Extracellular Vesicles and Bacteriophages: New Directions in Environmental Biocolloid Research.

There is a long-standing appreciation among environmental engineers and scientists regarding the importance of biologically derived colloidal particles and their environmental fate. This interest has been recently renewed in considering bacteriophages and extracellular vesicles, which are each poised to offer engineers unique insights into fundamental aspects of environmental microbiology and novel approaches for engineering applications, including advances in wastewater treatment and sustainable agricultural practices. Challenges persist due to our limited understanding of interactions between these nanoscale particles with unique surface properties and their local environments. This review considers these biological particles through the lens of colloid science with attention given to their environmental impact and surface properties. We discuss methods developed for the study of inert (nonbiological) particle-particle interactions and the potential to use these to advance our understanding of the environmental fate and transport of extracellular vesicles and bacteriophages.

Characterizing the Transport and Surface Affinity of Extracellular Vesicles Isolated from Yeast and Bacteria in Well-Characterized Porous Media.

Extracellular vesicles (EVs) are membrane-bounded, nanosized particles, produced and secreted by all biological cell types. EVs are ubiquitous in the environment, operating in various roles including intercellular communication and plant immune modulation. Despite their ubiquity, the role of EV surface chemistry in determining transport has been minimally investigated. Using the zeta (ζ)-potential as a surrogate for surface charge, this work considers the deposition of EVs from the yeast, Saccharomyces cerevisiae, and two bacterial species, Staphylococcus aureus and Pseudomonas fluorescens, in well-characterized porous medium under various background conditions shown to influence the transport of other environmental colloidal particles: ionic strength and humic acid concentration. The affinity of S. cerevisiae EVs for the porous medium (glass beads) appeared to be sensitive to changes in ionic strength, as predicted by colloid stability (Derjaguin, Landau, Verwey, and Overbeek or DLVO) theory, and humic acid concentration, while P. fluorescens EVs deviated from DLVO predictions, suggesting that mechanisms other than charge stabilization may control the deposition of P. fluorescens. Calculations of attachment efficiency from these deposition studies were used to estimate EV transport using a clean-bed filtration model. Based on these calculations, EVs could be transported through such homogeneous porous media up to 15 m.

Investigating and Modeling the Regulation of Extracellular Antibiotic Resistance Gene Bioavailability by Naturally Occurring Nanoparticles.

Extracellular antibiotic resistance genes (eARGs) are widespread in the environment and can genetically transform bacteria. This work examined the role of environmentally relevant nanoparticles (NPs) in regulating eARG bioavailability. eARGs extracted from antibiotic-resistant B. subtilis were incubated with nonresistant recipient B. subtilis cells. In the mixture, particle type (either humic acid coated nanoparticles (HASNPs) or their micron-sized counterpart (HASPs)), DNase I concentration, and eARG type were systematically varied. Transformants were counted on selective media. Particles decreased bacterial growth and eARG bioavailability in systems without nuclease. When DNase I was present (≥5 µg/mL), particles increased transformation via chromosomal (but not plasmid-borne) eARGs. HASNPs increased transformation more than HASPs, indicating that the smaller nanoparticle with greater surface area per volume is more effective in increasing eARG bioavailability. These results were also modeled via particle aggregation theory, which represented eARG-bacteria interactions as transport leading to collision, followed by attachment. Using attachment efficiency as a fitting factor, the model predicted transformant concentrations within 35% of experimental data. These results confirm the ability of NPs to increase eARG bioavailability and suggest that particle aggregation theory may be a simplified and suitable framework to broadly predict eARG uptake.

Relationship between Atomic Force Microscopy and Centrifugation Measurements for Dust Fractions Implicated in Solar Panel Soiling.

This work tests the reliability of a simple, rapid centrifugal technique to estimate the removal force necessary to detach common airborne particles from the surface of a photovoltaic panel. Previously, we have used atomic force microscopy (AFM) to obtain the surface-particle adhesion force for different pollutant types that generally contribute to panel soiling. To overcome the limitations of AFM, the same particles were studied as a population using an ultracentrifuge. Detachment was quantified at speeds between 1000 and 10,000 rpm, both as individual particle counts and as projected surface area coverage. The force of centrifugal detachment for each particle type followed a similar trend as the adhesion force given by AFM. Organic and carbon-based materials needed higher centrifugal speeds to be removed, suggesting a stronger attachment to the surface. However, the technique also highlighted the importance of particle diameter, aggregates, and individual particle characteristics, which should be considered when predicting the probability of detachment. We have identified the relationship between AFM-derived adhesion and centrifugal detachment forces using model particle fractions of materials commonly found to soil solar panels, demonstrating the utility in using the more easily applied to centrifugal method to obtain information that can be calibrated to direct measurements of the force of particle attachment. This technique could be applied effectively in further studies on the influence of dust composition on long-term soiling and its reversibility.

