A new UHPLC-HRMS metabolomics approach for the rapid and comprehensive analysis of phenolic compounds in blueberry, raspberry, blackberry, cranberry and cherry fruits.

Phenolic compounds are considered an important group of bioactive molecules that are present in abundant quantities in fruits such as berries and cherries; hence, the analysis and quantification of these compounds are of significant interest to the scientific community. The current study aimed to develop a novel analytical method using liquid chromatography and high-resolution mass spectrometry (UHPLC-HRMS) for the rapid, comprehensive and simultaneous analysis of 66 phenolic compounds optimized for the selected five types of fruits commercially available in Canada. Bioactive compounds that could potentially be metabolite markers for each berry were identified. Various phenolic compounds were identified and quantified in all five selected fruits. Notably, blackberries were rich in anthocyanins such as cyanidin-3-glucoside (368.4 ± 6 µg/g), while blueberries were rich in peonidin-3-glucoside (1083 ± 9 µg/g). In addition, raspberries and cherries contained significant amounts of cyanidin-3-rutinoside, at 3156 ± 36 µg/g and 301.3 ± 2 µg/g, respectively, while cranberries contained the highest concentrations of petunidin at 829.7 ± 3 µg/g. The newly developed and validated UHPLC-HRMS method proved helpful in comprehensively analyzing phenolic compounds in blueberry, raspberry, cranberry, blackberry and cherry. Identifying and quantifying bioactives can lead to applications in neutraceutical and pharmaceutical industries by using phenolic-rich berry extracts in functional foods, supplements, or pharmaceutical products.

Veterinary antimicrobials in cattle feedlot environs and irrigation conveyances in a high-intensity agroecosystem in southern Alberta, Canada.

The South Saskatchewan River Basin (SSRB) is considered one of the most intensively farmed regions in Canada, with high densities of livestock and expansive areas of irrigated cropland. We measured concentrations of seven veterinary antimicrobials (VAs) in 114 surface water samples from feedlot environs and 219 samples from irrigation conveyances in the SSRB. Overall, detection frequencies in feedlot environs were 100% for chlortetracycline (CTC) and tetracycline (TC), 94% for monensin (MON), 84% for tylosin (TYL), 72% for lincomycin (LIN), 66% for erythromycin (ERY), and 23% for sulfamethazine (SMZ). For irrigation conveyances, detection frequencies for CTC and TC remained high (94-100%), but dropped to 18% for ERY, 15% for TYL, 10% for MON, and 4% for SMZ. Lincomycin was not detected in irrigation conveyance water. Maximum concentrations of VAs ranged from 1384 µg L-1 (TC) to 17 ng L-1 (SMZ) in feedlot environs while those in irrigation conveyances were 155 ng L-1 (TC) to 29 ng L-1 (ERY). High detection frequencies and median concentrations of VAs in both feedlot environs and irrigation conveyances were associated with high amounts of precipitation. However, an irrigation district (ID) with high livestock density (Lethbridge Northern) did not exhibit higher concentrations of VAs compared to IDs with less livestock, while levels of VAs in irrigation conveyances were less influenced by the degree of surface runoff. The ubiquity of CTC and TC in our study is likely a reflection of its widespread use in intensive livestock operations. Additional investigation is required to link environmental concentrations of VAs with livestock densities and increase our understanding of potential antimicrobial resistance in high-intensity agroecosystems.

Implications of Crop Rotation and Fungicide on Fusarium and Mycotoxin Spectra in Manitoba Barley, 2017-2019.

Fusarium head blight (FHB) is one of the most important diseases of barley in Manitoba province (western Canada), and other major barley producing regions of the world. Little is known about the Fusarium species and mycotoxin spectra associated with FHB of barley in Manitoba. Hence, barley grain samples were collected from 149 commercial fields from 2017 to 2019, along with information on respective cropping history, and analyzed with respect to Fusarium species spectra, abundance, chemotype composition, and mycotoxin profiles. Fusarium poae was the predominant Fusarium species associated with FHB of barley in Manitoba, followed by F. graminearum, and F. sporotrichioides; F. equiseti and F. avenaceum were also detected but at low levels. F. poae strains with the nivalenol (NIV) chemotype and F. graminearum strains with 3-acetyl deoxynivalenol (3-ADON) and 15-acetyl deoxynivalenol (15-ADON) chemotypes were commonly detected in the barley grain samples. Nivalenol (597.7, 219.1, and 412.4 µg kg-1) and deoxynivalenol (DON) (264.7, 56.7, and 65.3 µg kg-1) were the two most prevalent mycotoxins contaminating Manitoba barley in 2017, 2018 and 2019, respectively. A substantially higher DON content was detected in grain samples from barley fields with cereals as a preceding crop compared to canola and flax. Furthermore, F. poae proved less sensitive to four triazole fungicides (metconazole, prothioconazole+tebuconazole, tebuconazole, and prothioconazole) than F. graminearum. Findings from this research will assist barley producers with improved understanding of FHB threat levels and optimizing practices for the best management of FHB in barley.

