Direct investigation of N2O production and fate in the soil profile has not advanced far, and is only beginning in the fertigation context. It is increasingly clear that much, if not most, soil N2O might be reduced before emission from surface. It is commonly assumed that the upper 20 cm play the central role in surface emissions ; in this study, production of emitted N2O was centered at the 10–15 cm depth. Calculations of net N2O production at different depths in the soil showed more overall N2O consumption than production at 20 cm and below, helping to explain lower emissions under NO3 treatments, where points of production were deeper. N2O production near surface has been difficult to measure, but is a missing link of great importance. The measurement and simulation of soil O2 availability, varying as it does at different points in soil aggregates, remains an obstacle that impedes comparison of field and laboratory soils. Basic questions also persist over fertilizer N availability in its different stages of transformation. And field trials have not verified whether fertilizer management can affect the completeness of N reduction in classical or nitrifier denitrification.In the past decade, nanoscale fertilizers and pesticides have been increasingly proposed and used in agriculture.In particular copper-containing nanopesticides are being introduced to the market due to their excellent antimicrobial and anti-fungal properties.The increasing use of nanopesticides in agriculture has motivated researchers to consider their environmental fate, bio-availability, and toxicity to edible plants.
Extensive investigations of bio-accumulation and phytotoxicity of copper-based nano pesticides 2) on a variety of crop plants, for example, radish , ryegrass , cilantro , zucchini , bean , wheat , duck weeds , lettuce , alfalfa , have been conducted.However, most studies spiked NPs in soil or via water in hydroponic systems,hydroponic growing system whereas in commercial agriculture most copper related pesticides are applied through foliar spray.Compared with the soil-root transfer, the leaf-root transfer pathway and toxicity mechanism have rarely been investigated and are poorly understood.Terrestrial vascular plants have the ability to uptake metals, organic contaminants, even nanoparticles through the leaves employing different pathways.Ions are able to penetrate the leaf cuticle and enter the cytosol of epidermal cells or mesophyll cells.For fine particles , stomatal uptake is an important pathway.For example, Uzu et al. reported that lettuce leaves contained 335 ± 50 mg Pb kg−1 after exposure to Pb-enriched fine particles for 43 days.Hong et al. showed that CeO2 NPs were taken up through cucumber leaves and translocated to root tissues.Lettuce is a widely cultivated vegetable and usually used as a model plant in contaminant transfer studies.Furthermore, since the leaves are the edible part of lettuces, investigating their foliar uptake of copper based nanopesticides is of high interest for risk assessment of human and ecological health. Copper is a redox active transition metal and is involved in redox reaction in cells, generating O2 •− and ·OH via the Haber Weiss and Fenton reactions.Once copper particles/ions enter into plant tissues, no matter where they are localized, they may induce oxidative stress and affect several metabolic processes.Metabolomics is a powerful approach for gaining a comprehensive understanding of biological mechanisms, including toxicity, generally by monitoring low molecular weight metabolites.In recent years, various technologies have been employed for metabolic investigations of organism responses to environmental stressors.
