Mapping Phosphorus Availability in Soil at a Large Scale and High Resolution Using Novel Diffusive Gradients in Thin Films Designed for X-ray Fluorescence Microscopy.

A novel binding layer (BL) as part of the diffusive gradients in thin films (DGT) technique was developed for the two-dimensional visualization and quantification of labile phosphorus (P) in soils. This BL was designed for P detection by synchrotron-based X-ray fluorescence microscopy (XFM). It differs from the conventional DGT BL as the hydrogel is eliminated to overcome the issue that the fluorescent X-rays of P are detected mainly from shallow sample depths. Instead, the novel design is based on a polyimide film (Kapton) onto which finely powdered titanium dioxide-based P binding agent (Metsorb) was applied, resulting in superficial P binding only. The BL was successfully used for quantitative visualization of P diffusion from three conventional P fertilizers applied to two soils. On a selection of samples, XFM analysis was confirmed by quantitative laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The XFM method detected significant differences in labile P concentrations and P diffusion zone radii with the P fertilizer incubation, which were explained by soil and fertilizer properties. This development paves the way for fast XFM analysis of P on large DGT BLs to investigate in situ diffusion of labile P from fertilizers and to visualize large-scale P cycling processes at high spatial resolution.

Repeated applications of fipronil, propyzamide and flutriafol affect soil microbial functions and community composition: A laboratory-to-field assessment.

Pesticides play an important role in conventional agriculture by controlling pests, weeds, and plant diseases. However, repeated applications of pesticides may have long lasting effects on non-target microorganisms. Most studies have investigated the short-term effects of pesticides on soil microbial communities at the laboratory scale. Here, we assessed the ecotoxicological impact of fipronil (insecticide), propyzamide (herbicide) and flutriafol (fungicide) on (i) soil microbial enzymatic activities, (ii) potential nitrification, (iii) abundance of the fungal and bacterial community and key functional genes (nifH, amoA, chiA, cbhl and phosphatase) and (iii) diversity of bacteria, fungi, ammonia oxidizing bacteria (AOB) and archaea (AOA) after repeated pesticide applications in laboratory and field experiments. Our results showed that repeated applications of propyzamide and flutriafol affected the soil microbial community structure in the field and had significant inhibitory effects on enzymatic activities. The abundances of soil microbiota affected by pesticides recovered to levels similar to the control following a second application, suggesting that they might be able to recover from the pesticide effects. However, the persistent pesticide inhibitory effects on soil enzymatic activities suggests that the ability of the microbial community to cope with the repeated application was not accompanied by functional recovery. Overall, our results suggest that repeated pesticide applications may influence soil health and microbial functionalities and that more information should be collected to inform risk-based policy development.

Impact of twenty pesticides on soil carbon microbial functions and community composition.

Pesticides are known to affect non-targeted soil microorganisms. Still, studies comparing the effect of multiple pesticides on a wide range of microbial endpoints associated with carbon cycling are scarce. Here, we employed fluorescence enzymatic assay and real-time PCR to evaluate the effect of 20 commercial pesticides, applied at their recommended dose and five times their recommended dose, on soil carbon cycling related enzymatic activities (α-1,4-glucosidase, ß-1,4-glucosidase, ß-d-cellobiohydrolase and ß-xylosidase), and on the absolute abundance of functional genes (cbhl and chiA), in three different South Australian agricultural soils. The effects on cellulolytic and chitinolytic microorganisms, and the total microbial community composition were determined using shotgun metagenomic sequencing in selected pesticide-treated and untreated samples. The application of insecticides significantly increased the cbhl and chiA genes absolute abundance in the acidic soil. At the community level, insecticide fipronil had the greatest stimulating effect on cellulolytic and chitinolytic microorganisms, followed by fungicide metalaxyl-M and insecticide imidacloprid. A shift towards a fungal dominated microbial community was observed in metalaxyl-M treated soil. Overall, our results suggest that the application of pesticides might affect the soil carbon cycle and may disrupt the formation of soil organic matter and structure stabilisation.

Foliar-applied manganese and phosphorus in deficient barley: Linking absorption pathways and leaf nutrient status.

