Food Safety and Environmental Stewardship

Glyphosate in Wheat-Finding a Chemical Marker

This project is an example of the FSES program responding to the needs of consumer and growers. Overspray and carryover of agrochemicals occasionally needs to be evaluated on a rapid turn-around bases, whether it is for crop damage or environmental assessment. In the case of glyphosate non-target applications, this can be an especially difficult task. Although glyphosate is a commonly used herbicide, the ability to test for this analyte is analytical challenging. The analysis must be performed within hours or a few days of the application event; however, often crops may not show damage for 5-10 days after exposure or longer. Consequently, by the time the damage is visually recognized, the glyphosate is gone or present at very low levels. In addition, because of the analytical difficulties with the typical glyphosate methods (a long 2-3 day extraction and complicated high performance liquid chromatography analysis) the procedure is expensive, detection limits are modest and typical turn-around time is slow. If one could find an alternative chemical marker , , for glyphosate analysis that was:
1) selective to glyphosate applications,
2) remains in the plant tissue for a longer period of time (especially through the first visible signs of damage), and
3) easier chemical extraction and analysis method,

this project would have value to both consumers and growers. A selective, automated, reliable and fast method for evaluation of plant response upon glyphosate treatment would be of value. At the biochemical level the primary mode of glyphosate action is inhibition of the enzyme 5-enolpyruvlyshikimiate-3-phosphate synthase5. This results in the blockage of the shikimate pathway causing a reduction in aromatic amino acid synthesis, reduced protein synthesis, reduced growth and premature cellular death6. Blockage of the shikimate pathway results in accumulation of high levels of shikimic acid8. The investigations of shikimic acid build-up have been limited to a few studies and a few commodities.

The reported extraction procedures for shikimic acid have been varied and in some instances labor intensive. All of the studies have been limited to a maximum of 100 hours after application. Build-up of shikimic acid in wheat has not been reported in the literature. The analysis developed at the FSES laboratory would be selectively indicative of a glyphosate application (via chemical marker) in wheat over a 20 day field trial study.

Project Findings:
The modified shikimic acid analytical method , performed well in the analysis of wheat samples as demonstrated with the quality control and spikes samples results. Spiked recoveries ranged from 67% to 105%, averaging 96%. The normal shikimic acid for wheat samples as received from this study ranged from 60-250 ug/kg dry wt and averaged 180 ug/kg dry wt. Analyses were performed on a dry-weight bases to eliminate the variable of moisture content as the plants died or 'stressed' from the application. The dry weight bases for analysis therefore, eliminated the moisture content from masking (or swamping) the shikimic acid trends. The sample extraction is a simple water/shake extraction, avoiding labor intensive procedures found in the literature. The low treat rate (4oz) shikimic acid samples were found to be significantly elevated by the day-5 sampling, with a ca. 400% increase in shikimic acid. The day-10 samples were returning to normal shikimic acid levels, although still ca. 200% above normal, and by day-15 the wheat shikimic acid levels where within normal range. The higher treat rate (14oz) shikimic acid samples were found to be significantly elevated by the day-5 samples with a >600% increase in shikimic acid. The shikimic acid level continued to increase with the day-10 shikimic acid levels a factor of 10 above normal. The shikimic acid remains elevated through the study period of 20 days.

Within this study's parameters, there appears to be strong evidence that shikimic acid is an excellent chemical maker for glyphosate applications in wheat. There remains however, at low applications levels, only a window of 5-15 days where shikimic acid remains elevated above normal prior to the plant "recovering" from the treatment. Future work will include looking at a more defined area of plant sub-sample to possible exploit a longer "window" for shikimic acid elevation. For instance, sampling only the 'growing tip' that was exposed to glyphosate might capitalize on the shikimic acid elevation for a longer period of time for the low application rates. This limited research project illustrates how the FSES program "Science Supporting Society" can function. Here, the FSES program was able to corroborate and illustrate a proof-of-concept that a chemical marker may be used for glyphosate applications in wheat. The method is easy (water extraction/shake and uncomplicated HPLC analysis) and can be completed in less than 2-3 hours. The FSES program will make this method available to the public. Future work in the FSES program will focus on several new classes of agrochemicals. One class of agrochemicals, sulfonylureas, will be investigated. If a reasonable approach can be found this will be one focus area for method development in the FSES program. This class of pesticide can not be readable analyzed by conventional instrumentation (e.g., GC, GC/MS or HPLC).