Using the Pesticide Toxicity Index to show the potential ecosystem benefits of on-farm biobeds.

The influent and effluent of two single-cell biobeds (Province of Alberta, Canada) and two dual cell-biobeds (Province of Saskatchewan, Canada) were monitored during a number of growing seasons. A total of 59 unique pesticide active ingredients were detected, with all biobed influent samples (n = 54) and 93% of effluent samples (n = 54) containing pesticide mixtures. About one-half of the effluent samples in both single-cell (56%) and dual-cell (45%) biobeds contained active ingredients that have Groundwater Ubiquity Score (GUS) values >2.8 and so were more likely to move through the biomatrix materials into effluent. The Pesticide Toxicity Index (PTI) calculated for aquatic indicator species (i.e., vascular and nonvascular plants, invertebrates, and fish) was always larger for influent samples (e.g., median PTI >500 for invertebrates in dual-cell biobed) than effluent samples (i.e., median PTI <1). As such, this study demonstrates the potential ecosystem benefits of the broad adoption of on-farm biobeds in the Canadian Prairies for recycling tank rinsate as a strategy to accelerate a green economy. Although biobeds were highly effective in reducing the concentrations for pesticides with a wide range of soil organic carbon coefficient and half-life values, the biobed effectiveness was relatively poor for the herbicides clopyralid, diclofop, fluroxypyr, and imazethapyr. Clopyralid (3.02), fluroxypyr (3.70), and imazethapyr (3.90) all have relatively high GUS values (>2.8) and are thus more likely to be detected in effluent than active ingredients with smaller GUS values. This suggests that further improvements in biosystem design need to be made for optimizing the recycling of these pesticides.

Both single-cell and dual-cell biobeds performed remarkably in colder climates (Canadian Prairies). Pesticides detected in effluents were more likely to be pesticides that have greater GUS values. The Pesticide Toxicity Index (PTI) was drastically smaller for biobed effluent than for influent. The broad adoption of biobeds for recycling tank rinsate will contribute to a green economy. The biobed system design must be refined to broaden its effectiveness to treat all pesticides.

Metagenomic and metatranscriptomic analysis reveals enrichment for xenobiotic-degrading bacterial specialists and xenobiotic-degrading genes in a Canadian Prairie two-cell biobed system.

Biobeds are agriculture-based bioremediation tools used to safely contain and microbially degrade on-farm pesticide waste and rinsate, thereby reducing the negative environmental impacts associated with pesticide use. While these engineered ecosystems demonstrate efficient pesticide removal, the microbiomes in these environments remain largely understudied both taxonomically and functionally. This study used metagenomic and metatranscriptomic techniques to characterize the microbial community in a two-cell Canadian biobed system before and after a field season of pesticide application. These culture-independent approaches identified an enrichment of xenobiotic-degrading bacteria, such as Afipia, Sphingopyxis and Pseudomonas, and enrichment and transcription of xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; we were able to directly link the transcription of these genes to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa.

Pesticide Mixtures in the Water-Column Versus Bottom-Sediments of Prairie Rivers.

River water-column and bottom-sediments samples were screened for 160 pesticide compounds to compare the types of pesticides present in the water-column versus bottom-sediments, and between segments of rivers flowing through intensively-managed versus semi-natural habitats. Of the 35 pesticide compounds detected, current-use pesticides accounted for 96% (water) and 76% (bottom sediments). Pesticide mixtures were present in 72% (water) and 51% (sediment) of the total samples. Only the river flowing through the most intensively managed habitat showed a wide range of pesticides in sediments, and many of these pesticides were also present in the water-column of that river. Current-use fungicides were detected in both the water-column and bottom-sediments but not in samples taken from rivers flowing predominantly through semi-natural habitats. The study period (May to August) corresponds to the peak time of regional pesticide applications and hence the time period that is most likely to show elevated concentrations of current-use pesticides in the water-column. The environmental concentrations of pesticide mixtures detected in the water-column were used to calculate Pesticide Toxicity Index (PTI) values as it applies to non-vascular or vascular plants, invertebrates, and fish. The PTI values were largest for non-vascular and vascular plants, reflecting that the pesticide mixtures in water-column were dominated by herbicides.

