Observations of High Nitrate Concentrations in Forage Bermudagrass during Severe to Exceptional Drought Years

With the prediction of climate change-induced increases in drought frequency and severity in the southeastern USA, it is important to better understand the risks that drought may pose to NO3 accumulation in bermudagrass [Cynodon dactylon (L.) Pers.] forage. This report offers observations of NO3 concentration in Bermudagrass forage samples submitted to the University of Georgia’s Feed and Environmental Water Lab (FEWL) during the extreme to exceptional drought of 2007, the severe drought of 2008, and the four preceding seasons when drought stress was minimal or absent. The probability (P) of a sample being at high risk for nitrate toxicosis was the greatest for the extreme to exceptional drought of 2007 (P = 0.160), slightly lower in the severe drought year of 2008 (P = 0.105), and the lowest for samples from the 2003-2006 growing seasons (P = 0.082) when drought stress was minimal or absent.


Introduction
Global climate change is expected to cause an increase in frequency and severity of drought in the southeastern United States during the next century [1].One of the risks posed to forage production by an increased frequency and severity of drought in this region would be the potential for a toxic build-up of nitrate-nitrogen (NO 3 -N) concentrations in the forage crops common to this region [2].Ruminants that consume a diet consisting of more than 2500 mg NO 3 kg −1 are at risk of developing abnormally high concentrations of nitrite in their bloodstream, leading to the formation of methemoglobinemia.This, consequently, can result in condition commonly referred to as nitrate toxicosis.Nitrate levels higher than 4500 mg NO 3 kg −1 pose a risk of chronic nitrate toxicosis and, in some situations, animal death [2].
Bermudagrass [Cynodon dactylon (L.) Pers.] is the most widely used warm season perennial grass for pasture and hay production in the southern USA, and it is noted for its drought tolerance and high water use efficiency [3].Further, bermudagrass is highly efficient at using N and most of the nitrate reductase activity occurs in its roots [4].Though this may reduce its risk in accumulating toxic concentrations of NO 3 [5], there is sufficient evidence to suggest that toxic NO 3 levels in bermudagrass do frequently occur [6]- [10].However, it is unclear how frequent one might expect to find bermudagrass forage samples to be high in nitrates, especially during severe to exceptionally severe drought.
Recent weather conditions in Georgia (USA) have granted a unique opportunity to make observations about the propensity for NO 3 accumulation in bermudagrass.The National Drought Mitigation Center's U.S. Drought Monitor classified a large majority of the state of Georgia in their extreme (D3) to exceptional (D4) drought categories during the 2007 growing season and at least a severe (D2) drought during 2008 ([11]; Figure 1).These years had been preceded by four years of relatively little prolonged drought stress.In each of these years, a substantial number of forage samples were being submitted for forage quality and nitrate analysis at the University of Georgia's Feed and Environmental Water Lab (FEWL).As a result, a very large and robust database exists that could provide insight into the nitrate concentration characteristics of forage submissions made during drought years and years when drought stress during the growing season was absent or much less pronounced.Thus, the objective of this work was to offer in actu observations from the University of Georgia's FEWL on the nitrate concentration of bermudagrass forage samples submitted during the drought years of 2007 and 2008 and the four preceding seasons when drought stress was minimal or absent.

Materials and Methods
This study uses observations obtained from the analyses of bermudagrass forage samples submitted to the University of Georgia's FEWL from 1 July 2003 through 31 March 2009.The data were first screened to include only those samples that had been analyzed for NO 3 concentration, which is determined in the FEWL by a colorimetric analysis of the nitration of salicylic acid [12].Data were further screened to eliminate samples from areas that had not experienced similar levels of drought stress during 2007 and 2008.Pasture condition reports ( ) In order to compare the frequency of having moderate or highly risky NO 3 concentrations within the growing season categories, the probabilities of these occurrences during the individual growing seasons were calculated.This was done by first grouping the bermudagrass samples into three categories of toxicity risk based on their NO 3 concentration: Low Risk (LR), where nitrate levels were less than 2500 mg NO 3 kg −1 ; Moderate Risk (MR), where nitrate levels were 2500 -4500 mg NO 3 kg −1 ; and High Risk (HR), where nitrate levels greater than 4500 mg NO 3 kg −1 .Because the three levels are ordinal, a cumulative logistic model [14] was used to estimate the log (base e) probability (P) of a bermudagrass forage sample being in a particular risk category in the 2003-2006, where the cumulative logits are defined as Equations (( 5) and ( 6)).  ; and where it is given, as defined in Equation (7), that the probabilities for each risk category in a given year sum to equal 1.

