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.

Effect of contrasting phosphorus levels on nitrous oxide and carbon dioxide emissions from temperate grassland soils.

Agricultural practices such as repeated fertilization impact carbon (C), nitrogen (N) and phosphorus (P) cycling and their relationships in the plant-soil continuum, which could have important implications for the magnitude of greenhouse gas emissions. However, little is known about the effect of C and N additions under contrasting soil P availability status on nitrous oxide (N2O) and carbon dioxide (CO2) emissions. In this study, we conducted a field-based experiment that investigated the impact of long-term (23 years) P management (no (P0, 0 kg P ha-1), low (P15, 15 kg P ha-1) and high (P45, 45 kg P ha-1) P inputs) on N2O and CO2 emissions following two C + N application events in two managed grassland ecosystems with loam and sandy loam soils. The magnitude of fluxes varied between the soil P availability levels. Cumulative N2O emission was significantly higher in P0 soils (1.08 ± 0.09 g N2O-N m-2) than P45 soils (0.63 ± 0.03 g N2O-N m-2), with the loam soil (1.04 ± 0.04 g N2O-N m-2) producing significantly higher emissions than the sandy loam soil (0.88 ± 0.05 g N2O-N m-2). We conclude that P-limitation stimulates N2O emissions, whereas P-enrichment promotes soil respiration in these temperate grassland sites. Our findings inform effective nutrient management strategies underpinning optimized use of N and P inputs to agricultural soils as mitigation measures for both food security and reducing greenhouse gas emissions.

Quantitative microbial risk assessment associated with ready-to-eat salads following the application of farmyard manure and slurry or anaerobic digestate to arable lands.

Farmyard manure and slurry (FYM&S) and anaerobic digestate are potentially valuable soil conditioners providing important nutrients for plant development and growth. However, these organic fertilisers may pose a microbial health risk to humans. A quantitative microbial risk assessment (QMRA) model was developed to investigate the potential human exposure to pathogens following the application of FYM&S and digestate to agricultural land. The farm-to-fork probabilistic model investigated the fate of microbial indicators (total coliforms and enterococci) and foodborne pathogens in the soil with potential contamination of ready-to-eat salads (RTEs) at the point of human consumption. The processes examined included pathogen inactivation during mesophilic anaerobic digestion (M-AD), post-AD pasteurisation, storage, dilution while spreading, decay in soil, post-harvest washing processes, and finally, the potential growth of the pathogen during refrigeration/storage at the retail level in the Irish context. The QMRA highlighted a very low annual probability of risk (Pannual) due to Clostridium perfringens, norovirus, and Salmonella Newport across all scenarios. Mycobacterium avium may result in a very high mean Pannual for the application of raw FYM&S, while Cryptosporidium parvum and pathogenic E. coli showed high Pannual, and Listeria monocytogenes displayed moderate Pannual for raw FYM&S application. The use of AD reduces this risk; however, pasteurisation reduces the Pannual to an even greater extent posing a very low risk. An overall sensitivity analysis revealed that mesophilic-AD's inactivation effect is the most sensitive parameter of the QMRA, followed by storage and the decay on the field (all negatively correlated to risk estimate). The information generated from this model can help to inform guidelines for policymakers on the maximum permissible indicator or pathogen contamination levels in the digestate. The QMRA can also provide the AD industry with a safety assessment of pathogenic organisms resulting from the digestion of FYM&S.

Quantitative microbial human exposure model for faecal indicator bacteria and risk assessment of pathogenic Escherichia coli in surface runoff following application of dairy cattle slurry and co-digestate to grassland.