Foliar fertilization delivers essential nutrients directly to plant tissues, reducing excessive soil fertilizer applications that can lead to eutrophication following nutrient leaching. Foliar nutrient absorption is a dynamic process affected by leaf surface structure and composition, plant nutrient status, and ion physicochemical properties. We applied multiple methods to study the foliar absorption behaviors of manganese (Mn) and phosphorus (P) in nutrient-deficient spring barley (Hordeum vulgare) at two growth stages. Nutrient-specific chlorophyll a fluorescence assays were used to visualize leaf nutrient status, while laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to visualize foliar absorption pathways for P and Mn ions. Rapid Mn absorption was facilitated by a relatively thin cuticle with a low abundance of waxes and a higher stomatal density in Mn-deficient plants. Following absorption, Mn accumulated in epidermal cells and in the photosynthetically active mesophyll, enabling a fast (6 h) restoration of Mn-dependent photosynthetic processes. Conversely, P-deficient plants developed thicker cuticles and epidermal cell walls, which reduced the penetration of P across the leaf surface. Foliar-applied P accumulated in trichomes and fiber cells above leaf veins without reaching the mesophyll and, as a consequence, no restoration of P-dependent photosynthetic processes was observed. This study reveals new links between leaf surface morphology, foliar-applied ion absorption pathways, and the restoration of affected physiological processes in nutrient-deficient leaves. Understanding that ions may have different absorption pathways across the leaf surface is critical for the future development of efficient fertilization strategies for crops in nutrient-limited soils.

Tandem Probe Analysis Mode for Synchrotron XFM: Doubling Throughput Capacity.

Synchrotron-based X-ray fluorescence microscopy (XFM) analysis is a powerful technique that can be used to visualize elemental distributions across a broad range of sample types. Compared to conventional mapping techniques such as laser ablation inductively coupled plasma mass spectrometry or benchtop XFM, synchrotron-based XFM provides faster and more sensitive analyses. However, access to synchrotron XFM beamlines is highly competitive, and as a result, these beamlines are often oversubscribed. Therefore, XFM experiments that require many large samples to be scanned can penalize beamline throughput. Our study was largely driven by the need to scan large gels (170 cm2) using XFM without decreasing beamline throughput. We describe a novel approach for acquiring two sets of XFM data using two fluorescence detectors in tandem; essentially performing two separate experiments simultaneously. We measured the effects of tandem scanning on beam quality by analyzing a range of contrasting samples downstream while simultaneously scanning different gel materials upstream. The upstream gels were thin (<200 µm) diffusive gradients in thin-film (DGT) binding gels. DGTs are passive samplers that are deployed in water, soil, and sediment to measure the concentration and distribution of potentially bioavailable nutrients and contaminants. When deployed on soil, DGTs are typically small (2.5 cm2), so we developed large DGTs (170 cm2), which can be used to provide extensive maps to visualize the diffusion of fertilizers in soil. Of the DGT gel materials tested (bis-acrylamide, polyacrylamide, and polyurethane), polyurethane gels were most suitable for XFM analysis, having favorable handling, drying, and analytical properties. This gel type enabled quantitative (>99%) transmittance with minimal (<3%) flux variation during raster scanning, whereas the other gels had a substantial effect on the beam focus. For the first time, we have (1) used XFM for mapping analytes in large DGTs and (2) developed a tandem probe analysis mode for synchrotron-based XFM, effectively doubling throughput. The novel tandem probe analysis mode described here is of broad applicability across many XFM beamlines as it could be used for future experiments where any uniform, highly transmissive sample could be analyzed upstream in the "background" of downstream samples.

Pesticide effects on nitrogen cycle related microbial functions and community composition.

