective assessment of nitrate reaction. Seven of the available samples contained less than 575 ppm nitrate-nitrogen, 7 analyzed between 575 and 1,150 ppm nitrate-nitrogen, and 9 analyzed 1,150 ppm or greater nitrate-nitrogen. Sixteen of the tested samples were categorized as safe based on their nitrate-nitrogen level. Seven tested samples were categorized as caution and 24 samples were categorized as dangerous. The subjective assessment differed among categories (P < 0.01). Within the danger category, there were no false negatives and within the safe category, 2 of 16 were false positive. The 7 samples that were categorized as caution had a 71% probability of being called negative and 29% probability of being called positive. In conclusion, samples that are either low or high in nitrate can accurately be assessed. The qualitative assessment is an area of concern with samples between 575 and 1,150 ppm nitratenitrogen and producers and county agents should take animal type and feeding precautions into consideration when making management decisions based on a qualitative assessment. Introduction: Nitrate toxicity is a concern for beef production systems that directly (sorghum-sudangrass grasses) or indirectly (johnsongrass) incorporate nitrate accumulating plants. Nitrate accumulation can also occur with forage species that aren t cataloged as nitrate accumulators, such as fescue, when conditions of high fertilization rates followed by poor growing conditions result in nitrate uptake by the plant but delayed conversion to plant proteins. From 1987 to 2007; 15,713 grass hay samples were analyzed at the University of Arkansas, Agricultural Diagnostics Service Laboratory. Of these, 37% were analyzed for nitrate-nitrogen concentration. This indicates nitrate is an area of concern among producers. Six percent of the hay samples were identified as either johnsongrass or a sorghum-sudangrass, whereas, 86% was bermudagrass, fescue or mixed grass hay. Fifty-five percent of samples submitted were below 575 ppm nitratenitrogen which is considered safe for beef cattle (Ball et al., 2002). The use of a diphenylamine reaction test through local county extension offices may help producers with forage management and quantitative testing decisions. The objective of this project was to introduce the qualitative nitrate test to county extension agents attending inservice trainings and assess their interpretation of the reaction to a sample of known nitrate-nitrogen concentration. 1 University of Arkansas, Cooperative Extension Service, Little Rock, AR

2 Materials and Methods: County extension agents attending 1 of 4 different in-service trainings conducted in 2008 and 2009 were introduced to qualitative nitrate testing based on the diphenylamine in sulfuric acid reaction (Garner, 1993). The test kit included the test reagent stored in an amber dropper bottle, rubber gloves, and safety glasses. The test kits and 2 trainings were funded by CSREES-USDA-Project No Beef cattle supply chain impacts of novel endophyte fescue: tradeoffs between animal and forage performance in the tall fescue region. Agents were given these kits to take back to their county office. Reaction color development was demonstrated by placing a drop of reagent into test tube containing ammonium nitrate dissolved in water. In addition, fresh plant material from forage that had been heavily fertilized with ammonium nitrate was used to demonstrate cut-stem testing. During the exercise, agents selected 1 of 23 available ground hay samples for subjective assessment of the nitrate reaction. The ground hay samples were assembled from a set of samples that were previously processed through the University of Arkansas, Agricultural Diagnostics Service Laboratory. Seven of the available samples contained less than 575 ppm nitrate-nitrogen, 7 analyzed between 575 and 1,150 ppm nitratenitrogen, and 9 analyzed 1,150 ppm or greater nitrate-nitrogen. For reference, these samples were categorized into management areas as either safe, caution, or dangerous, respectively (Ball et al., 2002). The exercise within training location was without sample replacement resulting in no shared samples among agents. Each agent was asked to indicate a positive or negative reaction upon testing their respective sample. Agent response and sample reference category was subjected to a chi-square analysis for independence using R statistical software (The R Foundation for Statistical Computing, Results and Discussion: Fort-seven agents completed the nitrate test assessment and were provided a nitrate test kit for use in their county. Sixteen of the tested samples were categorized as safe based on their nitrate-nitrogen level (Table 1). Seven tested samples were categorized as caution and 24 samples were categorized as dangerous. The subjective assessment differed among categories (P < 0.01). Within the danger category, there were no false negatives, meaning all 24 of the samples were correctly identified as being nitrate positive. Within the safe category, 2 of 16 were false positive. There was a 95% accuracy rate across the two groups. The 7 samples that were categorized as caution had a 71% probability of being called negative and 29% probability of being called positive. Samples that contained between 575 and 1,150 ppm nitrate-nitrogen were most difficult to discern by agents. Selk et. al. (1992) categorized sudangrass samples below or above 6,000 ppm nitrate (1,380 ppm nitrate-nitrogen). Samples below 1,380 ppm had a 60.6% false positive and samples above 1,380 ppm were false negatives 10.7% of the time. False positive percentage for samples below 1,150 ppm in this project was lower than the false positive reported by Selk et al. (1992). The false positive below 1,150 ppm