Animal waste contains high numbers of microorganisms and therefore can present a potential biological threat to human health. During episodic rainfall events resulting in runoff, microorganisms in the waste and soil may migrate into surface runoff, contaminating surface water resources. A probabilistic human exposure (HE) model was created to determine exposure to faecal indicator bacteria (FIB): total coliforms (TC), E. coli and enterococci following application of bio-based fertiliser (dairy cattle slurry, digestate) to grassland; using a combination of experimental field results and literature-based data. This step was followed by a quantitative microbial risk assessment (QMRA) model for pathogenic E. coli based on a literature-based dose-response model. The results showed that the maximum daily HE (HEdaily) is associated with E. coli for unprocessed slurry (treatment T1) on day 1, the worst-case scenario where the simulated mean HEdaily was calculated as 2.84 CFU day -1. The results indicate that the overall annual probability of risk (Pannual) of illness from E. coli is very low or low based on the WHO safe-limit of Pannual as 10 -6. In the worst-case scenario, a moderate risk was estimated with simulated mean Pannual as 1.0 × 10 -5. Unpasteurised digestate application showed low risk on day 1 and 2 (1.651 × 10 -6, 1.167 × 10 -6, respectively). Pasteurised digestate showed very low risk in all scenarios. These results support the restriction imposed on applying bio-based fertiliser if there is any rain forecast within 48 h from the application time. This study proposes a future extension of the probabilistic model to include time, intensity, discharge, and distance-dependant dilution factor. The information generated from this model can help policymakers ensure the safety of surface water sources through the quality monitoring of FIB levels in bio-based fertiliser.

Evaluation of pathogen concentration in anaerobic digestate using a predictive modelling approach (ADRISK).

Farmyard manure and slurry (FYM&S) is a valuable feedstock for anaerobic digestion (AD) plants. However, FYM&S may contain high concentrations of pathogens, and complete inactivation through the AD process is unlikely. Thus, following land application of digestate, pathogens may contaminate a range of environmental media posing a potential threat to public health. The present study aimed to combine primary laboratory data with literature-based secondary data to develop an Excel-based exposure assessment model (ADRISK) using a gamma generalised linear model to predict the final microorganism count in the digestate. This research examines the behaviour of a suite of pathogens (Cryptosporidium parvum, norovirus, Mycobacterium spp., Salmonella spp., Listeria monocytogenes, Clostridium spp., and pathogenic Escherichia coli) and indicators (total coliforms, E. coli, and enterococci) during mesophilic anaerobic digestion (M-AD) at 37 °C, pre/post-AD pasteurisation, and after a period of storage (with/without lime) for different feedstock proportions (slurry:food waste: 0:1, 1:3, 2:1, and 3:1). ADRISK tool simulations of faecal indicator bacteria levels across all scenarios show that the digestate can meet the EU standard without pasteurisation if the AD runs at 37 °C or a higher temperature with a higher C:N ratio (recipe 3) and a hydraulic retention time ≥ 7 days. The storage of digestate also reduced levels of microorganisms in the digestate. The Irish pasteurisation process (60 °C for 4 days), although more energy-intensive, is more effective than the EU pasteurisation (70 °C for 1 h) specification. Pre-AD pasteurisation was more effective for C. parvum, norovirus, Mycobacterium thermoresistibile. However, post-AD literature-based pasteurisation is most likely to assure the safety of the digestate. The information generated from this model can inform policy-makers regarding the optimal M-AD process parameters necessary to maximise the inactivation of microorganisms, ensuring adverse environmental impact is minimised, and public health is protected.

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.

Risk assessment of Escherichia coli in bioaerosols generated following land application of farmyard slurry.

Transfer of Escherichia coli in bioaerosols to humans during and shortly after the land application of farmyard slurry may pose human health hazards, but it has not been extensively explored to date. The present study developed a quantitative risk assessment model for E. coli through the air exposure route. The probabilistic model assessed the predicted number of microorganisms in the air (PNair) to which humans may be exposed. A Gaussian air dispersion model was used to calculate the concentration of E. coli transmitted through aerosols. Human exposure (HE) to E. coli was estimated using a Monte Carlo simulation approach. This research predicted the mean HE as 26 CFU day-1 (95th percentile 263 CFU day-1) and suggests the importance of keeping a distance of at least 100 m for the residential population from land spreading activities. However, the simulated mean daily or annual (once a year application) risk of 2.65 × 10-7 person-1 year-1 due to land application of slurry indicates very low occupational risk for farmworkers not equipped with the personal protective equipment (PPE), who are potentially exposed to E. coli indirectly. The model found that the decay constant of E. coli in air, duration of decay, and bio-aerosolisation efficiency factor (top three) could influence HE to airborne E. coli. Furthermore, this research recommends an average time lag of at least 2.5 h following the application of farmyard slurry to the field before humans access the field again without PPE, allowing the airborne pathogen to decay, thereby ensuring occupational safety. The model suggested that the bio-aerosolisation efficiency factor (E) for other pathogens requires further investigation. The information generated from this model can help to assess likely exposure from bioaerosols triggered by land application of farmyard slurry.