Degradation of antimicrobial resistance genes within stockpiled beef cattle feedlot manure.

Degradation of antimicrobial resistance genes (ARG) in manure from beef cattle administered (kg-1 feed) 44 mg of chlortetracycline (CTC), 44 mg of chlortetracycline plus sulfamethazine (CTCSMZ), 11 mg of tylosin (TYL), or no antimicrobials (Control) was examined. Manure was stockpiled and quantitative PCR (qPCR) was used to assess tetracycline [tet(C), (L), (M), (W)], erythromycin [erm(A), (B), (F), (X)], and sulfamethazine [sul(1), (2)] ARG and 16S rDNA. After 102 d, copies of all ARG decreased by 0.3 to 1.5 log10 copies (g dry matter)-1. Temperature in the interior of piles averaged ≥ 55 °C for 10 d, except for CTCSMZ, but did not reach 55 °C at pile exteriors. Compared to Control, CTCSMZ increased (P < 0.05) tet(C), tet(M), tet(W), sul(1), and sul(2) in stockpiled manure. Copies of 16S rDNA remained higher (P < 0.05) in CTCSMZ than Control for the first 26 d. Levels of most ARG did not differ between the interior and exterior of stockpiles. Our results suggest that stockpiled manure would still introduce ARG to land upon manure application, but at levels lower than if manure was applied fresh.

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada.

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016-2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.

Dissipation of antimicrobial resistance genes in compost originating from cattle manure after direct oral administration or post-excretion fortification of antimicrobials.

Dissipation of antimicrobial resistance genes (ARG) during composting of cattle manure generated through fortification versus administration of antimicrobials in feed was compared. Manure was collected from cattle fed diets containing (kg-1) dry matter (DM): (1) 44 mg chlortetracycline (CTC), (2) a mixture of 44 mg each of chlortetracycline and sulfamethazine (CTCSMZ), (3) 11 mg tylosin (TYL) or (4) Control, no antimicrobials. Manures were composted for 30 d with a single mixing after 16 d to generate the second heating cycle. Quantitative PCR (qPCR) was used to measure 16S rDNA and tetracycline (tet), erythromycin (erm) and sulfamethazine (sul) genes. Temperature peaks ranged from 48 to 68°C across treatments in the first composting cycle, but except for the control, did not exceed 55°C in the second cycle. Copy numbers of 16S rDNA decreased (P < 0.05) during composting, but were not altered by antimcrobials. Except tet(L), all ARG decreased by 0.1-1.6 log10 g DM-1 in the first cycle, but some genes (tet[B], tet[L], erm[F], erm[X]) increased (P < 0.05) by 1.0-3.1 log10 g DM-1 in the second. During composting, levels of tet(M) and tet(W) in CTC, erm(A), erm(B) and erm(X) in TYL, and sul(1) in CTCSMZ remained higher (P < 0.05) in fed than fortified treatments. The dissipation of ARG during composting of manure fortified with antimicrobials differs from manure generated by cattle that are administered antimicrobials in feed, and does not always align with the dissipation of antimicrobial residues.

Transport of Three Antimicrobials in Runoff from Windrows of Composting Beef Cattle Manure.

Rain runoff from windrowed or stockpiled manure may contain antimicrobials with the potential to contaminate surface and ground water. To quantify the concentration of antimicrobials transported in runoff from windrowed manure, antimicrobials were administered continuously in feed to beef cattle () as follows: 44 mg of chlortetracycline kg feed (dry weight), a 1:1 mixture of 44 mg of chlortetracycline and 44 mg sulfamethazine kg feed, and 11 mg of tylosin kg feed. Cattle in a fourth treatment group received no antimicrobials (control). Manure from the cattle was used to construct two windrows per treatment. On Days 2 and 21 of composting, a portable Guelph Rainfall Simulator II was used to apply deionized water at an intensity of 127 mm h to each windrow, and the runoff was collected. Manure samples were collected before rain simulations on Days 2 and 21 of composting for antimicrobial analysis. On Day 2, average concentrations of chlortetracycline, sulfamethazine, and tylosin in manure were 2580, 450, and 120 µg kg, respectively, with maximum concentrations in runoff of 2740, 3600, and 4930 µg L, respectively. Concentrations of all three antimicrobials in runoff were higher ( < 0.05) on Day 2 than on Day 21, reflecting the higher concentrations in manure on Day 2. Maximum estimated masses of chlortetracycline, sulfamethazine, and tylosin that could be transported in runoff from a windrow (3 m long, 2.5 m wide, 1.5 m high) were approximately 0.87 to 0.94, 1.57, and 1.23 g, respectively. This study demonstrates the importance of windrow composting in reducing antimicrobial concentrations in manure. The runoff from windrows can be a source of antimicrobials and demonstrates the need for containment of runoff from composting facilities to mitigate antimicrobial contamination of surface and groundwater resources.