Effects of Agricultural Stressors on Growth and an Immune Status Indicator in Wood Frog (Lithobates sylvaticus) Tadpoles and Metamorphs.

Like many amphibians, wood frog (Lithobates sylvaticus) populations have likely declined or experienced local extirpations as a result of habitat alterations. Despite this, wood frogs are still present and breeding in altered landscapes, like the agricultural Prairie Pothole Region of central Canada, and are exposed to a variety of anthropogenic impacts. As tadpoles, water contamination can have negative effects on growth, development, and immune systems. To investigate the potential effects of agricultural land use on tadpole growth and immune system stress, we used boosted regression trees to model body mass, body condition, and neutrophil to lymphocyte ratios, a measure of immune stress, against 32 variables including water quality, wetland habitat, and landscape-level measures. Developmental stage strongly influenced all 3 endpoints, and body mass was negatively influenced by higher levels of total dissolved solids (>600-700 mg/L) and at the first sign of pesticide detection (>0.01 proportion pesticides detected of those screened). While correlative, these data suggest that tadpoles developing in agricultural environments may experience survival and reproductive disadvantages if they metamorphose at smaller body sizes. Given the potential impacts this can have on adult frogs and frog populations, these results provide an impetus for further field-based investigation into the effects that pesticides, and especially total dissolved solids, may have on tadpoles. Environ Toxicol Chem 2021;40:2269-2281. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A triticale tapetal non-specific lipid transfer protein (nsLTP) is translocated to the pollen cell wall.

KEY MESSAGE: A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.

Evaluation of interactive effects of UV light and nano encapsulation on the toxicity of azoxystrobin on zebrafish.

The use of nanotechnology to enhance pesticide formulations holds the promise of reduced pesticide use, reduced mobility in soils, and overall improvements in agricultural practices while simultaneously maintaining yields. However, the toxicity of nano-enabled pesticides, including azoxystrobin (Az), has not been well studied compared with their conventional form. This study investigates both lethal and sub-lethal endpoints in zebrafish embryos up to 120 h post-fertilization (hpf) under either laboratory light or simulated UV light. The median lethal concentration (LC50) value of nano-enabled Az (nAz) was significantly lower than the conventional form (Az). Interestingly, artificial UV light significantly increased toxicity (decreased LC50) of both Az and nAz. Malformations were not observed but the remaining yolk sac volume was significantly increased in both types of Az at both light conditions. This decreased yolk consumption is in agreement with reduced oxygen consumption and heart rate. Catalase enzyme activity was only reduced to UV light while superoxide dismutase activity was significantly reduced by co-exposure of UV light, and either type of Az at a nominal concentration of 100 µg L-1. The co-exposure of Az at 100 µg L-1 and UV light significantly upregulated sod1, sod2, and gpx1b expression and both types of Az significantly reduced gpx1a expression. Lipid peroxidation was significantly increased in nAz and Az at 100 µg L-1 under laboratory light, while UV light induced even higher level of lipid peroxidation. The results will provide important information on the toxicity of nAz under ecologically realistic conditions.

Detecting Amphibians in Agricultural Landscapes Using Environmental DNA Reveals the Importance of Wetland Condition.