Beneficial effects of multi-species mixtures on N2O emissions from intensively managed grassland swards.

In a field experiment, annual nitrous oxide (N2O) emissions and grassland yield were measured across different plant communities, comprising systematically varying combinations of monocultures and mixtures of three functional groups (FG): grasses (Lolium perenne, Phleum pratense), legumes (Trifolium pratense, Trifolium repens) and herbs (Cichorium intybus, Plantago lanceolata). Plots received 150 kg ha-1 year-1 nitrogen (N) (150 N), except L. perenne monocultures which received two N levels: 150 N and 300 N. The effect of plant diversity on N2O emissions was derived from linear combinations of species performances' in monoculture (species identity) and not from strong interactions between species in mixtures. Increasing from 150 N to 300 N in L. perenne resulted in a highly significant increase in cumulative N2O emissions from 1.39 to 3.18 kg N2O-N ha-1 year-1. Higher N2O emissions were also associated with the legume FG. Emissions intensities (yield-scaled N2O emissions) from multi-species mixture communities around the equi-proportional mixture were lowered due to interactions among species. For N2O emissions scaled by nitrogen yield in forage, the 6-species mixture was significantly lower than L. perenne at both 300 N and 150 N. In comparison to 300 N L. perenne, the same N yield or DM yield could have been produced with the equi-proportional 6-species mixture (150 N) while reducing N2O losses by 63% and 58% respectively. Compared to 150 N L. perenne, the same N yield or DM yield could have been produced with the 6-species mixture while reducing N2O losses by 41% and 24% respectively. Overall, this study found that multi-species grasslands can potentially reduce both N2O emissions and emissions intensities, contributing to the sustainability of grassland production.

A Bayesian inference approach to quantify average pathogen loads in farmyard manure and slurry using open-source Irish datasets.

Farm-to-fork quantitative microbial risk assessments (QMRA) typically start with a preliminary estimate of initial concentration (Cinitial) of microorganism loading at farm level, consisting of an initial estimate of prevalence (P) and the resulting pathogen levels in animal faeces. An average estimation of the initial concentration of pathogens can be achieved by combining P estimates in animal populations and the levels of pathogens in colonised animals' faeces and resulting cumulative levels in herd farmyard manure and slurry (FYM&S). In the present study, 14 years of data were collated and assessed using a Bayesian inference loop to assess the likely P of pathogens. In this regard, historical and current survey data exists on P estimates for a number of pathogens, including Cryptosporidium parvum, Mycobacterium avium subspecies paratuberculosis (MAP), Salmonella spp., Clostridium spp., Campylobacter spp., pathogenic E. coli, and Listeria monocytogenes in several species (cattle, pigs, and sheep) in Ireland. The results revealed that Cryptosporidium spp. has potentially the highest mean P (Pmean) (25.93%), followed by MAP (15.68%) and Campylobacter spp. (8.80%) for cattle. The Pmean of E. coli is highest (7.42%) in pigs, while the Pmean of Clostridium spp. in sheep was estimated to be 7.94%. Cinitial for Cryptosporidium spp., MAP., Salmonella spp., Clostridium spp., and Campylobacter spp. in cattle faeces were derived with an average of 2.69, 4.38, 4.24, 3.46, and 3.84 log10 MPN g -1, respectively. Average Cinitial of Cryptosporidium spp., Salmonella spp., Clostridium spp., and E. coli in pig slurry was estimated as 1.27, 3.12, 3.02, and 4.48 log10 MPN g -1, respectively. It was only possible to calculate the average Cinitial of Listeria monocytogenes in sheep manure as 1.86 log10 MPN g -1. This study creates a basis for future farm-to-fork risk assessment models to base initial pathogen loading values for animal faeces and enhance risk assessment efforts.

