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Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n  = 18) being confirmed compared to those from a combination of CH-STEC and BVCC (n  = 42). A significant (P  < .05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone. [ABSTRACT FROM AUTHOR]

The main target of bone tissue engineering is to design biomaterials that support bone regeneration and vascularization. Nanostructured membranes of (MMA)1-co-(HEMA)1/(MA)3-co-(HEA)2 loaded with 5% wt of SiO2-nanoparticles (Si-M) were doped with zinc (Zn-Si-M) or doxycycline (Dox-Si-M). Critical bone defects were effectuated on six New Zealand-bred rabbit skulls and then they were covered with the membranes. After six weeks, a histological analysis (toluidine blue technique) was employed to determine bone cell population as osteoblasts, osteoclasts, osteocytes, M1 and M2 macrophages and vasculature. Membranes covering the bone defect determined a higher count of bone cells and blood vessels than in the sham group at the top regions of the defect. Pro-inflammatory M1 appeared in a higher number in the top regions than in the bottom regions, when Si-M and Dox-Si-M were used. Samples treated with Dox-Si-M showed a higher amount of anti-inflammatory and pro-regenerative M2 macrophages. The M1/M2 ratio obtained its lowest value in the absence of membranes. On the top regions, osteoblasts were more abundant when using Si-M and Zn-Si-M. Osteoclasts were equally distributed at the central and lateral regions. The sham group and samples treated with Zn-Si-M attained a higher number of osteocytes at the top regions. A preferential osteoconductive, osteoinductive and angiogenic clinical environment was created in the vicinity of the membrane placed on critical bone defects. [ABSTRACT FROM AUTHOR]

Invertebrate-pathogenic fungi in the genus Metarhizium are commonly found throughout the world on a wide range of different arthropod hosts. Some strains have been developed as biopesticides and many others have shown potential for controlling important insect pests. Given the current interest in using these fungi, precise identification at species level is crucial, both for understanding their diversity and ecology and for regulatory approval and monitoring of new biopesticides. Metarhizium has undergone extensive systematic revision over the last decade, resulting in a large number of name changes and new species. This has created regulatory issues in New Zealand and importation of biocontrol strains has become difficult due to a lack of knowledge of which Metarhizium species are present in the country. This study identified Metarhizium isolates held in culture collections in New Zealand following analysis of sequence data from the ef-1α, β-tubulin, rpb1, and ITS regions. Our results show that M. anisopliae, M. brunneum, M. frigidum, M. novozealandicum, M. pemphigi, M. rileyi and M. robertsii occur naturally in New Zealand on native and introduced insect hosts. Exotic Metarhizium species from several countries, stored in New Zealand collections, include M. acridum, M. lepidiotae, M. majus and M. pingshaense. [ABSTRACT FROM AUTHOR]

A pilot survey was performed to determine the prevalence of Campylobacter jejuni and Campylobacter coli on three age classes (lamb, hogget, and mutton) of ovine carcass trim postdressing and prechill. Sampling of hogget carcasses was undertaken 6 months before sampling of lamb and mutton carcasses. A total of 120 trim samples were collected from 11 processing plants across New Zealand. All samples were enriched and screened using PCR for the presence of C. jejuni and C. coli, and isolation was attempted for all screen-positive samples. Enumeration of Campylobacter from lamb trim samples showed that Campylobacter bacteria were present in very low numbers (< 10 CFU/g). The overall prevalence of Campylobacter for ovine trim based on PCR detection was 33% (39 of 120 samples), with prevalences for hogget, lamb, and mutton carcass trim of 56% (28 of 50), 11% (4 of 35), and 20% (7 of 35), respectively. Whole genome sequencing was performed on a selection of C. jejuni and C. coli isolates, and the data were used to subtype using multilocus sequence typing (MLST) and whole genome MLST. Twenty-five MLST sequence types (STs) were identified among 44 isolates, including ST42, ST50, ST3222, and ST3072, which have been previously reported to be associated with ruminant sources. Four novel STs were also identified. Whole genome MLST analysis further discriminated isolates within a single ST type and demonstrated a genetic diversity among the ovine isolates collected. Genes associated with the oxacillinase class of β-lactamase enzymes were identified in 41 of 44 Campylobacter isolates. This study provides preliminary data that can be incorporated into existing source attribution models to assist in determining the potential contribution of ovine sources to the burden of campylobacteriosis in New Zealand. Campylobacter prevalence for ovine carcass trim based on PCR detection was 33%. Enumeration of Campylobacter bacteria was < 10 CFU/g on lamb trim. Twenty-five multilocus sequence types (STs) were identified among 44 isolates. Whole genome sequence analysis discriminated isolates within a single ST. [ABSTRACT FROM AUTHOR]

