Joan E. McLean

Silver Nanoparticles Disrupt Wheat (Triticum aestivum L.) Growth in a Sand Matrix

Hydroponic plant growth studies indicate that silver nanoparticles (Ag NPs) are phytotoxic. In this work, the phytotoxicity of commercial Ag NPs (10 nm) was evaluated in a sand growth matrix. Both NPs and soluble Ag were recovered from water extracts of the sand after growth of plants challenged with the commercial product; the surface charge of the Ag NPs in this extract was slightly reduced compared to the stock NPs. The Ag NPs reduced the length of shoots and roots of wheat in a dose-dependent manner. Furthermore, 2.5 mg/kg of the NPs increased branching in the roots of wheat (Triticum aestivum L.), thereby affecting plant biomass. Micron-sized (bulk) Ag particles (2.5 mg/kg) as well as Ag ions (63 μg Ag/kg) equivalent to the amount of soluble Ag in planted sand with Ag NPs (2.5 mg/kg) did not affect plant growth compared to control. In contrast, higher levels of Ag ions (2.5 mg/kg) reduced plant growth to a similar extent as the Ag NPs. Accumulation of Ag was detected in the shoots, indicating an uptake and transport of the metal from the Ag NPs in the sand. Transmision electron microscopy indicated that Ag NPs were present in shoots of plants with roots exposed to the Ag NPs or high levels of Ag ions. Both of these treatments caused oxidative stress in roots, as indicated by accumulation of oxidized glutathione, and induced expression of a gene encoding a metallothionein involved in detoxification by metal ion sequestration. Our findings demonstrate the potential effects of environmental contamination by Ag NPs on the metabolism and growth of food crops in a solid matrix.

Fate of CuO and ZnO Nano- and Microparticles in the Plant Environment

The environmental fate of metal oxide particles as a function of size was assessed by comparing the behavior of CuO or ZnO nanoparticles (NPs) to that of the corresponding microparticles (MPs) in a sand matrix, with and without wheat (Triticum aestivum L.) growth. After 14 days of incubation in the planted sand, the CuO and ZnO NPs were increased from their nominal sizes of <50 nm and <100 nm, to ~317 nm and ~483 nm, respectively. Accordingly, the negative surface charge of colloids present in aqueous extracts from the sand amended with CuO (-27.0 mV) and ZnO (-10.0 mV) NPs was reduced by the presence of plants, to -19.8 mV and -6.0 mV, respectively. The surface charge of the MPs was not influenced by plants. Plant growth increased dissolution of NPs and MPs of both metal oxides in the sand from <0.3 mg/kg to about 1.0 mg/kg for the CuO products, and from ≤0.6 mg/kg to between 1.0 and 2.2 mg/kg for the Zn products. The NP or MP products reduced wheat root length by ~60% or ~50% from control levels; CuO was more toxic than ZnO. X-ray absorption spectroscopy (XAS) analysis showed that treatments with MPs or NPs of ZnO led to similar accumulations of Zn-phosphate species in the shoots, likely from dissolution of ZnO. Exposure to CuO NPs or MPs resulted in similar XAS spectra for Cu in the shoots explained by plant accumulation of both CuO and Cu(I)-sulfur complexes. These findings demonstrate the similarities between commercial NPs and MPs of CuO or ZnO in wheat plants, with greater root toxicity correlating with smaller particle size. Factors from the sand and the plant modified the aggregation or dissolution of both types of particles, thus, influencing their environmental fates.

Interaction of silver nanoparticles with an environmentally beneficial bacterium, Pseudomonas chlororaphis

This study explores the potential antimicrobial mechanisms of commercial silver nanoparticles (Ag NPs) in the environmental bacterium, Pseudomonas chlororaphis O6. The 10nm size NPs aggregated in water, as demonstrated by atomic force microscopy. Solubility of the NPs at 10mg/L was 0.28 mg/L (pH 6) and 2.3mg/L (pH 7); release from 10mg/L bulk Ag was below detection. The NPs eliminated cell culturability at 3mg/L, whereas no effect was observed at 10mg/L bulk Ag. Zeta potential measurements revealed that the NPs were negatively charged; unlike Ag ions, their addition to the negatively charged cells did not change cell charge at pH 6, but showed a trend to reduce cell charge at pH 7. Isolated extracellular polymeric substances (EPS) from PcO6 was polydisperse, with negative charge that was neutralized by Ag ions, but not by the NPs. Addition of EPS eliminated Ag NPs toxicity in cells lacking EPS. Intracellular accumulation of OH was not detected in NP-treated cells; however, the use of scavengers suggested the NPs caused extracellular H(2)O(2) production. No evidence was found for loss of membrane integrity upon treatment with the NPs. Our findings indicate that growth of environmental bacteria could be impaired by Ag NPs, depending on the extent of EPS production.