Competition and community succession link N transformation and greenhouse gas emissions in urine patches.

Nitrous oxide (N2O) is a strong greenhouse gas produced by biotic/abiotic processes directly linked to both fungal and prokaryotic communities that produce, consume or create conditions leading to its emission. In soils exposed to nitrogen (N) in the form of urea, an ecological succession is triggered resulting in a dynamic turnover of microbial populations. However, knowledge of the mechanisms controlling this succession and the repercussions for N2O emissions remain incomplete. Here, we monitored N2O production and fungal/prokaryotic community changes (via 16S and 18S amplicon sequencing) in soil microcosms exposed to urea. Contributions of microbes to emissions were determined using biological inhibitors. Results confirmed that urea leads to shifts in microbial community assemblages by selecting for certain microbial groups (fast growers) as dictated through life history strategies. Urea reduced overall community diversity by conferring dominance to specific groups at different stages in the succession. The diversity lost under urea was recovered with inhibitor addition through the removal of groups that were actively growing under urea indicating that species replacement is mediated in part by competition. Results also identified fungi as significant contributors to N2O emissions, and demonstrate that dominant fungal populations are consistently replaced at different stages of the succession. These successions were affected by addition of inhibitors which resulted in strong decreases in N2O emissions, suggesting that fungal contributions to N2O emissions are larger than that of prokaryotes.

Source partitioning using N2O isotopomers and soil WFPS to establish dominant N2O production pathways from different pasture sward compositions.

Nitrous oxide (N2O) is a potent greenhouse gas (GHG) emitted from agricultural soils and is influenced by nitrogen (N) fertiliser management and weather and soil conditions. Source partitioning N2O emissions related to management practices and soil conditions could suggest effective mitigation strategies. Multispecies swards can maintain herbage yields at reduced N fertiliser rates compared to grass monocultures and may reduce N losses to the wider environment. A restricted-simplex centroid experiment was used to measure daily N2O fluxes and associated isotopomers from eight experimental plots (7.8 m2) post a urea-N fertiliser application (40 kg N ha-1). Experimental pastures consisted of differing proportions of grass, legume and forage herb represented by perennial ryegrass (Lolium perenne), white clover (Trifolium repens) and ribwort plantain (Plantago lanceolata), respectively. N2O isotopomers were measured using a cavity ring down spectroscopy (CRDS) instrument adapted with a small sample isotope module (SSIM) for the analysis of gas samples ≤20 mL. Site preference (SP = δ15Nα - δ15Nß) and δ15Nbulk ((δ15Nα + δ15Nß) / 2) values were used to attribute N2O production to nitrification, denitrification or a mixture of both nitrification and denitrification over a range of soil WFPS (%). Daily N2O fluxes ranged from 8.26 to 86.86 g N2O-N ha-1 d-1. Overall, 34.2% of daily N2O fluxes were attributed to nitrification, 29.0% to denitrification and 36.8% to a mixture of both. A significant diversity effect of white clover and ribwort plantain on predicted SP and δ15Nbulk indicated that the inclusion of ribwort plantain may decrease N2O emission through biological nitrification inhibition under drier soil conditions (31%-75% WFPS). Likewise, a sharp decline in predicted SP indicates that increased white clover content could increase N2O emissions associated with denitrification under elevated soil moisture conditions (43%-77% WFPS). Biological nitrification inhibition from ribwort plantain inclusion in grassland swards and management of N fertiliser source and application timing to match soil moisture conditions could be useful N2O mitigation strategies.