Amphibians are declining worldwide, in part because of large-scale degradation of habitat from agriculture and pervasive pathogens. Yet a common North American amphibian, the wood frog (Lithobates sylvaticus), ranges widely and persists in agricultural landscapes. Conventional survey techniques rely on visual encounters and dip-netting efforts, but detectability limits the ability to test for the effects of environmental variables on amphibian habitat suitability. We used environmental DNA to determine the presence of wood frogs and an amphibian pathogen (ranavirus) in Prairie Pothole wetlands and investigated the effects of 32 water quality, wetland habitat, and landscape-level variables on frog presence at sites representing different degrees of agricultural intensity. Several wetland variables influenced wood frog presence, the most influential being those associated with wetland productivity (i.e., nutrients), vegetation buffer width, and proportion of the surrounding landscape that is comprised of other water bodies. Wood frog presence was positively associated with higher dissolved phosphorus (>0.4 mg/L), moderate dissolved nitrogen (0.1-0.2 mg/L), lower chlorophyll a (≤15 µg/L), wider vegetation buffers (≥10 m), and more water on the landscape (≥0.25). These results highlight the effects of environmental factors at multiple scales on the presence of amphibians in this highly modified landscape-namely the importance of maintaining wetland water quality, vegetation buffers, and surrounding habitat heterogeneity. Environ Toxicol Chem 2019;38:2750-2763. © 2019 SETAC.

Auxin Herbicides and Pesticide Mixtures in Groundwater of a Canadian Prairie Province.

Groundwater samples were collected from piezometers and water table wells in both dryland and irrigated agricultural regions of Alberta, Canada, to examine the occurrence of pesticide mixtures. Fourteen current-use pesticides and two historical compounds were detected over a 3-yr sampling period. Pesticide mixtures were detected in ∼3% of the groundwater samples, and the frequency of detection increased from spring (1.5%) to summer (3.8%) and fall (4.8%). Pesticide mixtures always consisted of at least one of two auxin herbicides: 2,4-dichlorophenoxyacetic acid (2,4-D) or 2-methyl-4-chlorophenoxyacetic acid (MCPA). 19% of all samples contained a single pesticide, with auxin herbicides 2,4-D (7.3%), MCPA (4.4%), and clopyralid (3.9%) being most prevalent. We detected 2,4-D predominantly in the fall (72% of 2,4-D detections) and less in spring and summer (28%). We detected MCPA mostly in summer (85% of MCPA detections) and less in spring and fall (15%). Clopyralid was more evenly detected across spring (30%), summer (25%), and fall (45%). Since the auxin herbicides above are typically applied in summer, results suggest that each herbicide may have different mobility and persistence characteristics in prairie soils. Guidelines for Canadian Drinking Water Quality have been set for a range of individual pesticides, but not for pesticide mixtures. If Canada is to establish such guidelines, this study demonstrates that auxin herbicides should be prioritized. In addition, only 7 of the 16 compounds detected in this study have established maximum acceptable concentrations (MACs), excluding clopyralid, which was detected in all three sampling years.

Integrated assessment of climate change impact on surface runoff contamination by pesticides.

Pesticide transport by surface runoff depends on climate, agricultural practices, topography, soil characteristics, crop type, and pest phenology. To accurately assess the impact of climate change, these factors must be accounted for in a single framework by integrating their interaction and uncertainty. This article presents the development and application of a framework to assess the impact of climate change on pesticide transport by surface runoff in southern Québec (Canada) for the 1981-2040 period. The crop enemies investigated were: weeds for corn (Zea mays); and for apple orchard (Malus pumila), 3 insect pests (codling moth [Cydia pomonella], plum curculio [Conotrachelus nenuphar], and apple maggot [Rhagoletis pomonella]), 2 diseases (apple scab [Venturia inaequalis], and fire blight [Erwinia amylovora]). A total of 23 climate simulations, 19 sites, and 11 active ingredients were considered. The relationship between climate and phenology was accounted for by bioclimatic models of the Computer Centre for Agricultural Pest Forecasting (CIPRA) software. Exported loads of pesticides were evaluated at the edge-of-field scale using the Pesticide Root Zone Model (PRZM), simulating both hydrology and chemical transport. A stochastic model was developed to account for PRZM parameter uncertainty. Results of this study indicate that for the 2011-2040 period, application dates would be advanced from 3 to 7 days on average with respect to the 1981-2010 period. However, the impact of climate change on maximum daily rainfall during the application window is not statistically significant, mainly due to the high variability of extreme rainfall events. Hence, for the studied sites and crop enemies considered, climate change impact on pesticide transported in surface runoff is not statistically significant throughout the 2011-2040 period. Integr Environ Assess Managem 2016;12:559-571. © Her Majesty the Queen in Right of Canada 2015; Published 2015 SETAC.