Soil components mitigate the antimicrobial effects of silver nanoparticles towards a beneficial soil bacterium, Pseudomonas chlororaphis O6

Silver nanoparticles (Ag NPs) are widely used for their antimicrobial activity and consequently the particles will become environmental contaminants. This study evaluated in sand and soil matrices the toxicity of 10nm spherical Ag NPs (1 and 3 mg Ag/L) toward a beneficial soil bacterium, Pseudomonas chlororaphis O6. In sand, both NP doses resulted in loss in bacterial culturability whereas in a loam soil, no cell death was observed. Amendments of sand with clays (30% v/v kaolinite or bentonite) did not protect the bacterium when challenged with Ag NPs. However, culturability of the bacterium was maintained when the Ag NP-amended sand was mixed with soil pore water or humic acid. Imaging by atomic force microscopy revealed aggregation of single nanoparticles in water, and their embedding into background material when suspended in pore water and humic acids. Zeta potential measurements supported aggregation and surface charge modifications with pore water and humic acids. Measurement of soluble Ag in the microcosms and geochemical modeling to deduce the free ion concentration revealed bacterial culturability was governed by the predicted free Ag ion concentrations. Our study confirmed the importance of Ag NPs as a source of ions and illustrated that processes accounting for protection in soil against Ag NPs involved distinct NP- and ion-effects. Processes affecting NP bioactivity involved surface charge changes due to sorption of Ca²⁺ from the pore water leading to agglomeration and coating of the NPs with humic acid and other organic materials. Removal of bioactive ions included the formation of soluble Ag complexes with dissolved organic carbon and precipitation of Ag ions with chloride in pore water. We conclude that mitigation of toxicity of Ag NPs in soils towards a soil bacterium resides in several interactions that differentially involve protection from the Ag NPs or the ions they produce.

The phytotoxicity of ZnO nanoparticles on wheat varies with soil properties.

Zn is an essential element for plants yet some soils are Zn-deficient and/or have low Zn-bioavailability. This paper addresses the feasibility of using ZnO nanoparticles (NPs) as soil amendments to improve Zn levels in the plant. The effects of soil properties on phytotoxicity and Zn bioavailability from the NPs were studied by using an acidic and a calcareous alkaline soil. In the acid soil, the ZnO NPs caused dose-dependent phytotoxicity, observed as inhibition of elongation of roots of wheat, Triticum aestivum. Phytotoxicity was mitigated in the calcareous alkaline soil although uptake of Zn from the ZnO NPs occurred doubling the Zn level compared to control plants. This increase occurred with a low level of Zn in the soil solution as expected from the interactions of Zn with the soil components at the alkaline pH. Soluble Zn in the acid soil was 200-fold higher and shoot levels were tenfold higher than from the alkaline soil correlating with phytotoxicity. Mitigation of toxicity was not observed in plants grown in sand amended with a commercial preparation of humic acid: growth, shoot uptake and solubility of Zn from the NPs was not altered by the humic acid. Thus, variation in humic acid between soils may not be a major factor influencing plant responses to the NPs. These findings illustrate that formulations of ZnO NPs to be used as a soil amendment would need to be tuned to soil properties to avoid phytotoxicity yet provide increased Zn accumulations in the plant.

Production of indole-3-acetic acid via the indole-3-acetamide pathway in the plant-beneficial bacterium, Pseudomonas chlororaphis O6, is inhibited by ZnO nanoparticles but enhanced by CuO nanoparticles

ABSTRACT The beneficial bacterium Pseudomonas chlororaphis O6 produces indole-3-acetic acid (IAA), a plant growth regulator. However, the pathway involved in IAA production in this bacterium has not been reported. In this paper we describe the involvement of the indole-3-acetamide (IAM) pathway in IAA production in P. chlororaphis O6 and the effects of CuO and ZnO nanoparticles (NPs). Sublethal levels of CuO and ZnO NPs differentially affected the levels of IAA secreted in medium containing tryptophan as the precursor. After 15 h of growth, CuO NP-exposed cells had metabolized more tryptophan than the control and ZnO NP-challenged cells. The CuO NP-treated cells produced higher IAA levels than control cultures lacking NPs. In contrast, ZnO NPs inhibited IAA production. Mixing of CuO and ZnO NPs resulted in an intermediate level of IAA production relative to the levels in the separate CuO and ZnO NP treatments. The effect of CuO NPs on IAA levels could be duplicated by ions at the concentrations released from the NPs. However, ion release did not account for the inhibition caused by the ZnO NPs. The mechanism underlying changes in IAA levels cannot be accounted for by effects on transcript accumulation from genes encoding a tryptophan permease or the IAM hydrolase in 15-h cultures. These findings raise the issue of whether sublethal doses of NPs would modify the beneficial effects of association between plants and bacteria.