Application of plasma activated water for decontamination of alfalfa and mung bean seeds.

Microbial contamination of fresh produce is a major public health concern, with the number of associated disease outbreaks increasing in recent years. The consumption of sprouted beans and seeds is of particular concern, as these foodstuffs are generally consumed raw, and are produced in conditions favourable for the growth of zoonotic pathogens, if present in seeds prior to sprouting or in irrigation water. This work aimed to evaluate the activity of plasma activated water (PAW) as a disinfecting agent for alfalfa (Medicago sativa) and mung bean (Vigna radiata) seeds, during seed soaking. Each seed type was inoculated with Escherichia coli O157, E. coli O104, Listeria monocytogenes or Salmonella Montevideo, and treated with PAW for different times. A combination of PAW and ultrasound treatment was also evaluated. The germination and growth rate of both seeds were assessed after PAW treatments. PAW was demonstrated to have disinfecting ability on sprouted seeds, with reductions of up to Log10 1.67 cfu/g in alfalfa seeds inoculated with E. coli O104, and a reduction of Log10 1.76 cfu/g for mung bean seeds inoculated with E. coli O157 observed. The germination and growth rate of alfalfa and mung bean sprouts were not affected by the PAW treatments. The combination of a PAW treatment and ultrasound resulted in increased antimicrobial activity, with a reduction of Log10 3.48 cfu/g of S. Montevideo in mung bean seeds observed. These results demonstrate the potential for PAW to be used for the inactivation of pathogenic microorganisms which may be present on sprouted seeds and beans, thereby providing greater assurance of produce safety.

Development and verification of a novel isotopic N2 O measurement technique for discrete static chamber samples using cavity ring-down spectroscopy.

RATIONALE: N2 O isotopomers are a useful tool to study soil N cycling processes. The reliability of such measurements requires a consistent set of international N2 O isotope reference materials to improve inter-laboratory and inter-instrument comparability and avoid reporting inaccurate results. All these are the more important given the role of N2 O in anthropogenic climate change and the pressing need to develop our understanding of soil N cycling and N2 O emission to mitigate such emissions. Cavity ring-down spectroscopy (CRDS) could potentially overcome resource requirements and technical challenges, making N2 O isotopomer measurements more feasible and less expensive than previous approaches (e.g., gas chromatography [GC] and isotope ratio mass spectrometry [IRMS]). METHODS: A combined laser spectrometer and small sample isotope module (CRDS & SSIM) method enabled N2 O concentration, δ15 Nbulk , δ15 Nα , δ15 Nß and site preference (SP) measurements of sample volumes <20 mL, such as static chamber samples. Sample dilution and isotopic mixing as well as N2 O concentration dependence were corrected numerically. A two-point calibration procedure normalised δ values to the international isotope-ratio scales. The CRDS & SSIM repeatability was determined using a reference gas (Ref Gas). CRDS & SSIM concentration measurements were compared with those obtained by GC, and the isotope ratio measurements from two different mass spectrometers were compared. RESULTS: The repeatability (mean ± 1σ; n = 10) of the CRDS & SSIM measurements of the Ref Gas was 710.64 ppb (± 8.64), 2.82‰ (± 0.91), 5.41‰ (± 2.00), 0.23‰ (± 0.22) and 5.18‰ (± 2.18) for N2 O concentration, δ15 Nbulk , δ15 Nα , δ15 Nß and SP, respectively. The CRDS & SSIM concentration measurements were strongly correlated with GC (r = 0.99), and they were more precise than those obtained using GC except when the N2 O concentrations exceeded the specified operating range. Normalising CRDS & SSIM δ values to the international isotope-ratio scales using isotopic N2 O standards (AK1 and Mix1) produced accurate results when the samples were bracketed within the range of the δ values of the standards. The CRDS & SSIM δ15 Nbulk and SP precision was approximately one order of magnitude less than the typical IRMS precision. CONCLUSIONS: CRDS & SSIM is a promising approach that enables N2 O concentrations and isotope ratios to be measured by CRDS for samples <20 mL. The CRDS & SSIM repeatability makes this approach suitable for N2 O "isotopomer mapping" to distinguish dominant source pathways, such as nitrification and denitrification, and requires less extensive lab resources than the traditionally used GC/IRMS. Current study limitations highlighted potential improvements for future users of this approach to consider, such as automation and physical removal of interfering trace gases before sample analysis.