P LR P MR P HR
For each growing season category (2003-2006, 2007, and 2008), the estimated parameters from Equation (4) were used in Equation ( 5) to calculate the probability of MR or greater [P(≥MR)] concentrations of NO 3 in the submitted forage samples.Similarly, these model parameters were then used in Equation ( 6) to calculate the probability of HR [P(HR)] concentrations of NO 3 in the sample submissions for each growing season category.The probability of MR [P(MR)] NO 3 concentrations was derived by subtracting P(HR) from P(≥MR).Then the probability of LR [P(LR)] NO 3 concentrations was derived from Equation (7) using the estimates of P(HR) and P(MR).

In Actu Observations of Nitrate Concentrations in Bermudagrass
The results of the analysis of maximum likelihood estimates for β 0 , β 1 , and β 2 indicated that all parameters were significantly (P < 0.01) greater than zero (Table 2).Since β 0 , β 0 + β 1 , and β 0 + β 2 are estimates of the means of the true natural log transformed NO 3 concentrations during the 2003-2006, 2007, and 2008 growing season categories, respectively, these estimates were calculated and transformed back to the original units (Table 3).The mean concentration of NO 3 in samples submitted during the 2003-2006 growing seasons (512 mg NO 3 kg −1 ) was significantly lower (P < 0.05) than in samples submitted from the drought-stressed season of 2008 (626 mg NO 3 kg −1 ), and both were significantly lower than those from the extreme to exceptional drought-stressed season in 2007 (1170 mg NO 3 kg −1 ).Though these mean values are considerably lower than those expected to pose a moderate (2500 mg NO 3 kg −1 ) or high (4500 mg NO 3 kg −1 ) risk of nitrate toxicosis [2], each growing season had a substantial number of samples with NO 3 concentrations that would be considered moderate or highly risky (Table 4).

Estimates of the Probability of Low, Moderate, and High Nitrate Concentrations
To compare the frequency of moderate or highly risky NO 3 concentrations across growing season categories, the probabilities of these occurrences during the individual growing seasons were calculated.The results of the analysis of maximum likelihood estimates for parameters α 1 , α 2 , τ 1 , and τ 2 used in Equation ( 4) indicated that each of the parameters was significantly different (P < 0.01) from zero (Table 5).These parameters were used to estimate the probabilities of LR, MR, and HR concentrations of NO 3 in bermudagrass forage sample submissions during the 2003-2006, 2007, and 2008 growing seasons (Table 6).
In each of the three growing seasons, there existed a high probability (P > 0.65) that a bermudagrass forage sample would be classified as LR.However, this probability differed between each growing season category,

Limitations, Additional Key Findings, and the Need for Additional Research
It is important to note that our results should not be interpreted as establishing the expected mean NO 3 concentration of samples from similar seasons or number of observations in LR, MR, and HR categories during drought.
Our analysis was performed on producer-submitted bermudagrass forage samples.It is likely that producers were more apt to identify and submit forage samples from bermudagrass lots that may have been at an increased risk of nitrate toxicity during the droughts of 2007 and 2008.This may have increased the observed mean and altered the distribution of NO 3 concentrations in the samples from these seasons.Even so, this analysis has shown that high risk samples do frequently occur and that this may even occur in samples from growing seasons that were not severely drought stressed (Table 1, Table 4, and Table 6).
Moreover, these observations indicate that NO 3 may accumulate in bermudagrass to a much higher level than previously thought.Of the published work that reports NO 3 concentration in forage bermudagrass [6]- [9], the highest reported level was 8350 mg NO 3 kg −1 [8].In the 2003-2006 growing seasons when relatively little prolonged drought stress was present, we observed that 1.86% (47/2533) of the samples had NO 3 concentrations greater than 8350 mg NO 3 kg −1 .In the extreme to exceptional drought of 2007 and the severe drought season of 2008, we observed that 3.47% (35/1010) and 2.30% (20/869) of the bermudagrass samples submitted from those respective growing seasons exceeded this previously reported high.
The degree to which drought stress influences NO 3 concentrations in forage bermudagrass will need to be assessed in well-designed field trials.For example, this additional research should include an assessment of the influence of N fertilizer application strategies (e.g., splitting the applied N equally across multiple applications, the use of "enhanced-efficiency" and slow/controlled-released fertilizers, etc.) so that an assessment can be made of the degree to which N management interacts with the growing season conditions to influence the development of toxic NO 3 concentrations in bermudagrass.