Rather than target a limited number of metabolites or physiological parameters, nontargeted metabolomics can provide information on a large number of metabolites, which results in a deeper insight into the molecular mechanisms underlying the physiological and biochemical changes. In addition, metabolomics can be used to reveal the mechanism of plant defense and detoxification of contaminants.Pidatala et al.performed metabolomics studies and revealed detoxification and tolerance mechanism of Vetiver to Pb. Our recent study on cucumber plant root exudate metabolomics revealed that exposure to nano-Cu up-regulated a number of amino acids that bind with copper NPs and ions, likely to detoxify Cu from its nearby environment.The primary aim of this work was to determine the metabolic profile changes in plants exposed to Cu2 nanopesticides using GC-TOF-MS. The objective is to elucidate the toxicity and detoxification mechanisms underlying up- or down regulated metabolites. In addition, knowledge on the uptake and translocation of Cu2 nanopesticides and released Cu ions in lettuce leaves, through foliar application, is of high interest for risk assessment.The Cu2 nanopesticide used in this study were in the form of a commercial biocide . Detailed physicochemical properties of Kocide 3000 have been reported before.Specifically, the primary particle size is ∼50 to >1000 nm. The hydrodynamic diameter is 1532 ± 580 nm and the zeta potential is −47.6 ± 43 mV, measured via Dynamic Light Scattering , in NanoPure water at pH 7. Although Kocide 3000 particles are mainly micron-sized, these micronized particles are made up of nanosheets of Cu2 that are bound together by an organic composite and can potentially redissociate in water.For this reason the pesticide is considered “nano”. Copper content in Kocide 3000 is 26.5 ± 0.9%, while other elements including C, O, Na, Al, Si, S, Zn compose 73.5% of mass.Lettuce seeds were purchased from Seed Savers Exchange . The soil was collected from the Natural Reserve System of UC Santa Barbara , from the top 20 cm. The soil texture is sandy loamy grassland with sand/silt/clay percentage of 54.0%, 29.0% and 17.0%. Soil pH is 5.90 ± 0.04. Loss-onignition organic matter is 3.11 ± 0.07%. Cation exchange capacity is 25.8 ± 0.1 mequiv 100 g−1 .
More information regarding the soil composition was reported in a previous study.Lettuce seeds were planted in pots containing 250 g of soil. Each pot contained one seed. Plants were grown in a greenhouse, which was maintained at 28 °C by day and 20 °C by night. The daily light integral was 17.3 ± 3.6 mol m−2 d−1 . When plants were 24 days old, we began to spray them with Kocide 3000 suspended in NanoPure water at 105, 155, and 210 mg Kocide/100 mL. The doses were selected generally following the manufacturer’s recommendation . Before spraying, the suspension was sonicated for 30 min in cooled water. A hand-held spray bottle was used for spraying. The lettuces were sprayed a total of 8 times during 4 weeks; the amount sprayed each time was 8.75 , 12.9 and 17.5 mg/pot. Each spray was ∼1 mL. The seven treatments were: control; low, medium, and high NPs in uncovered soil; low, medium and high NPs in covered soil. Each treatment was replicated five times. In covered samples, the soil was covered with filter paper. In covered soil, Cu detected in root should be only from leaf translocation. In uncovered soil, Cu in root not only comes from translocation, but also from soil, because the soil was also exposed to some Cu2 nanopesticide during spraying. This allows us to determine whether the Cu NPs were translocated from leaf to root. At 52 days after planting, all plants were harvested.At harvest, the lettuce plants were gently removed from the soil, thoroughly rinsed with tap water for 5 min and then rinsed with NanoPure water three times. Leaf tissue was carefully separated from vascular and mesophyll tissues Figure S1. Mesophyll and root tissues were ground in liquid nitrogen and lyophilized for 5 days. Part of the freeze-dried mesophyll tissues were sent to UC Davis for metabolomics analysis, and another portion was ovendried at 70 °C for ICP-MS analysis. Since only a small amount of vascular tissue was available,vertical grow table it was only oven-dried for metal analysis.Dried plant tissues were digested with a mixture of 4 mL of H2O2 and 1 mL of plasma pure HNO3 using a microwave oven system at 165 °C for 1 h. The standard reference materials NIST 1547 and 1570a were also digested and analyzed as samples. The recoveries for all elements were between 90 and 99%. Cu and other six important mineral elements were analyzed using inductively coupled plasma mass spectrometry .The freeze-dried lettuce tissues samples were subjected to GC-TOF-MS analysis at the Genome Center Core Services, University of California Davis to identify the metabolites present in lettuce tissues. A description of sample pretreatment, analytical method and instrument has been described by Fiehn et al.Briefly, an Agilent 6890 gas chromatograph containing a Rtx-5Sil MS column with an additional 10 mm integrated guard column was used to run the samples, controlled using Leco ChromaTOF software version 2.32 . quantification was reported as peak height using the unique ion as default. Metabolites were unambiguously assigned by the BinBase identifier numbers using retention index and mass spectrum as the two most important identification criteria. More details regarding data acquisition, data processing and data reporting are provided in the SI.Partial least-squares discriminant analysis is a supervised clustering method, which uses a multiple linear regression technique to maximize the separation between groups and helps to understand which variables carry the class separating information.PLS-DA was run based on GC-TOFMS data using online resources.