Sward composition and soil moisture conditions affect nitrous oxide emissions and soil nitrogen dynamics following urea-nitrogen application.

Increased emissions of N2O, a potent greenhouse gas (GHG), from agricultural soils is a major concern for the sustainability of grassland agriculture. Emissions of N2O are closely associated with the rates and forms of N fertilisers applied as well as prevailing weather and soil conditions. Evidence suggests that multispecies swards require less fertiliser N input, and may cycle N differently, thus reducing N loss to the environment. This study used a restricted simplex-centroid experimental design to investigate N2O emissions and soil N cycling following application of urea-N (40 kg N ha-1) to eight experimental swards (7.8 m2) with differing proportions of three plant functional groups (grass, legume, herb) represented by perennial ryegrass (PRG, Lolium perenne), white clover (WC, Trifolium repens) and ribwort plantain (PLAN, Plantago lanceolata), respectively. Swards were maintained under two contrasting soil moisture conditions to examine the balance between nitrification and denitrification. Two N2O peaks coincided with fertiliser application and heavy rainfall events; 13.4 and 17.7 g N2O-N ha-1 day-1 (ambient soil moisture) and 39.8 and 86.9 g N2O-N ha-1 day-1 (wet soil moisture). Overall, cumulative N2O emissions post-fertiliser application were higher under wet soil conditions. Increasing legume (WC) proportions from 0% to 60% in multispecies swards resulted in model predicted N2O emissions increasing from 22.3 to 96.2 g N2O-N ha-1 (ambient soil conditions) and from 59.0 to 219.3 g N2O-N ha-1 (wet soil conditions), after a uniform N application rate. Soil N dynamics support denitrification as the dominant source of N2O especially under wet soil conditions. Significant interactions of PRG or WC with PLAN on soil mineral N concentrations indicated that multispecies swards containing PLAN potentially inhibit nitrification and could be a useful mitigation strategy for N loss to the environment from grassland agriculture.

Ranking hazards pertaining to human health concerns from land application of anaerobic digestate.

Anaerobic digestion (AD) has been identified as one of the cleanest producers of green energy. AD typically uses organic materials as feedstock and, through a series of biological processes, produces methane. Farmyard manure and slurry (FYM&S) are important AD feedstock and are typically mixed with agricultural waste, grass and/or food wastes. The feedstock may contain many different pathogens which can survive the AD process and hence also possibly be present in the final digestate. In this study, a semi-quantitative screening tool was developed to rank pathogens of potential health concern emerging from AD digestate. A scoring system was used to categorise likely inactivation during AD, hazard pathways and finally, severity as determined from reported human mortality rates, number of global human-deaths and infections per 100,000 populations. Five different conditions including mesophilic and thermophilic AD and three different pasteurisation conditions were assessed in terms of specific pathogen inactivation. In addition, a number of scenarios were assessed to consider foodborne incidence data from Ireland and Europe and to investigate the impact of raw FYM&S application (without AD and pasteurisation). A sensitivity analysis revealed that the score for the mortality rate (S3) was the most sensitive parameter (rank coefficient 0.49) to influence the final score S; followed by thermal inactivation score (S1, 0.25) and potential contamination pathways (S2, 0.16). Across all the scenarios considered, the screening tool prioritised Cryptosporidium parvum, Salmonella spp., norovirus, Streptococcus pyogenes, enteropathogenic E. coli (EPEC), Mycobacterium spp., Salmonella typhi (followed by S. paratyphi), Clostridium spp., Listeria monocytogenes and Campylobacter coli as the highest-ranking pathogens of human health concern resulting from AD digestate in Ireland. This tool prioritises potentially harmful pathogens which can emerge from AD digestate and highlights where regulation and intervention may be required.