The extensive application of pesticides in agriculture raises concerns about their potential negative impact on soil microorganisms, being the key drivers of nutrient cycling. Most studies have investigated the effect of a single pesticide on a nutrient cycling in single soil type. We, for the first time, investigated the effect of 20 commercial pesticides with different mode of actions, applied at their recommended dose and five times their recommended dose, on nitrogen (N) microbial cycling in three different agricultural soils from southern Australian. Functional effects were determined by measuring soil enzymatic activities of ß-1,4-N-acetyliglucosaminidase (NAG) and l-leucine aminopeptidase (LAP), potential nitrification (PN), and the abundance of functional genes involved in N cycling (amoA and nifH). Effects on nitrifiers diversity were determined with amplicon sequencing. Overall, the pesticides effect on N microbial cycling was dose-independent and soil specific. The fungicides flutriafol and azoxystrobin, the herbicide chlorsulfuron and the insecticide fipronil induced a significant reduction in PN and ß-1,4-N-acetylglucosaminidase activity (P < 0.05) (NAG) in the alkaline loam soil with low organic carbon content i.e. a soil with properties which typically favors pesticide bioavailability and therefore potential toxicity. For the nitrifier community, the greatest pesticide effects were on the most dominant Nitrososphaeraceae (ammonia-oxidizing archaea; AOA) whose abundance increased significantly compared to the less dominant AOA and other nitrifiers. The inhibiting effects were more evident in the soil samples treated with fungicides. By testing multiple pesticides in a single study, our findings provide crucial information that can be used for pesticide hazard assessment.

Zinc Accumulates in the Nodes of Wheat Following the Foliar Application of 65Zn Oxide Nano- and Microparticles.

Using zinc (Zn) foliar fertilizers to enhance the grain quality of wheat (Triticum aestivum) can be an effective alternative or supplement to Zn soil fertilizers. However, knowledge about the mechanisms of Zn absorption and translocation following foliar application is scarce. Here, autoradiography and γ-spectrometry were used to investigate the behavior of 65Zn applied to wheat leaves as soluble 65Zn chloride (65ZnCl2), chelated 65Zn (65ZnEDTA), 65Zn oxide nanoparticle (65ZnO-NP) suspensions, and 65ZnO microparticle (65ZnO-MP) suspensions. The largest amount of 65Zn absorption occurred in 65ZnCl2 treated leaves. However, this treatment (65ZnCl2) also had the lowest proportion of absorbed 65Zn translocated away from the treated leaf after 15 d due to leaf scorching (p = 0.0007). Foliar-applied 65ZnO-NPs and 65ZnO-MPs had the lowest absorption, but 65ZnO-NPs had the highest relative translocation. 65Zinc EDTA was intermediate, with higher 65Zn absorption than 65ZnO treatments but similar translocation. Regardless, the majority of the foliar-applied 65Zn remained in the treated leaf for all treatments. Furthermore, 65ZnO-NPs and 65ZnO-MPs accumulated in plant nodes, suggesting that Zn was absorbed as dissolved 65Zn and particulate 65ZnO. Overall, the form and amount of absorbed 65Zn affected translocation.

Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes.

Our knowledge of uptake, toxicity and detoxification mechanisms as related to nanoparticles' (NPs') characteristics remains incomplete. Here we combine the analytical power of three advanced techniques to study the cellular binding and uptake and the intracellular transformation of silver nanoparticles (AgNPs): single-particle inductively coupled mass spectrometry, mass cytometry and synchrotron X-ray absorption spectrometry. Our results show that although intracellular and extracellularly bound AgNPs undergo major transformation depending on their primary size and surface coating, intracellular Ag in 24 h AgNP-exposed human lymphocytes exists in nanoparticulate form. Biotransformation of AgNPs is dominated by sulfidation, which can be viewed as one of the cellular detoxification pathways for Ag. These results also show that the toxicity of AgNPs is primarily driven by internalized Ag. In fact, when toxicity thresholds are expressed as the intracellular mass of Ag per cell, differences in toxicity between NPs of different coatings and sizes are minimized. The analytical approach developed here has broad applicability in different systems where the aim is to understand and quantify cell-NP interactions and biotransformation.

Development and evaluation of a new colorimetric DGT technique for the 2D visualisation of labile phosphate in soils.

