Measurement of grass uptake of the urease inhibitor NBPT and of the nitrification inhibitor dicyandiamide co-applied with granular urea.

Grass uptake and phytoaccumulation factors of N-(n-butyl) thiophosphoric triamide (NBPT) and dicyandiamide (DCD) were quantified. Following the application of urea fertilizer treated with the inhibitors in Irish grassland, grass samples were collected at 5, 10, 15, 20, and 30 day time intervals following five application cycles. Uptake of NBPT by grass was below the limit of quantitation of the analytical method (0.010 mg NBPT kg-1). Dicyandiamide concentrations in grass ranged from 0.004 to 28 mg kg-1 with the highest concentrations measured on days 5 and 10. A reducing trend in concentration was found after day 15. The DCD phytoaccumulation factor was ranged from 0.004% to 1.1% showing that DCD can be taken up by grass at low levels when co-applied with granular urea. In contrast, NBPT was not detected indicating that grass uptake is unlikely when co-applied with granular urea fertilizer. The contrasting results are likely due to very different longevity of DCD and NBPT along with the much lower rate of NBPT, which is used compared with DCD.

Development of One-Step Non-Solvent Extraction and Sensitive UHPLC-MS/MS Method for Assessment of N-(n-Butyl) Thiophosphoric Triamide (NBPT) and N-(n-Butyl) Phosphoric Triamide (NBPTo) in Milk.

N-(n-butyl) thiophosphoric triamide (NBPT) is a urease inhibitor utilised in urea-based fertilizers. In Ireland, fertilizer treated with NBPT is applied to pasture to mitigate both ammonia and nitrous oxide emissions, but concerns arise as to the potential for residues in milk products. A quick ultrafiltration extraction and ultra-high performance liquid chromatography coupled with mass spectrometry triple quadrupole (UHPLC-MS/MS) quantitation method was developed and validated in this study. The method was applied in the analysis of samples collected from a field study investigating potential transfer of NBPT residues into milk. NBPT and NBPTo residues, were extracted from fortified milk samples and analysed on a UHPLC-MS/MS with recoveries ranging from 74 to 114%. Validation of the UHPLC-MS/MS method at low (0.0020 mg kg-1) and high (0.0250 mg kg-1) concentration levels in line with SANTE/12682/2019 showed overall trueness in the range of 99 to 104% and precision between 1 and 10%, RSD for both compounds. The limit of quantitation (LOQ) was 0.0020 mg kg-1 and other tested parameters (linearity, sensitivity, specificity, matrix effect, robustness, etc.) satisfied acceptance criteria. Stability assessment using spiked samples revealed the compounds were stable in raw and pasteurised milk for 4 weeks at -80 °C storage temperature. Maintaining samples at pH 8.5-9.0 further improved stability. Analysis of 516 milk samples from the field study found that NBPT and NBPTo concentrations were below the LOQ of 0.0020 mg kg-1, thus suggesting very low risk of residues occurring in the milk. The method developed is quick, robust, and sensitive. The method is deemed fit-for-purpose for the simultaneous determination of NBPT and NBPTo in milk.

Impact of postharvest processing on the fungal population contaminating African walnut shells (Tetracarpidium conophorum Mull. Arg) at different maturity stages and potential mycotoxigenic implications.

African walnut (Tetracarpidium conophorum Mull. Arg) is commonly processed by boiling or toasting and consumed as a snack or used as a thickener in many West African soup preparations. The nuts are usually exposed to both high temperatures and high relative humidity in open markets which predisposes them to fungal growth. Hence, the dangers of spore inhalation and resultant mycosis cannot be over-emphasized as retailers and consumers are always in direct contact with these nuts during harvest, processing and consumption. So far, there is no reported research on potential mycotoxin contamination of African walnut and whether this risk might be accentuated by processing. African walnut, at early and late maturity stages, were processed by toasting, boiling or left unprocessed before being stored at 25°C and 37°C, respectively under controlled relative humidity for 7days. Nuts were cracked and shell pieces cultured in malt extract agar (MEA) and Dichloran Glycerol 18 (DG18) media and incubated at 25°C for 7days. Results revealed that potential mycotoxigenic species - Aspergillus section Nigri, Aspergillus flavus/parasiticus, Fusarium spp. and Penicillium spp. - were frequently isolated. When compared with unprocessed nuts, toasting completely prevented fungal contamination in shell pieces from nuts in the non-stored (NSN) group at the early maturity stage, while boiling significantly reduced the level of contamination to about 58% (p<0.05). In general, simulating open market conditions caused 100% fungal contamination in all boiled samples and toasted samples at early maturity. However, contamination in toasted samples at late maturity was increased to 90 and 70% at 25°C in DG18 and MEA, respectively, while at 37°C contamination was 40 and 60% in DG18 and MEA, respectively. Mycotoxin analysis using Yeast Extract Sucrose (YES) agar and High Performance Liquid Chromatography (HPLC)-Fluorescence detection (FLD) showed that Aflatoxins - G1 (AFG1), B1 (AFB1), G2 (AFG2), and B2 (AFB2) were produced by 20 isolates with both AFG1 and AFB1 being predominant at concentration ranges 4.33-32,200 and 4.20-22,700ng/g plug weight, respectively. No ochratoxin A (OTA) was detected out of 23 isolates analysed. From these findings, it is suggested that toasting of nuts, preferably at early maturity is a safer processing option than boiling in terms of prevention of possible fungal growth on nut shells and risk of mycotoxin contamination.