Background: We describe the investigation of a Campylobacter outbreak linked to contamination of an untreated, groundwater derived drinking water supply.Methods: We analysed epidemiological data collected from clinician-confirmed diarrheal cases and estimated the total burden of Havelock North cases using an age-adjusted cross-sectional telephone survey. Campylobacter isolates from case fecal specimens, groundwater samples, and sheep fecal specimens from paddocks adjacent to the drinking water source were whole genome sequenced.Findings: We estimate between 6260 and 8320 cases of illness including up to 2230 who lived outside the reticulation area, were linked to the contaminated water supply. Of these, 953 cases were physician reported, 42 were hospitalized, three developed Guillain-Barré syndrome, and Campylobacter infection contributed to at least four deaths. Of the 12 genotypes observed in cases, four were also observed in water, three were also observed in sheep and one was also observed in both water and sheep.Interpretation: The contamination of the untreated reticulated water supply occurred following a very heavy rainfall event which caused drainage of sheep feces into a shallow aquifer. The existence of a routine clinical surveillance system for campylobacteriosis facilitated identification of the outbreak, recovery of clinical isolates, and early testing of the water for pathogens. Genotyping of the Campylobacter jejuni helped define the source of the outbreak and confirm outbreak periods and cases. Expected increases in heavy rainfall events and intensification of agriculture mean that additional safeguards are needed to protect populations from such drinking water outbreaks.Funding: NZ Ministry of Health, Health Research Council, ESR SSIF, Royal Society. [ABSTRACT FROM AUTHOR]

The objective of this study was to undertake a microbiological survey of foods, animal faeces and wastewater samples for Clostridium difficile, and determine the genotypes and antimicrobial susceptibilities of isolates. A total of 211 samples were tested for C. difficile using culture methods. Thirteen toxigenic C. difficile isolates were obtained; ten from wastewater samples, one each from pig and duck faeces and another from a raw meat product. Eight PCR‐ribotypes (RTs) were identified, including two novel RTs (878 and 879). Single‐nucleotide polymorphism analysis using WGS data for all isolates provided greater discrimination between C. difficile isolates within the same RT and multilocus sequence typing (MLST) profiles. All C. difficile isolates were found to be susceptible to the first‐line human antimicrobials used to treat C. difficile infection. Significance and Impact of the Study: This is the first study to report the isolation of Clostridium difficile from animals, food and wastewater in New Zealand (NZ) and provides important data with respect to ribotypes and multilocus sequence typing profiles, whole genome sequence and antimicrobial susceptibilities. The results highlight the need for further investigations into the epidemiology of C. difficile in NZ and to elucidate the role of the environmental and food sources as transmission routes of human infection. [ABSTRACT FROM AUTHOR]