Copper and cadmium: responses in Pseudomonas putida KT2440

Aims:  To compare responses of a soil bacterium to Cu and Cd.

Polycyclic Aromatic Hydrocarbon Biodegradation as a Function of Oxygen Tension in Contaminated Soil

Abstract Laboratory tests were conducted to determine the effect of soil gas oxygen concentration on the degradation and mineralization of spiked 14C-pyrene and nonspiked 16 priority pollutant polycyclic aromatic hydrocarbons (PAH) present in the soil. The soil used for the evaluation was taken from a prepared-bed land treatment unit at the Champion International Superfund Site in Libby, Montana. This soil was contaminated with wood preserving wastes including creosote (composed primarily of polycyclic aromatic hydrocarbons and pentachlorophenol). Degradation rates of 14C-pyrene and PAH compounds were found to be enhanced under soil gas oxygen concentrations between 2% and 21% in the contaminated soil. Between 45% and 55% of 14C-pyrene spiked onto the soil was mineralized after 70 days at soil gas oxygen levels between 2% and 21%. No statistically significant mineralization was found to occur at 0% oxygen concentrations. Mineralization of 14C-pyrene in contaminated soil poisoned with mercuric chloride was determined to be less than 0.5%. Degradation of indigenous nonradiolabeled PAH in non-poisoned soil was statistically significantly greater than in poisoned soil. These results indicated that the degradation of 14C-pyrene and PAH compounds was biological and would occur under low oxygen concentrations. For example, the use of soil aeration technology in order to achieve continued treatment for buried lifts of soil while new lifts are added will decrease the total time for soil remediation of the prepared-bed.

CuO and ZnO nanoparticles differently affect the secretion of fluorescent siderophores in the beneficial root colonizer, Pseudomonas chlororaphis O6

Abstract To understand the impact of environmental deposition of CuO and ZnO nanoparticles (NPs) on the production of bacterial metabolites, we examined the effects of their sub-lethal levels on the production of a pyoverdine (PVD) siderophore in a plant-beneficial bacterium, Pseudomonas chlororaphis O6 (PcO6). The NPs were characterized for size, shape, surface charge and ion release. Production of PVD by the cells was evaluated by fluorimetry and the expression of genes for PVD biosynthesis and export. The level of PVD in the presence of ZnO NPs was similar to that with Zn ions at 24 h, but the NPs maintained higher level than control at 48 h. In contrast, the reduction in PVD by CuO NPs was not duplicated by Cu ions. Expression of a gene encoding an inner membrane PVD transporter was inhibited by CuO NPs, supporting the observed low amounts of external PVD detected in those cells. These findings revealed NP-specific effect for CuO NPs on PcO6 metabolism, while for ZnO NPs, the release of ions was important. Because siderophores are part of the chemical communication between soil microbes and plants, the outcomes of plant–microbe interactions could be affected by responses to NPs.

Sorption of copper and cadmium from the water soluble fraction of an acid mine waste by two calcareous soils

We reacted the water extract of a milling waste, containing concentrations of Cu, Cd, and Zn in excess of USEPA (1974) drinking water limits in batch systems with two calcareous soils to determine the mechanisms and rates of Cu and Cd sorption. The pH of the extract was 4 to 4.5, and the predominant anion was SO4. The computer program GEOCHEM was used to calculate metal activities in the “equilibrated” suspensions. Calculated activities of Cu and Cd indicated that the suspensions of soil containing 30% carbonate were undersaturated with respect to oxide and carbonate pure solid phases at the lower levels of metal addition. As additions of Cu and Cd increased, the activities approached the solubility of tenorite (CuO) and octavite (CdCO3), respectively. At low levels of addition adsorption on carbonate surfaces apparently controlled solution concentrations. Carbonates dissolved from the soil containing 0.2% carbonates, and desorption with 0.01 M CaCl2 suggested that cation exchange processes were involved in metal sorption. The sorption of Cu, Cd, and Zn required 14 d to reach time-invariant concentrations in suspensions of the soil containing 30% carbonates, but only about 1 d in the 0.2% carbonate soil suspension.