Development of a real-time immuno-PCR assay for the quantification of 17ß-estradiol in water.

A competitive real-time (RT) immuno-polymerase chain reaction (iPCR) (RT-iPCR) assay was developed for the sensitive quantification of 17ß-estradiol in water. Using a universal iPCR method and polyclonal antibodies, 17ß-estradiol was accurately quantified at concentrations ranging from 1 pg mL(-1) to 10 µg mL(-1). The RT-iPCR assay's limit of detection was 0.7 pg mL(-1). The RT-iPCR assay provided an 800-fold increase in sensitivity as well as an expanded working range compared with the corresponding enzyme-linked immunosorbent assay. Assay cross-reactivity to estrone and estriol, two structurally related estrogens, was below 8%. Water samples spiked with 17ß-estradiol were analyzed by RT-iPCR to determine the assay's potential as a rapid screen for the monitoring of manure-borne estrogens in the environment. The assay showed recoveries of 82, 102 and 103% for Milli-Q, tap, and irrigation water, respectively, without requiring sample extraction or concentration prior to analysis.

A coupled stochastic/deterministic model to estimate the evolution of the risk of water contamination by pesticides across Canada.

Periodic assessments of the risk of water contamination by pesticides help decision makers improve the sustainability of agricultural management practices. In Canada, when evaluating the risk of water contamination by pesticides, 2 main constraints arise. First, because the area of interest is large, a pesticide transport model with low computational running time is mandatory. Second, some relevant input data for simulations are not known, and most are known only at coarse scale. This study aims to develop a robust methodology to estimate the evolution of the risk of water contamination by pesticides across Canada. To circumvent the 2 aforementioned issues, we constructed a stochastic model and coupled it to the 1-dimensional pesticide fate model Pesticide Root Zone Model (PRZM). To account for input data uncertainty, the stochastic model uses a Monte Carlo approach to generate several pesticide application scenarios and to randomly select PRZM parameter values. One hundred different scenarios were simulated for each of over 2000 regions (Soil Landscapes of Canada [SLC] polygons) for the years 1981 and 2006. Overall, the results indicated that in those regions in which the risk increased from 1981 to 2006, the increase in risk was mainly attributable to the increased area treated by pesticides or an increase in the number of days with runoff. More specifically, this work identifies the areas at higher risk, where further analyses with finer-scale input data should be performed. The model is specific for Canadian data, but the framework could be adapted for other large countries.

Solid beef cattle manure application impacts on soil properties and 17ß-estradiol fate in a clay loam soil.

Livestock manure applied to agricultural land is one of the ways natural steroid estrogens enter soils. To examine the impact of long-term solid beef cattle (Bos Taurus) manure on soil properties and 17ß-estradiol sorption and mineralization, this study utilized a soil that had received beef cattle manure over 35 years. The 17ß-estradiol was strongly sorbed and sorption significantly increased (P < 0.05) with increasing soil organic carbon content (SOC) and with an increasing annual rate of beef cattle manure. The 17ß-estradiol mineralization half-life was significantly negatively correlated, and the total amount of 17ß-estradiol mineralized at 90 days (MAX) was significantly positively correlated with 17ß-estradiol sorption. The long-term rate of manure application had no significant effect on MAX, but the addition of fresh beef cattle manure in the laboratory resulted in significantly (P < 0.05) smaller MAX values. None of the treatments showed MAX values exceeding one-third of the 17ß-estradiol applied.

Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

Development of competitive ELISAs for 17beta-estradiol and 17beta-estradiol +estrone+estriol using rabbit polyclonal antibodies.

Estrogens are a family of feminizing hormones that are excreted by vertebrates. It has been documented that their presence in surface waters, even in the ng/L range, can have detrimental impacts on fish reproduction. Two competitive enzyme-linked immunosorbent assays using rabbit polyclonal antibodies were developed: one for 17beta-estradiol and a second one for 17beta-estradiol (E2)+estrone (E1)+estriol (E3). Two different conjugates were synthesized using the Mixed-anhydride (for the 17beta-estradiol ELISA) and the Mannich (for the E1 + E2 + E3 ELISA) reactions. The 17beta-estradiol ELISA was highly specific with an IC(50) of 243 ng/mL for 17beta-estradiol. The E1 + E2 + E3 ELISA exhibited cross-reactivity with estrone (85%) and estriol (62%) with an IC(50) of 18 ng/mL for 17beta-estradiol. Cross-reactivity was tested against 13 chemically related compounds and both immunoassays showed significant cross-reactivity with two estradiol conjugates: beta estradiol-17-valerate and beta estradiol-3-benzoate (from 57 to 84 %) for which, to our knowledge, there are currently no commercially available ELISA. Characteristics (sensitivity, inter and intra assay variation, and cross-reactivity) of the E1 + E2 + E3 ELISA were further compared to those from a commercial Estriol ELISA. The commercial ELISA was more specific, sensitive and its inter-assay variation was less (9.5% compared to 10% for the E1 + E2 + E3 ELISA) but the E1 + E2 + E3 ELISA had less intra-assay variation (4% compared to 5% for the commercial ELISA). Finally, a solid-phase extraction method compatible with the E1 + E2 + E3 immunoassay demonstrated that this combined approach of extraction and immunoassay had good potential for determining estrogen concentrations in environmental samples such as surface water in urban and agricultural ecosystems.

Isolation and affinity maturation of hapten-specific antibodies.

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.

Selection, characterization, and CDR shuffling of naive llama single-domain antibodies selected against auxin and their cross-reactivity with auxinic herbicides from four chemical families.

Indoleacetic acid (IAA)-binding single-domain antibodies (sdAbs) were isolated from a naive phage-display library constructed from the heavy chain antibody repertoire of a Ilama. The highest-affinity sdAb isolated (CSF2A) had a K(D) of 5-20 microM for two IAA-protein conjugates and a K(D) of 20 microM for free IAA. This sdAb also bound to a synthetic auxin analogue, 1-naphthaleneacetic acid (NAA), and to six auxinic herbicides (K(D) values of 0.5-2 mM), but not to serotonin and tryptophan, which are structurally similar to IAA but have no auxinic activity. To understand how sdAb CSF2A binds IAA and to determine which complementary-determining region(s) (CDR) participate(s) most in binding IAA, CSF2A was shuffled with four other sdAb clones by staggered extension process (StEP). After panning against IAA, two shuffled sdAbs were found: sdAb CSB1A, which originated from three different parental clones, and sdAb CSE8A, derived from two parental clones. These shuffled sdAbs and CSF2A were each fused to the B subunit of the Escherichia coli verotoxin, resulting in the formation of the pentamerized sdAbs V2NCSB1A, V2NCSE8A, and V2NCSF2A, which were analyzed by surface plasmon resonance (SPR) along with the sdAbs previously isolated. The shuffled clones had affinity for IAA (20 microM) similar to that of the highest affinity parental clone CSF2A, but much lower affinity for the auxinic herbicides. CDR2 was instrumental in binding IAA, whereas hydrophobic CDR3 was important for binding the auxinic herbicides. A novel SPR methodology is also described for specific immobilization of pentamerized sdAbs, allowing determination of K(D) values of Ab interaction with underivatized, low molecular weight haptens.

Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.

Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.