Four cases of listeriosis in a hospital (A) in New Zealand were identified in 2012. Pulsed‐field gel electrophoresis (PFGE) used at the time identified four pulsotypes amongst the clinical isolates. Two of the pulsotypes matched to Listeria monocytogenes isolates obtained from ready‐to‐eat (RTE) meat samples from a RTE producer tested during a nationwide microbiological survey the month prior. The outbreak investigation confirmed that the RTE producer had supplied product to the hospital and additional testing confirmed the presence of L.  monocytogenes in RTE meats from the hospital kitchen. Two further listeriosis cases presented in another hospital (B) with one clinical isolate identified as the same pulsotype as identified for one case in hospital A, but the epidemiology information concluded that the clinical cases from hospital B were not linked to the outbreak. Retrospective whole‐genome sequencing confirmed that epidemiologically linked isolates belonging to three different genotypes for clinical cases from hospital A and RTE meats samples from the hospital kitchen differed by 0‐1 core‐genome locus or single nucleotide polymorphisms (SNP). The use of core‐genome multilocus sequence typing and SNP analysis provided a greater degree of discrimination between isolates compared to PFGE. Significance and Impact of the Study: This study describes a listeriosis outbreak associated with a hospital in New Zealand and attributed to contaminated ready‐to‐eat (RTE) meat supplied to the hospital by a single producer. Retrospective whole‐genome sequence analysis of outbreak isolates was found to provide a greater degree of discrimination between isolates compared to pulsed‐field gel electrophoresis and supported the conclusions made at the time of the outbreak. The multiple genotypes identified from clinical cases and the RTE meats obtained during the outbreak highlight the importance of epidemiological concordance alongside genotyping. [ABSTRACT FROM AUTHOR]

Background Shiga toxin–producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. Methods We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. Results The common ancestor of this set of isolates occurred around 1890 (1845–1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909–1958), to the United States in 1941 (1921–1962), to Canada in 1960 (1943–1979), and from Australia to New Zealand in 1966 (1943–1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. Conclusions Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally. [ABSTRACT FROM AUTHOR]

A microbiological survey was undertaken on packaged ready-to-eat red meats available at retail in New Zealand. A total of 1,485 samples (297 lots of five samples each) were collected according to a sampling plan based on market share and regulatory regimes (Animal Products Act 1999 and Food Act 1981) and were tested against the microbiological limits specified in Food Standards Code (FSC) 1.6.1 applicable at the time of sampling. Each lot was tested as a composite for the presence or absence of Salmonella spp., coagulase-producing staphylococci, Listeria monocytogenes, and other Listeria spp. at the end of the manufacturer's stated shelf life. Individual samples within a positive lot were subsequently enumerated for L. monocytogenes. None of the samples contained Salmonella spp. or had coagulase-producing staphylococci counts above the acceptable level specified in FSC 1.6.1 (>100 CFU/g). Data showed that 93.6% (278 of 297 lots) of ready-to-eat red meat complied with the FSC 1.6.1 criteria applicable at the time of the survey. The failure of 19 lots (6.4%) was due to the presence of L. monocytogenes from product obtained from 8 of 33 producers tested. Thirteen samples of 95 positive samples were found to contain between 50 and 500 CFU/g L. monocytogenes, but all of these samples were manufactured by the same operator. Pulsed-field gel electrophoresis typing of all of the L. monocytogenes isolates obtained from the survey identified 12 different pulsotypes. Different pulsotypes were often identified in samples from the same operator sampled on separate occasions. A total of 46 lots (15.5%) contained Listeria spp. (including L. monocytogenes). The detection of Listeria in samples may highlight the existence of problems in operator processing and/or packaging processes and suggests that improvements in good hygienic practice and implementation of more effective risk mitigation strategies are needed. [ABSTRACT FROM AUTHOR]