In Vivo Effects of Silver Nanoparticles on Development, Behavior, and Mitochondrial Function are Altered by Genetic Defects in Mitochondrial Dynamics.

Silver nanoparticles (AgNPs) are extensively used in consumer products and biomedical applications, thus guaranteeing both environmental and human exposures. Despite extensive research addressing AgNP safety, there are still major knowledge gaps regarding AgNP toxicity mechanisms, particularly in whole organisms. Mitochondrial dysfunction is frequently described as an important cytotoxicity mechanism for AgNPs; however, it is still unclear if mitochondria are the direct targets of AgNPs. To test this, we exposed the nematodeCaenorhabditis elegans to sublethal concentrations of AgNPs and assessed specific mitochondrial parameters as well as organismal-level endpoints that are highly reliant on mitochondrial function, such as development and chemotaxis behavior. All AgNPs tested significantly delayed nematode development, disrupted mitochondrial bioenergetics, and blocked chemotaxis. However, silver was not preferentially accumulated in mitochondria, indicating that these effects are likely not due to direct mitochondria-AgNP interactions. Mutant nematodes with deficiencies in mitochondrial dynamics displayed both greater and decreased susceptibility to AgNPs compared to wild-type nematodes, which was dependent on the assay and AgNP type. Our study suggests that AgNPs indirectly promote mitochondrial dysfunction, leading to adverse outcomes at the organismal level, and reveals a role of gene-environment interactions in the susceptibility to AgNPs. Finally, we propose a novel hypothetical adverse outcome pathway for AgNP effects to guide future research.

Effect of Dust Composition on the Reversibility of Photovoltaic Panel Soiling.

Eight types of common airborne particles were used to investigate whether the composition of dust influences its soiling potential on photovoltaic panels. Chosen model particles were roughly spherical, 10-30 µm in diameter to minimize the differences in size and shape. While the predicted van der Waals forces were lower than the adhesion forces measured with an atomic force microscope (AFM), the adhesion potential as a function of surface energy did follow the theoretical pattern. The organic and carbon-based materials, namely the pollen grains and spherical graphite, exhibited a significantly larger adhesion force to the glass surface, indicating high attachment efficiency. The developed generalized linear model confirmed that the type of material should be included in soiling models as a variable, as it provides information on the likelihood of particles sticking to and remaining on the surface. The adhesion force between soiled particles and the surface can be estimated based on the local ambient dust composition to predict the short-term fate of the depositing particles and develop cleaning schedules and techniques accordingly. The results also highlight the need to study dust composition to understand long-term soiling, where chemical characteristics and changing environmental conditions may lead to cementation.

Discovery of Welcome Biopolymers in Surface Water: Improvements in Drinking Water Production.

The presence of biopolymers in surface waters and their significance for potable water supply have received little attention previously owing to their low concentrations. In this paper, we present the results of an extensive study that has investigated the role and benefits of biopolymers during the purification of surface water with reference to their specific biological and physico-chemical properties. Using samples collected from two representative surface waters in China and the United Kingdom, macromolecular biopolymers were separated and concentrated for subsequent investigation of their role in coagulation, metal ion adsorption, and membrane separation. Our results show that biopolymers significantly improve the antifouling capability of membrane nanofiltration, in combination with the enhanced conventional coagulation performance and additional security against several unhealthy metal pollutants (e.g., Fe, Al, and Cr). We believe this is the first study that reveals the versatile benefits and the fate of natural biopolymers in surface water purification processes.

Quantifying Mechanical Abrasion of MWCNT Nanocomposites Used in 3D Printing: Influence of CNT Content on Abrasion Products and Rate of Microplastic Production.

Manufactured nanomaterials (MNMs) are incorporated as "nanofillers" into consumer products to enhance properties of interest. Multiwalled carbon nanotubes (MWCNTs) are known for their unique properties and have many applications in polymers. However, the release of MWCNTs during the nanoenabled product life cycle is concerning. During the use phase, mechanical stresses can produce fragmented materials containing MNMs. The degree of MNM release, the resulting exposure to these materials, and the potential impacts of their release are active research topics. In this study, we describe methodological improvements to study the abrasion of plastics containing MNMs (nanocomposites) and report on characteristics of abrasion products produced and rates of microplastic production. The abrasion device developed for this work allows for the measurement of power inputs to determine scaled release rates. Abrasion rates for plastics used in 3D printing were found to be 0.27 g/m2/s for the PETG polymer and 0.3 g/m2/s for the 2% MWCNT-PETG nanocomposite. Embedded and protuberant MWCNTs appeared to impact the particle size, shape, hydrophobicity, and surface charge of the microplastics, while the inclusion of MWCNTs had a small effect on microplastic production. Measurements of power input to the abrasion process provided a basis for estimating microplastic production rates for these nanocomposites.