A new colorimetric technique for the measurement of labile phosphate in soils using the diffusive gradients in thin films (DGT) technique was developed in this study. This technique can determine the mass of phosphate accumulated on the precipitated Zr-oxide based binding gel by forming the blue colour following the standard molybdate-ascorbic acid method. The optimal reaction temperature and coloration time were 20 °C (room temperature) and 26 min. After determining a well-fitted calibration equation, the technique was able to measure phosphate concentration up to 2.5 mg/L for 24 h deployment with a detection limit of 10.1 µg/L. Two-dimensional quantitative visualisation of phosphate diffusion in three phosphorus (P) fertilised soils were obtained using the colorimetric technique. The results from the colorimetric DGT technique were compared to the elution DGT technique and Colwell P extraction. The DGT techniques (colorimetric and elution) and Colwell P measurements demonstrated similar patterns of phosphate diffusion in soil. Both DGT techniques showed similar phosphate concentration along the concentric rings around the fertiliser application. A new, convenient, and fast DGT colorimetric technique was developed, and successfully used to measure the distribution of potentially available phosphate in soils. The new technique is less laborious than current techniques as it does not require any pre-treatment of the binding gel layers or heating during scanning, thus providing faster results. Therefore, the technique may be more suitable for in-field applications and can be used to investigate the in situ diffusion of potentially available phosphate from fertilisers, and relate this to the plant uptake of P.

Plant-Available Phosphorus in Highly Concentrated Fertilizer Bands: Effects of Soil Type, Phosphorus Form, and Coapplied Potassium.

Phosphorus (P) is increasingly being applied in concentrated bands to satisfy plant nutrient requirements. To quantify changes in plant-available P in the fertosphere of highly concentrated fertilizer bands, we conducted a soil-fertilizer incubation experiment using seven soil types, three highly water-soluble P sources [monocalcium phosphate (MCP), monoammonium phosphate (MAP), and diammonium phosphate (DAP)], and coapplication of potassium chloride (KCl). First, we found that soil properties were important in influencing P availability. For a calcareous soil, availability was generally low irrespective of treatment, presumably due to precipitation of the fertilizer as Ca-P minerals. For all six noncalcareous soils, fertosphere pH was critical in determining potential P availability, with decreasing pH values decreasing availability, presumably due to precipitation of Al- and Fe-P minerals. Second, given the importance of pH, we also found that the form of P supplied (MCP, MAP, or DAP) had a pronounced effect on P availability due to associated changes in fertosphere pH. Finally, we also found that the coapplication of K also decreased P availability in some soils. We conclude that the selection of the P source is of utmost importance when fertilizers are placed as highly concentrated bands and that soil properties also need to be considered.

Bioimaging Techniques Reveal Foliar Phosphate Uptake Pathways and Leaf Phosphorus Status.

Global demand for phosphorus (P) requires new agronomic practices to address sustainability challenges while increasing food production. Foliar P fertilization could increase P use efficiency; however, leaf entry pathways for inorganic phosphate ion (Pi) uptake remain unknown, and it is unclear whether foliar P applications can meet plant nutrient demands. We developed two techniques to trace foliar P uptake in P-deficient spring barley (Hordeum vulgare) and to monitor the effectiveness of the treatment on restoring P functionality. First, a whole-leaf P status assay was developed using an IMAGING PAM system; nonphotochemical quenching was a proxy for P status, as P-deficient barley developed nonphotochemical quenching at a faster rate than P-sufficient barley. The assay showed restoration of P functionality in P-deficient plants 24 h after foliar P application. Treated leaves reverted to P deficiency after 7 d, while newly emerging leaves exhibited partial restoration compared with untreated P-deficient plants, indicating Pi remobilization. Second, vanadate was tested as a possible foliar Pi tracer using high-resolution laser ablation-inductively coupled plasma-mass spectrometry elemental mapping. The strong colocalization of vanadium and P signal intensities demonstrated that vanadate was a sensitive and useful Pi tracer. Vanadate and Pi uptake predominantly occurred via fiber cells located above leaf veins, with pathways to the vascular tissue possibly facilitated by the bundle sheath extension. Minor indications of stomatal and cuticular Pi uptake were also observed. These techniques provided an approach to understand how Pi crosses the leaf surface and assimilates to meet plant nutrient demands.

Foliar application of zinc sulphate and zinc EDTA to wheat leaves: differences in mobility, distribution, and speciation.