A sound understanding of the effects of hydrogeological factors on loss, transport and transformation of farm nutrients is essential for predicting their impacts on ecosystem health of receiving waters. We assessed the potential of groundwater to attenuate nitrate through denitrification, and the distribution of this potential across the Tararua Groundwater Management Zone (GWMZ) in the Manawatu River catchment, New Zealand. We combined a number of methods in an unprecedented manner to confirm findings and obtain supporting evidence for the features that determine the subsurface denitrification characteristics. Our results showed that the denitrification characteristics of groundwater varied considerably in the Tararua GWMZ. The southern part of the Tararua GWMZ contained mainly oxic groundwater with low potential to denitrify, whereas the middle and northern parts of the Tararua GWMZ contained reduced groundwater with high denitrification potential. The hydrogeological features that influence denitrification potential in groundwater were identified as soil texture and drainage class, and the aquifer material or rock type. Low dissolved oxygen levels and nitrate concentrations were found in groundwater where the combinations of soil and rock types had poor drainage characteristics as opposed to higher concentrations in groundwater under well-drained soils and rocks (e.g. gravels). Intensive pastoral farming over well-drained soils and rocks showed high nitrate concentration in groundwater. This spatial variability in denitrification potential of groundwater offers a targeted management of nutrients runoff and leaching from pastoral lands to reduce their impacts on receiving surface waters. [ABSTRACT FROM AUTHOR]

Background: Shiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results: Seventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx2c (vh-a) (91%) in Argentina, stx2 (89%) in New Zealand, and stx1/stx2 (30%) in Australia. No stx1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion: STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx-genotype and phage type. Additionally, no common PFGE patterns were found in strains isolated in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones. [ABSTRACT FROM AUTHOR]

The rate of yersiniosis in New Zealand (NZ) is high compared with other developed countries, and rates have been increasing over recent years. Typically, >99% of human cases in NZ are attributed to Yersinia enterocolitica (YE), although in 2014, a large outbreak of 220 cases was caused by Yersinia pseudotuberculosis. Up until 2012, the most common NZ strain was YE biotype 4. The emergent strain since this time is YE biotype 2/3 serotype O:9. The pathogenic potential of some YE biotypes remains unclear. Most human cases of yersiniosis are considered sporadic without an identifiable source. Key restrictions in previous investigations included insufficient sensitivity for the isolation of Yersinia spp. from foods, although foodborne transmission is the most likely route of infection. In NZ, YE has been isolated from a variety of sick and healthy domestic and farm animals but the pathways from zoonotic reservoir to human remain unproven. Whole-genome sequencing provides unprecedented discriminatory power for typing Yersinia and is now being applied to NZ epidemiological investigations. A "One-Health" approach is necessary to elucidate the routes of transmission of Yersinia and consequently inform targeted interventions for the prevention and management of yersiniosis in NZ [ABSTRACT FROM AUTHOR]

Subsurface denitrification plays a key role in the reduction or 'attenuation' of nitrate contamination of groundwater and surface waters. We investigated subsurface denitrification characteristics in the vadose zone and shallow groundwater at four sites under pastoral farming in the Manawatū River catchment, located in the lower part of North Island, New Zealand. The denitrification potential of the vadose zone was determined by the laboratory incubation assays measuring the denitrifying enzyme activity (DEA) in soil samples collected from different soil horizons (up to 2.1 m below ground surface), whereas denitrification rates in shallow groundwaters were measured in situ by single-well, push-pull tests conducted in piezometers installed at multiple depths at the study sites. Soils and underlying geology, defining hydrogeologic settings, appear to influence the spatial variability of subsurface denitrification characteristics at the study sites. Where the vadose zone is thin and composed of coarse-textured soils, percolation of nitrate was evident in observed high nitrate-nitrogen concentrations (>5 mg L−1) in oxic and young shallow groundwaters, but low nitrate-nitrogen concentrations (<0.05 mg L−1) were observed in the reduced shallow groundwater found underneath the fine textured soils and/or a thick vadose zone. This was confirmed by the push-pull tests measuring denitrification rates from 0.08 to 1.07 mg N L−1 h−1 in the reduced shallow groundwaters (dissolved oxygen or DO < 0.5 mg L−1), while negligible in the oxic groundwaters (DO > 5 mg L−1) found at the study sites. These contrasting subsurface denitrification characteristics determine the ultimate delivery of nitrate losses from agricultural soils to receiving waters, where the fine textured thick vadose zone and reducing groundwater conditions offer nitrate reduction in the subsurface environment. Image 1 • Subsurface denitrification was assessed in different hydrogeological settings. • Laboratory incubations measured denitrification potential in the vadose zone layers. • I n-situ push-pull tests measured denitrification rates in the shallow groundwaters. • Soils and underlying geology influence variability of subsurface denitrification. • Nitrate reduction was greatest in fine textured soils and reducing groundwaters. [ABSTRACT FROM AUTHOR]