Lack of Detectable Direct Effects of Silver and Silver Nanoparticles on Mitochondria in Mouse Hepatocytes.

Silver nanoparticles (AgNPs) are well-proven antimicrobial nanomaterials, yet little is elucidated regarding the mechanism underlying cytotoxicity induced by these nanoparticles. Here, we tested the hypothesis that mitochondria are primary intracellular targets of two AgNPs and silver ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based media. AML12 cells were more sensitive to mitochondrial uncoupling when grown with galactose rather than glucose. However, 24 h treatments with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 µg/mL) and AgNO3 (1 and 3 µg/mL), concentrations that resulted in either 10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells grown on galactose than glucose. Furthermore, colocalization analysis and subcellular Ag quantification did not show any enrichment of silver content in mitochondria in either medium. Finally, the effects of the same exposures on mitochondrial respiration were mild or undetectable, a result inconsistent with mitochondrial toxicity causing cell death. Our results suggest that neither ionic Ag nor the AgNPs that we tested specifically target mitochondria and are inconsistent with mitochondrial dysfunction being the primary cause of cell death after Ag exposure under these conditions.

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

Groundwater Chemistry Has a Greater Influence on the Mobility of Nanoparticles Used for Remediation than the Chemical Heterogeneity of Aquifer Media.

The application of nanoscale zerovalent iron (nano-ZVI) particles for groundwater remediation has spurred research into the influence of the collector heterogeneity on the nano-ZVI mobility. The chemical heterogeneity of surfaces within aquifer media affects their surface charge distribution and their affinity for nano-ZVI. The groundwater chemistry affects the properties of both aquifer surfaces and the nano-ZVI particles. Commercial poly(acrylic acid)-coated nano-ZVI (PAA-nano-ZVI) particles were tested in column experiments using two solution chemistries and silica collectors with different degrees of chemical heterogeneity, achieved by ferrihydrite coating. A porous media filtration model was used to determine the attachment efficiency of PAA-nano-ZVI particles, and the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was used to describe the interactions between PAA-nano-ZVI particles and the aquifer "collectors". The mobility of PAA-nano-ZVI particles suspended in ultrapure water depended on the extent of ferrihydrite coating on the collector surfaces. The mobility of PAA-nano-ZVI particles under environmentally relevant conditions was independent of the collector chemical heterogeneity. The size of PAA-nano-ZVI aggregates doubled, inducing gravitational sedimentation and possibly straining as mechanisms of particle deposition. There was no repulsive energy barrier between particles and collectors, and the DLVO theory was unable to explain the observed particle attachment. Our results suggest that the groundwater chemistry has a greater influence on the mobility of PAA-nano-ZVI particles than the collector chemical heterogeneity. A better understanding of polymer adsorption to nanoparticles and its conformation under natural groundwater conditions is needed to further elucidate nanoparticle-collector interactions.

Thermal Activation of Peracetic Acid in Aquatic Solution: The Mechanism and Application to Degrade Sulfamethoxazole.

Chemical oxidation using peracetic acid (PAA) can be enhanced by activation with the formation of reactive species such as organic radicals (R-O•) and HO•. Thermal activation is an alternative way for PAA activation, which was first applied to degrade micropollutants in this study. PAA is easily decomposed by heat via both radical and nonradical pathways. Our experimental results suggest that a series of reactive species including R-O•, HO•, and 1O2 can be produced through the thermal decomposition of PAA. Sulfamethoxazole (SMX), a typical sulfa drug, can be effectively removed by the thermoactivated PAA process under conditions of neutral pH. R-O• including CH3C(O)O• and CH3C(O)OO• has been shown to play a primary role in the degradation of SMX followed by direct PAA oxidation in the thermoactivated PAA process. Both higher temperature (60 °C) and higher PAA dose benefit SMX degradation, while coexisting H2O2 inhibits SMX degradation in the thermoactivated PAA process. With a variation of solution pH, conditions near a neutral value show the best performance of this process in SMX degradation. Based on the identified intermediates, transformation of SMX was proposed to undergo oxidation of the amine group and oxidative coupling reactions. This study definitively illustrates the PAA decomposition pathways at high temperature in aquatic solution and addresses the possibility of the thermoactivated PAA process for contaminant destruction, demonstrating this process to be a feasible advanced oxidation process.