Dissipation of Antimicrobials in Feedlot Manure Compost after Oral Administration versus Fortification after Excretion.

Fortification of manure with antimicrobials is one approach to studying their dissipation. However, fortified antimicrobials may not accurately model dissipation that occurs after antimicrobials have been administered to livestock in feed and excreted in manure. This study examined the dissipation of antimicrobials excreted in manure versus those added directly to manure (fortified). Steers were fed a diet containing (kg feed) (i) 44 mg chlortetracycline, (ii) 44 mg each of chlortetracycline and sulfamethazine, (iii) 11 mg tylosin, and (iv) no antimicrobials (control). Fortified antimicrobial treatments were prepared by adding antimicrobials to control manure. Manure was composted for 30 d, sampled every 2 to 3 d, and analyzed for antimicrobials and compost properties. Antimicrobial dissipation followed first-order kinetics. The dissipation rate constant was significantly greater (based on 95% confidence limit) for excreted (0.29-0.54 d) than for fortified chlortetracycline (0.11-0.13 d). In contrast, dissipation rate constants were significantly greater for fortified sulfamethazine (0.47 d) and tylosin (0.31 d) than when the same antimicrobials were excreted (0.08 and 0.07 d, respectively). On average, 85 to 99% of the initial antimicrobial concentrations in manure were dissipated after 30 d of composting. The degree of dissipation was greater ( < 0.0001) for fortified (99%) than for excreted tylosin (85%). Composting can be used to reduce environmental loading of antimicrobials before field application of beef cattle manure. Dissipation rates of fortified antimicrobials during manure composting may not accurately reflect those of antimicrobials that are consumed and excreted by cattle.

Dissipation of Antimicrobial Resistance Determinants in Composted and Stockpiled Beef Cattle Manure.

Windrow composting or stockpiling reduces the viability of pathogens and antimicrobial residues in manure. However, the impact of these manure management practices on the persistence of genes coding for antimicrobial resistance is less well known. In this study, manure from cattle administered 44 mg of chlortetracycline kg feed (dry wt. basis) (CTC), 44 mg of CTC and 44 mg of sulfamethazine kg feed (CTCSMZ), 11 mg of tylosin kg feed (TYL), and no antimicrobials (control) were composted or stockpiled over 102 d. Temperature remained ≥55°C for 35 d in compost and 2 d in stockpiles. Quantitative PCR was used to measure levels of 16S rRNA genes and tetracycline [(B), (C), (L), (M), (W)], erythromycin [(A), (B), (F), (X)], and sulfamethazine [(1), (2)] resistance determinants. After 102 d, 16S rRNA genes and all resistance determinants declined by 0.5 to 3 log copies per gram dry matter. Copies of 16S rRNA genes were affected ( < 0.05) by antimicrobials with the ranking of control > CTC = TYL > CTCSMZ. Compared with the control, antimicrobials did not increase the abundance of resistance genes in either composted or stockpiled manure, except (M) and (2) in CTCSMZ ( < 0.05). The decline in 16S rRNA genes and resistance determinants was higher ( < 0.05) in composted than in stockpiled manure. We conclude that composting may be more effective than stockpiling in reducing the introduction of antimicrobial resistance genes into the environment before land application of manure.

Transport of three veterinary antimicrobials from feedlot pens via simulated rainfall runoff.