While enabling economically viable use of poorly drained soils, artificial subsurface drainage has also been found to be a significant pathway for nutrient transfers from agricultural land to surface waters. Thus, mitigating the impacts of agriculture on surface water quality needs to address nutrient transfers via subsurface drainage. Woodchip bioreactors are a promising mitigation option as demonstrated under arable agriculture in the mid-west of the USA. However, research is needed to ascertain their efficiency in removing nutrients from very flashy drainage flows common in New Zealand (NZ) pastoral agriculture and any possible pollution swapping (e.g. reduction of leaching losses vs. greenhouse gas emissions). Accordingly, a lined 78-m3 woodchip bioreactor was constructed on a dairy farm in the Hauraki Plains (Waikato, NZ) with a drainage area of 0.65 ha. Rainfall, flow, hydrochemistry and dissolved gases in the inflow and outflow were monitored for two drainage seasons (part of 2017, 2018). Based on the nitrate-N fluxes, the estimated nitrate removal efficiency of the bioreactor was 99 and 48% in 2017 and 2018, respectively. The higher removal efficiency in 2017 could be attributed to two reasons. Firstly, the substantially longer hydraulic residence time (HRT) of the water in the bioreactor (mean = 21.1 days vs 4.7 days in 2018) provided more opportunity for microorganisms to reduce the nitrate. A strong positive relationship between HRT and removal efficiency was also observed within the 2018 drainage season. Secondly, denitrification was supported in 2017 by greater electron donor availability. Evidence of this was the higher mass of DOC discharge from the bioreactor (318 mg C L−1 of bioreactor volume vs 165 mg C L−1 in 2018). Removal rates in the bioreactor varied from 0.67–1.60 g N m−3 day−1 and were positively correlated with inflow nitrate loads. Pollution swapping was observed during the start-up phase of the bioreactor in both years (DOC, and DRP only in 2017) and during periods with very long HRTs (hydrogen sulphide (H 2 S) and methane (CH 4) production). Substantially elevated discharges of DOC and DRP, as compared to inlet conditions, occurred during the initial start-up phase of the bioreactor in 2017 (3 to 3.5 pore volumes of the bioreactor), but only slightly elevated DOC and decreased DRP discharges were observed when drainage flow resumed at the start of the 2018 drainage season. Unexpectedly, cumulative DRP removal during the 2018 drainage season amounted to 89% of the DRP inflow into the bioreactor. Long HRTs (>5 days) enabled high nitrate removal efficiency (≥59%) and promoted complete reduction of nitrate to harmless dinitrogen gas but also promoted strongly reduced conditions, resulting in the production of H 2 S and CH 4. On the other hand, short HRTs (<4 days) only allowed for moderate nitrate removal efficiency (≤43%) and constrained complete reduction of nitrate resulting in higher nitrous oxide concentrations in the outflow as compared to the inflow. Thus, nitrate removals above 50% were not able to be achieved without inducing H 2 S and CH 4 generation. However, it may be achievable when the microbial community is provided with an additional source of readily available carbon during the critical periods when hydraulic flow and concomitant N load peaks occur. Unlabelled Image • Hydraulic residence time, available organic carbon, and inflow nitrate load affected nitrate removal. • Long hydraulic residence time (>5 days) resulted in hydrogen sulphide production. • Short hydraulic residence time (<4 days) resulted in net nitrous oxide production. • Enhancing nitrate removal from dynamic drainage requires additional readily available carbon during high flows. • High retention (89%) of dissolved reactive phosphorus in 2nd year merits further investigation. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 Asc I and 29 Apa I PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Whole-genome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates. • The set of New Zealand Listeria monocytogenes food isolates formed unique genetic clusters. • There were 23 clonal complexes and 25 sequence types found in this study. • NZ horticulture and seafood L. monocytogenes isolates were predominantly lineage II accounting for 89%. • WGS-SNP gave unambiguous and more robust phylogenetic inferences than PFGE or MLST [ABSTRACT FROM AUTHOR]