Foliar application of zinc (Zn) to crops is an effective way to increase the grain concentration of Zn. However, the development of more efficient foliar Zn fertilizers is limited by a lack of knowledge regarding the distribution, mobility, and speciation of Zn in leaves once it is taken up by the plant. We performed an experiment using radiolabelled Zn (65Zn), and in situ time-resolved elemental imaging using synchrotron X-ray fluorescence microscopy (XFM), to investigate the behaviour of two commonly used Zn foliar fertilizers (Zn sulphate and ZnEDTA) in wheat (Triticum aestivum) leaves. Both experiments showed that Zn had limited mobility in leaves, moving <25 mm from the application point after 24 h. Although limited, the translocation of Zn occurred quickly for both treatments; moving more between 3 h and 12 h after application than between 12 h and 24 h. Speciation analysis using synchrotron-based X-ray absorption near-edge structure (XANES) showed that ZnEDTA was in fact taken up in chelated form and not as ionic Zn (Zn2+). The XANES data also showed that Zn, from both treatments, was then complexed by ligands in the leaf (e.g. phytate and citrate), potentially in response to localized Zn toxicity. The results of the present study provide important insights into the behaviour of commonly used foliar-applied Zn fertilizers, and can be used to optimize current fertilization strategies and contribute to the development of more efficient foliar Zn fertilizers.

Ecological Risk Assessment of Nano-enabled Pesticides: A Perspective on Problem Formulation.

Plant protection products containing nanomaterials that alter the functionality or risk profile of active ingredients (nano-enabled pesticides) promise many benefits over conventional pesticide products. These benefits may include improved formulation characteristics, easier application, better targeting of pest species, increased efficacy, lower application rates, and enhanced environmental safety. After many years of research and development, nano-enabled pesticides are starting to make their way into the market. The introduction of this technology raises a number of issues for regulators, including how does the ecological risk assessment of nano-enabled pesticide products differ from that of conventional plant protection products? In this paper, a group drawn from regulatory agencies, academia, research, and the agrochemicals industry offers a perspective on relevant considerations pertaining to the problem formulation phase of the ecological risk assessment of nano-enabled pesticides.

Quantifying the Sensitivity of Soil Microbial Communities to Silver Sulfide Nanoparticles Using Metagenome Sequencing.

Soils are a sink for sulfidised-silver nanoparticles (Ag2S-NPs), yet there are limited ecotoxicity data for their effects on microbial communities. Conventional toxicity tests typically target a single test species or function, which does not reflect the broader community response. Using a combination of quantitative PCR, 16S rRNA amplicon sequencing and species sensitivity distribution (SSD) methods, we have developed a new approach to calculate silver-based NP toxicity thresholds (HCx, hazardous concentrations) that are protective of specific members (operational taxonomic units, OTUs) of the soil microbial community. At the HC20 (80% of species protected), soil OTUs were significantly less sensitive to Ag2S-NPs compared to AgNPs and Ag+ (5.9, 1.4 and 1.4 mg Ag kg-1, respectively). However at more conservative HC values, there were no significant differences. These trends in OTU responses matched with those seen in a specific microbial function (rate of nitrification) and amoA-bacteria gene abundance. This study provides a novel molecular-based framework for quantifying the effect of a toxicant on whole soil microbial communities while still determining sensitive genera/species. Methods and results described here provide a benchmark for microbial community ecotoxicological studies and we recommend that future revisions of Soil Quality Guidelines for AgNPs and other such toxicants consider this approach.

Bioavailability of silver and silver sulfide nanoparticles to lettuce (Lactuca sativa): Effect of agricultural amendments on plant uptake.

Silver nanoparticles (AgNPs) can enter terrestrial systems as sulfidised AgNPs (Ag2S-NPs) through the application of biosolids to soil. However, the bioavailability of Ag2S-NPs in soils is unknown. The two aims of this study were to investigate (1) the bioavailability of Ag to lettuce (Lactuca sativa) using a soil amended with biosolids containing Ag2S-NPs and (2) the effect of commonly used agricultural fertilisers/amendments on the bioavailability of Ag, AgNPs and Ag2S-NPs to lettuce. The study used realistic AgNP exposure pathways and exposure concentrations. The plant uptake of Ag from biosolids-amended soil containing Ag2S-NPs was very low for all Ag treatments (0.02%). Ammonium thiosulfate and potassium chloride fertilisation significantly increased the Ag concentrations of plant roots and shoots. The extent of the effect varied depending on the type of Ag. Ag2S-NPs, the realistic form of AgNPs in soil, had the lowest bioavailability. The potential risk of AgNPs in soils is low; even in the plants that had the highest Ag concentrations (Ag(+)+thiosulfate), only 0.06% of added Ag was found in edible plant parts (shoots). Results from the study suggest that agricultural practises must be considered when carrying out risk assessments of AgNPs in terrestrial systems; such practises can affect AgNP bioavailability.