Figure 1 .
Figure 1.The percent of the state of Georgia (USA) that was classified by the national drought mitigation center's U.S. drought monitor (2009) as being in extreme to exceptional (D3-D4) and severe to exceptional (D2-D4) drought categories between 1 July 2003 and 30 June 2009.
of the true ln transformed NO 3 concentrations in the null, 2007, and 2008 growing season categories, respectively; I{year = 2007} is an indicator variable which is 1 for growing season 2007 and 0 for other growing seasons and I{year = 2008} is an indicator variable which is 1 for growing season 2008 and 0 for other growing seasons (i.e., for the null growing seasons, the two indicator variables are both zero).
α 1 and α 2 are associated with log odds 1 and log odds 2 (as defined above), respectively, and have the interpretation of log odds in the 2003-2006 growing seasons, τ 1 is the increment to the log odds in the 2007 growing season, and τ 2 is the increment to the log odds in the 2008 growing season; where I {year = 2007} is an indicator variable that equals 1 for the 2007 growing season and 0 for other growing seasons and I {year = 2008} is an indicator variable that equals 1 for growing season 2008 and 0 for other growing seasons (i.e., for the 2003-2006 growing seasons, the two indicator variables are both zero)

Table 1 .
Distribution characteristics of the observations used in the analysis of the NO 3 concentrations in bermudagrass forage samples submitted to the UGA-FEWL during the 2003-2006, 2007, and 2008 growing season categories., and 2008 growing seasons.Specifically, Equation (4) presents the cumulative logistic model for the log odds that was analyzed using the LOGISTIC procedure in SAS (SAS Institute, 2004).

Table 2 .
Results of the analysis of maximum likelihood estimates for 0 0 , which are parameters in the models (Equations (1) to (3)) that estimate the means of the true log (base e) transformed NO 3 concentrations during the 2003-2006 (null), 2007, and 2008 growing season categories, respectively. +

Table 3 .
Means and 95% confidence intervals for the NO 3 concentrations during the 2003-2006, 2007, and 2008 growing season categories.
† Same letters within column are not significantly different at P < 0.05.

Table 4 .
The frequency with which bermudagrass forage samples submitted to the UGA-FEWL were at low, moderate, and high risk for nitrate toxicosis during the2003-2006, 2007, and 2008growing seasons.

Table 5 .
Results of the analysis of maximum likelihood estimates for α 1 , α 2 , τ 1 , and τ 2 , which are parameters in the models (Equations (4) to (6)) used to estimate the probability of low, moderate, and high risk NO 3 concentrations during the2003-2006, 2007, and 2008growing season categories in Table6.

Table 6 .
The calculated probability (and 95% confidence intervals) of a submitted bermudagrass forage sample being categorized as a low, moderate, and high risk for nitrate toxicosis during the2003-2006, 2007, and 2008growing seasons.severedroughtseason of 2008 (0.757 ± 0.0549), and with the lowest probability occurring in the extreme to exceptional drought of 2007 (0.656 ± 0.0602).The most concerning issue revealed in this compari-son was that samples submitted from the 2007 growing season were 1.95 times (0.160/0.082) more likely to be in the high risk category than in the "normal" growing seasons of[2003][2004][2005][2006].Though this also tended (P < 0.10) to occur in 2008, it was not as likely as in 2007.