Woodchip bioreactors are a practical, low-cost technology for reducing nitrate (NO 3) loads discharged from agriculture. Traditional methods of quantifying their performance in the field mostly rely on low-frequency, time-based (weekly to monthly sampling interval) or flow-weighted sample collection at the inlet and outlet, creating uncertainty in their performance and design by providing incomplete information on flow and water chemistry. To address this uncertainty, two field bioreactors were monitored in the US and New Zealand using high-frequency, multipoint sampling for in situ monitoring of NO 3 –N concentrations. High-frequency monitoring (sub hourly interval) at the inlet and outlet of both bioreactors revealed significant variability in volumetric removal rates and percent reduction, with percent reduction varying by up to 25 percentage points within a single flow event. Time series of inlet and outlet NO 3 showed significant lag in peak concentrations of 1–3 days due to high hydraulic residence time, where calculations from instantaneous measurements produced erroneous estimates of performance and misleading relationships between residence time and removal. Internal porewater sampling wells showed differences in NO 3 concentration between shallow and deep zones, and "hot spot" zones where peak NO 3 removal co-occurred with dissolved oxygen depletion and dissolved organic carbon production. Tracking NO 3 movement through the profile showed preferential flow occurring with slower flow in deeper woodchips, and slower flow further from the most direct flowpath from inlet to outlet. High-frequency, in situ data on inlet and outlet time series and internal porewater solute profiles of this initial work highlight several key areas for future research. Image 1 • In situ, short term monitoring of nitrate removal for two field woodchip bioreactors. • High frequency measurements reduced uncertainty of performance. • Misleading relationships between flow and removal caused by lagged nitrate peaks. • Vertical and longitudinal gradients of nitrate, organic carbon, and dissolved oxygen. • High frequency solute tracking revealed zones where preferential flow was occurring. [ABSTRACT FROM AUTHOR]

Woodchip bioreactors have gained popularity in many countries as a conservation practice for reducing nitrate load to freshwater. However, current methods for assessing their performance may be inadequate when nitrate removal rates (RR) are determined from low-frequency (e.g., weekly) concurrent sampling at the inlet and outlet. We hypothesised that high-frequency monitoring data at multiple locations can help improve the accuracy of quantifying nitrate removal performance, enhance the understanding of processes occurring within a bioreactor, and therefore improve the design practice for bioreactors. Accordingly, the objectives of this study were to compare RRs calculated using high- and low-frequency sampling and assess the spatiotemporal variability of the nitrate removal within a bioreactor to unravel the processes occurring within a bioreactor. For two drainage seasons, we monitored nitrate concentrations at 21 locations on an hourly or two-hourly basis within a pilot-scale woodchip bioreactor in Tatuanui, New Zealand. A novel method was developed to account for the variable lag time between entry and exit of a parcel of sampled drainage water. Our results showed that this method not only enabled lag time to be accounted for but also helped quantify volumetric inefficiencies (e.g., dead zone) within the bioreactor. The average RR calculated using this method was significantly higher than the average RR calculated using conventional low-frequency methods. The average RRs of each of the quarter sections within the bioreactor were found to be different. 1-D transport modelling confirmed the effect of nitrate loading on the removal process as nitrate reduction followed Michaelis-Menten (MM) kinetics. These results demonstrate that high-frequency temporal and spatial monitoring of nitrate concentrations in the field allows improved description of bioreactor performance and better understanding of processes occurring within woodchip bioreactors. Thus, insights gained from this study can be used to optimise the design of future field bioreactors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)