3 would be 17% or 30%, depending on how samples between 575 and 1,150 are interpreted. In conclusion, these results suggest that samples that are either low or high in nitrate can accurately be assessed. The qualitative assessment is an area of concern with samples between 575 and 1,150 ppm nitrate-nitrogen. While this area is recognized to pose no major threat to beef cattle, it is suggested that nitrate-nitrogen at 1,150 ppm becomes a health risk to pregnant beef cattle (Merck Veterinary Manual, 2010). Producers and county agents should take animal type and feeding precautions into consideration when assessing the nitrate presence in harvested forages qualitatively. Literature Cited: Merck Veterinary Manual. Nitrate and nitrite poisoning. d=nitrate%2cpoisoning Accessed April 12, Ball, D.M., C.S. Hoveland, and G.D. Lacefield Southern Forages, third ed. PPI and FAR, Norcross, GA. Garner, G.B Qualitative nitrate detection for toxicity potential. Univ. of Missouri Agri. Publ. G Selk, G.E., D.G. Wagner, G.L. Strickland, and S.E. Hawkins Comparison of a qualitative field test versus laboratory analysis for nitrate concentration in hybrid sudangrass hays. OK Agri. Exp. Sta., Anim. Sci. Res. Rep

4 Table 1. County agent subjective response (counts) based on qualitative nitrate assessment by nitrate category for samples with a known nitrate concentration. Safe 0 to 575 ppm Nitrate-nitrogen category Caution 575 to 1,150 ppm Dangerous 1,150+ ppm Agent response a Positive reaction b Negative reaction a Response was dependent upon nitrate-nitrogen category (P < 0.01). b Positive reaction based on a nitrate reaction, blue to blue-black color development.

5 QUALITATIVE ASSESSMENT OF NITRATE PRESENCE BY COUNTY EXTENSION AGENTS UTILIZING A DIPHENYLAMINE/SULFURIC ACID SOLUTION K. Simon, M.S. Gadberry, and J. Jennings 2 From 1987 to 2007; 15,713 grass hay samples were analyzed at the University of Arkansas, Soil and Plant Diagnostics Laboratory. Of these, 37% were analyzed for nitrate-nitrogen concentration. This indicates nitrate is an area of concern among Arkansas livestock producers. The use of a diphenylamine reaction test for nitrates through local county extension offices may help producers with forage management and quantitative testing decisions. The objective of this project was to introduce the qualitative nitrate test to county extension agents attending in-service trainings and assess their interpretation of the reaction to a sample of known nitrate-nitrogen concentration. Forty-seven agents participated in the training. A comparison of qualitative assessment to management category indicated that agents were 95% accurate at identify safe and dangerous samples. Samples that fell within 575 to 1,150 ppm nitrate-nitrogen were more difficult to discern. These samples had a 71% probability of being called negative and 29% probability of being called positive. In conclusion, samples that are either low or high in nitrate can accurately be assessed. The qualitative assessment is troublesome with samples between 575 and 1,150 ppm nitrate-nitrogen and producers and county agents should take this into consideration when assessing the nitrate presence in harvested forages. 2 University of Arkansas, Cooperative Extension Service, Little Rock, AR