Impact of beef extract used for sample concentration on the detection of Escherichia coli DNA in water samples via qPCR.

There is increasing interest in methodologies for the simultaneous concentration and detection of multiple targets in individual samples. The aim of this study was to investigate the potential presence of E. coli DNA in beef extract powder used as part of a procedure to concentrate water samples for the simultaneous detection of bacteria, viruses and protozoa. DNA from E. coli was detected in five out of six beef extract lots tested, demonstrating the limitations of its inclusion when being used in assays that will be used for the detection of E. coli in water samples. Further work is required to clarify if this phenomenon also occurs for other microorganisms of interest in water.

Impact of nitrogen compounds on fungal and bacterial contributions to codenitrification in a pasture soil.

Ruminant urine patches on grazed grassland are a significant source of agricultural nitrous oxide (N2O) emissions. Of the many biotic and abiotic N2O production mechanisms initiated following urine-urea deposition, codenitrification resulting in the formation of hybrid N2O, is one of the least understood. Codenitrification forms hybrid N2O via biotic N-nitrosation, co-metabolising organic and inorganic N compounds (N substrates) to produce N2O. The objective of this study was to assess the relative significance of different N substrates on codenitrification and to determine the contributions of fungi and bacteria to codenitrification. 15N-labelled ammonium, hydroxylamine (NH2OH) and two amino acids (phenylalanine or glycine) were applied, separately, to sieved soil mesocosms eight days after a simulated urine event, in the absence or presence of bacterial and fungal inhibitors. Soil chemical variables and N2O fluxes were monitored and the codenitrified N2O fluxes determined. Fungal inhibition decreased N2O fluxes by ca. 40% for both amino acid treatments, while bacterial inhibition only decreased the N2O flux of the glycine treatment, by 14%. Hydroxylamine (NH2OH) generated the highest N2O fluxes which declined with either fungal or bacterial inhibition alone, while combined inhibition resulted in a 60% decrease in the N2O flux. All the N substrates examined participated to some extent in codenitrification. Trends for codenitrification under the NH2OH substrate treatment followed those of total N2O fluxes (85.7% of total N2O flux). Codenitrification fluxes under non-NH2OH substrate treatments (0.7-1.2% of total N2O flux) were two orders of magnitude lower, and significant decreases in these treatments only occurred with fungal inhibition in the amino acid substrate treatments. These results demonstrate that in situ studies are required to better understand the dynamics of codenitrification substrates in grazed pasture soils and the associated role that fungi have with respect to codenitrification.

Development and Optimisation of a Multiresidue Method for the Determination of 40 Anthelmintic Compounds in Environmental Water Samples by Solid Phase Extraction (SPE) with LC-MS/MS Detection.

A comprehensive multiresidue method was developed and validated for the determination of 40 anthelmintic compounds, including 13 transformation products, in surface and groundwater samples at sub nanogram per litre (ng L-1) levels. Anthelmintic residues were extracted from unfiltered water samples using polymeric divinylbenzene solid phase extraction (SPE) cartridges, and eluted with methanol: acetone (50:50, v/v). Purified extracts were concentrated, filtered and injected for UHPLC-MS/MS determination. The method recovery (at a concentration representative of realistic expected environmental water levels based on literature review) ranged from 83-113%. The method was validated, at three concentration levels, in accordance to Commission Decision 2002/657/EC and SANTE/11813/2017 guidelines. Trueness and precision, under within-laboratory reproducibility conditions, ranged from 88-114% and 1.1-19.4%, respectively. The applicability of the method was assessed in a pilot study whereby 72 different surface and groundwater samples were collected and analysed for the determination of these 40 compounds for the first time in Ireland. This is the most comprehensive method available for the investigation of the occurrence of both anthelmintic parent compounds and their transformation products in raw, unfiltered environmental waters.

Author Correction: Influence of soil moisture on codenitrification fluxes from a urea-affected pasture soil.

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