Veterinary antimicrobials are introduced to wider environments by manure application to agricultural fields or through leaching or runoff from manure storage areas (feedlots, stockpiles, windrows, lagoons). Detected in manure, manure-treated soils, and surface and ground water near intensive cattle feeding operations, there is a concern that environmental contamination by these chemicals may promote the development of antimicrobial resistance in bacteria. Surface runoff and leaching appear to be major transport pathways by which veterinary antimicrobials eventually contaminate surface and ground water, respectively. A study was conducted to investigate the transport of three veterinary antimicrobials (chlortetracycline, sulfamethazine, tylosin), commonly used in beef cattle production, in simulated rainfall runoff from feedlot pens. Mean concentrations of veterinary antimicrobials were 1.4 to 3.5 times higher in surface material from bedding vs. non-bedding pen areas. Runoff rates and volumetric runoff coefficients were similar across all treatments but both were significantly higher from non-bedding (0.53Lmin(-1); 0.27) than bedding areas (0.40Lmin(-1); 0.19). In keeping with concentrations in pen surface material, mean concentrations of veterinary antimicrobials were 1.4 to 2.5 times higher in runoff generated from bedding vs. non-bedding pen areas. Water solubility and sorption coefficient of antimicrobials played a role in their transport in runoff. Estimated amounts of chlortetracycline, sulfamethazine, and tylosin that could potentially be transported to the feedlot catch basin during a one in 100-year precipitation event were 1.3 to 3.6ghead(-1), 1.9ghead(-1), and 0.2ghead(-1), respectively. This study demonstrates the magnitude of veterinary antimicrobial transport in feedlot pen runoff and supports the necessity of catch basins for runoff containment within feedlots.

Effects of a herbicide mixture on primary and bacterial productivity in four prairie wetlands with varying salinities: an enclosure approach.

Wetlands in the Prairie pothole region of Saskatchewan and Manitoba serve an important role in providing wildlife habitat, water storage and water filtration. They display a wide range of water quality parameters such as salinity, nutrients and major ions with sulfate as the dominant ion for the most saline wetlands. The differences in these water quality parameters among wetlands are reflected in the composition of aquatic plant communities and their productivity. Interspersed within an intensely managed agricultural landscape where pesticides are commonly used, mixtures of herbicides are often detected in these wetlands as well as in rivers, and drinking water reservoirs. One freshwater and three wetlands of varying salinity in the St. Denis National Wildlife Area, Saskatchewan, Canada were selected to study the effects of a mixture of eight herbicides (2,4-D, MCPA, dicamba, clopyralid, bromoxynil, mecoprop, dichlorprop, and glyphosate) on wetland microbial communities using an outdoor enclosure approach. Six enclosures (three controls and three treatments) were installed in each wetland and the herbicide mixture added to the treatment enclosures. The concentration of each herbicide in the enclosure water was that which would have resulted from a direct overspray of a 0.5-m deep wetland at its recommended field application rate. After herbicide addition, primary and bacterial productivity, and algal biomass were measured in both planktonic and benthic communities over 28 days. The herbicide mixture had a stimulatory effect on primary productivity in the nutrient-sufficient freshwater wetland while no stimulatory effect was observed in the nutrient-deficient saline wetlands. The differences observed in the effects of the herbicide mixture appear to be related to the nutrient bioavailability in these wetlands.

Dissipation of Three Veterinary Antimicrobials in Beef Cattle Feedlot Manure Stockpiled over Winter.

Dissipation of veterinary antimicrobials is known to occur during aerated windrow composting of beef cattle manure. However, it is unclear if a similar dissipation occurs during stockpiling. Chlortetracycline, tylosin, and sulfamethazine are three of the most commonly used veterinary antimicrobials in beef cattle production in western Canada. Their dissipation in stockpiled manure was investigated over 140 d during winter in Alberta, Canada. Beef cattle housed in pens were administered 44 mg of chlortetracycline kg feed (dry weight), 44 mg of chlortetracycline + 44 mg sulfamethazine kg feed, 11 mg of tylosin kg feed, or feed without antimicrobials (control). Manure samples were extracted using pressurized liquid extraction, and the extracts were analyzed for chlortetracycline, sulfamethazine, and tylosin by LC-MS-MS. Dissipation of all three antimicrobials in the manure was explained by exponential decay kinetics. Times for 50% dissipation (DT) were 1.8 ± 0.1 d for chlortetracycline alone or 6.0 ± 0.8 d when mixed with sulfamethazine, 20.8 ± 3.8 d for sulfamethazine, and 4.7 ± 1.2 d for tylosin. After 77 d, <1% of initial chlortetracycline and <2% of sulfamethazine remained. Tylosin residues were more variable, decreasing to approximately 12% of initial levels after 28 d, with 20% present after 77 d and 13% after 140 d. Temperatures within stockpiles reached maximum values within 6 d of establishment and varied with location (bottom, 62.5°C; middle, 63.8°C; and top, 42.9°C). Antimicrobials in the manure did not inhibit microbial activity, as indicated by temperature and mass losses of carbon (C) and nitrogen (N). The C/N ratio in the manure decreased over the stockpiling period, indicating decomposition of manure to a more stable state. Dissipation of excreted residues with DT values 1.8 to 20.8 d showed that stockpiling can be as effective as windrow composting in mitigating the transfer of these three veterinary antimicrobials into the environment during land application of processed manure.