Variable Importance in Projection is the weighted sum of the squares of the PLS-DA analysis, which indicates the importance of a variable to the entire model.A variable with a VIP above 1 is regarded as significant.Biological pathway analysis was performed based on all detected metabolites data using MetaboAnalyst 2.0.The impact value threshold calculated for pathway identification was set at 0.1.Cu in leaves increased in a dose-dependent manner in both covered and uncovered soil . Cu increased 82−140 times in vascular and 115−184 times in mesophyll tissues relative to the control, which indicates a high bio-accumulation of copper/nanoparticles in leaf tissues. Even though the leaves were thoroughly washed, copper ions and NPs remained on the surface or were incorporated into leaf tissues; it is likely that washing was not 100% efficient in removing them. Leaf exudates can form weak acids in the presence of water,which can accelerate dissolution of Cu2 nanopesticide, releasing cupric ions as long as the water remains on the leaf. This may result in a pathway for cupric ions to penetrate the epidermis cells and translocate to other tissues. In addition to ionized Cu, nanoparticles smaller than the stomatal diameter may enter past the guard cells. Stomatal diameters range from 8 to 12 μm for several species.Even though trichomes are not abundant on lettuce leaf surfaces, ESEM images taken after 24 h exposure to Cu2 nanopesticides show that many small particles were deposited on the lettuce leaf surface and stomatal cavities. The typical diameter observed for lettuce stomata is 13.1 μm , which is large enough to permit entry to Cu2 nanopesticides aggregates with an observed hydrodynamic diameter of 1530 ± 580 nm. Eichert demonstrated that 43 nm NPs entered stomata and migrated along the surface of stomatal pores.After passing the stomatal guard cells, the NPs may either attach to cell walls or move between cell walls. For example, Stamenkovic and Gustin showed the majority of foliar Hg was located in epidermal and stomatal cell walls and was rarely found in mesophyll or vascular tissue.However, Hong showed that Ce was present in cucumber root phloem after foliar application of CeO2 NPs.As seen in Table 1, the average [Cu] in control roots is 6.0 mg/kg, while in treated plants, [Cu] in root is 17.5−26.1 mg/kg in covered soil and 34.2−56.9 mg/kg in uncovered soil. Statistical analysis showed all the NP treated plants have significantly higher [Cu] in roots compared to controls, even though application was only foliar. In covered soil, where no direct root uptake could occur, Cu in the roots was translocated from the leaves via phloem loading. Liao et al. showed that some xylem-transported Cu was recirculated to roots via the phloem in chicory and tomato plants.Even though we observed evidence of Cu translocation to the roots, 97−99% of Cu mass was retained in the leaves. In addition, the translocation rate in NP-treated plants was 0.009 to 0.014, which is far lower than that in the control . This indicates plants sequestered most of the Cu in leaves.The threshold level for Cu to induce toxicity in plants is 20−30 mg/kg.However, high concentrations of Cu in lettuce leaves did not cause any visible toxic symptoms throughout the entire exposure period . On the contrary, the leaf biomass significantly increased at low and medium levels for uncovered treatment and medium level for covered treatment . Since a high amount of Cu was retained in leaf tissues but did not induce any toxic symptoms, lettuces likely employ a detoxification mechanism to build tolerance to excess Cu.