Characterization and ecological risk assessment of nanoparticulate CeO2 as a diesel fuel catalyst.

Nanoparticulate cerium dioxide (nano-CeO2 ), when combusted as an additive to diesel fuel, was transformed from 6 nm to 14 nm sizes into particles near 43 nm, with no obvious change in the unit cell dimensions or crystalline form. Cerium sulfate, if formed during combustion, was below detection limits. Ceria nanoparticles were agglomerated within the soot matrix, with a mean aerodynamic diameter near 100 nm. The dissolution of cerium from the dried ceria catalyst in synthetic soft water was extremely small (<0.0006% or <0.2 µg Ce/L), with particles being highly agglomerated (<450 nm). Agglomeration was reduced in the presence of humic acid. In the combusted samples, soot was dominant, and the solubility of cerium in soft water showed an almost 100-fold increase in the <1 nm fraction compared to that before combustion. It appeared that the nano-CeO2 remained agglomerated within the soot matrix and would not be present as dispersed nanoparticles in aquatic or soil environments. Despite the increased dissolution, the solubility was not sufficient for the combusted ceria to represent a risk in aquatic ecosystems. The predicted environmental concentrations were still orders of magnitude below the predicted no effects concentration of near 1 mg/L. In the soil environment, any cerium released from soot materials would interact with natural colloids, decreasing cerium concentrations in soil solutions and further minimizing the potential risk to soil organisms.

Transformation of PVP coated silver nanoparticles in a simulated wastewater treatment process and the effect on microbial communities.

BACKGROUND: Manufactured silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials in consumer goods and consequently their concentrations in wastewater and hence wastewater treatment plants are predicted to increase. We investigated the fate of AgNPs in sludge that was subjected to aerobic and anaerobic treatment and the impact of AgNPs on microbial processes and communities. The initial identification of AgNPs in sludge was carried out using transmission electron microscopy (TEM) with energy dispersive X-ray (EDX) analysis. The solid phase speciation of silver in sludge and wastewater influent was then examined using X-ray absorption spectroscopy (XAS). The effects of transformed AgNPs (mainly Ag-S phases) on nitrification, wastewater microbial populations and, for the first time, methanogenesis was investigated. RESULTS: Sequencing batch reactor experiments and anaerobic batch tests, both demonstrated that nitrification rate and methane production were not affected by the addition of AgNPs [at 2.5 mg Ag L(-1) (4.9 g L(-1) total suspended solids, TSS) and 183.6 mg Ag kg (-1) (2.9 g kg(-1) total solids, TS), respectively]. The low toxicity is most likely due to AgNP sulfidation. XAS analysis showed that sulfur bonded Ag was the dominant Ag species in both aerobic (activated sludge) and anaerobic sludge. In AgNP and AgNO3 spiked aerobic sludge, metallic Ag was detected (~15%). However, after anaerobic digestion, Ag(0) was not detected by XAS analysis. Dominant wastewater microbial populations were not affected by AgNPs as determined by DNA extraction and pyrotag sequencing. However, there was a shift in niche populations in both aerobic and anaerobic sludge, with a shift in AgNP treated sludge compared with controls. This is the first time that the impact of transformed AgNPs (mainly Ag-S phases) on anaerobic digestion has been reported. CONCLUSIONS: Silver NPs were transformed to Ag-S phases during activated sludge treatment (prior to anaerobic digestion). Transformed AgNPs, at predicted future Ag wastewater concentrations, did not affect nitrification or methanogenesis. Consequently, AgNPs are very unlikely to affect the efficient functioning of wastewater treatment plants. However, AgNPs may negatively affect sub-dominant wastewater microbial communities.