Effects of herbicide mixture on microbial communities in prairie wetland ecosystems: a whole wetland approach.

Wetlands in the prairie pothole region of Saskatchewan and Manitoba serve an important role in providing wildlife habitat, water storage and water filtration. These wetlands are regularly interspersed among agricultural operations where multiple pesticides are commonly used. Although mixtures of pesticides are often detected in these important aquatic ecosystems, very little information is known, regarding their effects. In this study, a curtained wetland approach was used to investigate the effects of a herbicide mixture (2,4-D, MCPA, clopyralid, dicamba, dichlorprop, mecoprop, bromoxynil, and glyphosate) on the structure and function of microbial communities in an ephemeral wetland and a semi-permanent wetland. In the two studied wetlands, located in Manitoba Zero Till Research Association Farm, Brandon, Manitoba, Canada, herbicide treatment based on maximum-exposure scenarios had a significant effect on pelagic and biofilm phytoplankton productivity over relatively short time periods. The stimulation of phytoplankton productivity in the ephemeral wetland appeared to be the result of a hormonal effect of the auxin-type herbicides present in the mixture, similar to naturally occurring auxins. Herbicidal effects of auxin-type herbicides were also noticed in the semi-permanent wetland where phytoplankton productivity was suppressed during the first week as a result of the concentration addition effect of the auxin-type herbicides present in the mixture. BIOLOG and pigment profiles of the biofilm community suggested a change in the community structure in both wetlands.

Effects of glyphosate and two herbicide mixtures on microbial communities in prairie wetland ecosystems: a mesocosm approach.

A multitrophic outdoor mesocosm system was used to mimic a wetland ecosystem and to investigate the effects of glyphosate and two herbicide mixtures on wetland microbial communities. The glyphosate concentration used was 1000 times the environmentally relevant concentration (ERC). One herbicide mixture consisted of six auxin-type herbicides (2,4-D, MCPA, clopyralid, dicamba, dichlorprop, mecoprop), each at 1000 times the ERC. The second mixture was comprised of eight herbicides, including the six auxin-type herbicides as well as bromoxynil and glyphosate. For this mixture, a dose-response approach was used to treat mesocosms with the ERCs of each herbicide as the base concentration. Algal biomass and production and bacterial production and numbers for pelagic and attached communities were measured at different times over a 22-d period. The experimental results indicate that the eight-herbicide mixture, even at low concentrations, produced negative effects on microbial communities. Glyphosate on its own suppressed algal biomass and production for the duration of the study in pelagic and biofilm communities. Algal biomass and production, although initially depressed in the auxin-type herbicide treatment, were stimulated from Day 9 until experiment end. Due to their similar modes of action, the effects of this herbicide mixture appear to be a result of concentration addition. Such negative effects, however, were brief, and microbial communities recovered from herbicide exposure. Based on evidence presented in this study, it appears that glyphosate has a higher potential to inhibit primary production and chlorophyll content in pelagic and attached wetland algal communities than the auxin-type herbicide mixture.

Biodegradation of mono-alkyl phthalate esters in natural sediments.

Mono-alkyl phthalate esters (MPEs) are primary metabolites of di-alkyl phthalate esters (DPEs), a family of industrial chemicals widely used in the production of soft polyvinyl chloride and a large range of other products. To better understand the long term fate of DPEs in the environment, we measured the biodegradation kinetics of eight MPEs (-ethyl, -n-butyl, -benzyl, -i-hexyl, -2-ethyl-hexyl, -n-octyl, -i-nonyl, and -i-decyl monoesters) in marine and freshwater sediments collected from three locations in the Greater Vancouver area. After a lag period in which no apparent biodegradation occurred, all MPEs tested showed degradation rates in both marine and freshwater sediments at 22 degrees C with half-lives ranging between 16 and 39 h. Half-lives increased approximately 8-fold in incubations performed at 5 degrees C. Biodegradation rates did not differ between marine and freshwater sediments. Half-lives did not show a relationship with increasing alkyl chain length. We conclude that MPEs can be quickly degraded in natural sediments and that the similarity in MPE degradation kinetics among sediment types suggests a wide occurrence of nonspecific esterases in microorganisms from various locations, as has been reported previously.