Residues of glyphosate in food and dietary exposure.

Glyphosate is the active ingredient in Roundup® brand nonselective herbicides, and residue testing for food has been conducted as part of the normal regulatory processes. Additional testing has been conducted by university researchers and nongovernmental agencies. Presence of residues needs to be put into the context of safety standards. Furthermore, to appropriately interpret residue data, analytical assays must be validated for each food sample matrix. Regulatory agency surveys indicate that 99% of glyphosate residues in food are below the European maximum residue limits (MRLs) or U.S. Environmental Protection Agency tolerances. These data support the conclusion that overall residues are not elevated above MRLs/tolerances due to agricultural practices or usage on genetically modified (GM) crops. However, it is important to understand that MRLs and tolerances are limits for legal pesticide usage. MRLs only provide health information when the sum of MRLs of all foods is compared to limits established by toxicology studies, such as the acceptable daily intake (ADI). Conclusions from dietary modeling that use actual food residues, or MRLs themselves, combined with consumption data indicate that dietary exposures to glyphosate are within established safe limits. Measurements of glyphosate in urine can also be used to estimate ingested glyphosate exposure, and studies indicate that exposure is <3% of the current European ADI for glyphosate, which is 0.5 mg glyphosate/kg body weight. Conclusions of risk assessments, based on dietary modeling or urine data, are that exposures to glyphosate from food are well below the amount that can be ingested daily over a lifetime with a reasonable certainty of no harm.

Glyphosate and aminomethylphosphonic acid are not detectable in human milk.

BACKGROUND: Although animal studies have shown that exposure to glyphosate (a commonly used herbicide) does not result in glyphosate bioaccumulation in tissues, to our knowledge there are no published data on whether it is detectable in human milk and therefore consumed by breastfed infants. OBJECTIVE: We sought to determine whether glyphosate and its metabolite aminomethylphosphonic acid (AMPA) could be detected in milk and urine produced by lactating women and, if so, to quantify typical consumption by breastfed infants. DESIGN: We collected milk (n = 41) and urine (n = 40) samples from healthy lactating women living in and around Moscow, Idaho and Pullman, Washington. Milk and urine samples were analyzed for glyphosate and AMPA with the use of highly sensitive liquid chromatography-tandem mass spectrometry methods validated for and optimized to each sample matrix. RESULTS: Our milk assay, which was sensitive down to 1 µg/L for both analytes, detected neither glyphosate nor AMPA in any milk sample. Mean ± SD glyphosate and AMPA concentrations in urine were 0.28 ± 0.38 and 0.30 ± 0.33 µg/L, respectively. Because of the complex nature of milk matrixes, these samples required more dilution before analysis than did urine, thus decreasing the sensitivity of the assay in milk compared with urine. No difference was found in urine glyphosate and AMPA concentrations between subjects consuming organic compared with conventionally grown foods or between women living on or near a farm/ranch and those living in an urban or suburban nonfarming area. CONCLUSIONS: Our data provide evidence that glyphosate and AMPA are not detectable in milk produced by women living in this region of the US Pacific Northwest. By extension, our results therefore suggest that dietary glyphosate exposure is not a health concern for breastfed infants. This study was registered at clinicaltrials.gov as NCT02670278.

Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS.

Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89-107% at three separate fortification levels including the LOQ. Precision for replicates was ≤ 7.4% relative standard deviation (RSD) for milk and ≤ 11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.

The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G-->A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

Metabolically engineered oilseed crops with enhanced seed tocopherol.

Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol soybean with transgenic high alpha-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-fold higher than the average WT soybean seed vitamin E activity.