Application of Cobalt/Peracetic Acid to Degrade Sulfamethoxazole at Neutral Condition: Efficiency and Mechanisms.

An advanced oxidation process of combining cobalt and peracetic acid (Co/PAA) was developed to degrade sulfamethoxazole (SMX) in this study. The formed acetylperoxy radical (CH3CO3•) through the activation of PAA by Co (Co2+) was the dominant radical responsible for SMX degradation, and acetoxyl radical (CH3CO2•) might also have played a role. The efficient redox cycle of Co3+/Co2+ allows good removal efficiency of SMX even at quite low dosage of Co (<1 µM). The presence of H2O2 in the Co/PAA process has a negative effect on the degradation of SMX due to the competition for reactive radicals. The SMX degradation in the Co/PAA process is pH dependent, and the optimum reaction pH is near-neutral. Humic acid and HCO3- can inhibit SMX degradation in the Co/PAA process, while the presence of Cl- plays a little role in the degradation of SMX in this system. Although transformation products of SMX in the Co/PAA system show higher acute toxicity, the low Co dose and SMX concentration in aquatic solution can efficiently weaken the acute toxicity. After reaction in the Co/PAA process, numerous carbon sources that could be provided for bacteria and algae growth can be produced, suggesting that the proposed Co/PAA process has good potential when combined with the biotreatment processes.

Differential Reactivity of Copper- and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm.

Reliable predictions of the environmental fate and risk of engineered nanomaterials (ENMs) require a better understanding of ENM reactivity in complex, biologically active systems for chronic low-concentration exposure scenarios. Here, simulated freshwater wetland mesocosms were dosed with ENMs to assess how their reactivity and seasonal changes in environmental parameters influence ENM fate in aquatic systems. Copper-based ENMs (Kocide), known to dissolve in water, and gold nanoparticles (AuNPs), stable against dissolution in the absence of specific ligands, were added weekly to mesocosm waters for 9 months. Metal accumulation and speciation changes in the different environmental compartments were assessed over time. Copper from Kocide rapidly dissolved likely associating with organic matter in the water column, transported to terrestrial soils and deeper sediment where it became associated with organic or sulfide phases. In contrast, Au accumulated on/in the macrophytes where it oxidized and transferred over time to surficial sediment. A dynamic seasonal accumulation and metal redox cycling were found between the macrophyte and the surficial sediment for AuNPs. These results demonstrate the need for experimental quantification of how the biological and chemical complexity of the environment, combined with their seasonal variations, drive the fate of metastable ENMs.

Formulation and Validation of a Functional Assay-Driven Model of Nanoparticle Aquatic Transport.

Here, we present a model for the prediction of nanoparticle fate in aquatic environments, parametrized using functional assays that take into account conditions of the environmental media and nanoparticle properties. The model was used to explore scenarios for five nanomaterials in a freshwater wetland setting and compared with experimental results obtained in mesocosm studies. Material characteristics used in the model were size, density, dissolution rate constants, and surface attachment efficiencies. Model predictions and experimentally measured removal rate constants from the water column were strongly correlated, with Pearson correlation coefficient 0.993. Further, the model predicted removal rate constants quantitively very close to measured rates. Of particular importance for accurate predictions were two key processes beyond the usual heteroaggregation with suspended solids. These were homoaggregation of nanomaterials and nanomaterial attachment to aquatic plant surfaces. These results highlight the importance of including all relevant aggregation and deposition processes over short time scales for nanoparticle transport, while demonstrating the utility of functional assays for surface attachment as model inputs.

Selective Recovery of Rare Earth Elements from Coal Fly Ash Leachates Using Liquid Membrane Processes.

Coal combustion residues and other geological waste materials have been proposed as a resource for rare earth elements (REEs, herein defined as the 14 stable lanthanides, yttrium, and scandium). The extraction of REEs from residues often generate acidified leachates that require highly selective separation methods to recover the REEs from other major soluble ions in the leachates. Here, we studied two liquid membrane processes (liquid emulsion membranes, LEM, and supported liquid membranes, SLM) and compared them to standard solvent extraction techniques for selective recovery and concentration of REEs from a leachate of coal fly ash. All separation methods involved an organic solution of di(2-ethylhexyl)phosphoric acid dissolved in kerosene or mineral oil and an acid strippant solution of 5 M nitric acid for the liquid-based separations. The LEM configuration, which separated REEs by immersing an acid-in-oil emulsion in the ash leachate, resulted in similar recovery percentages of individual REEs as the conventional solvent extraction approach. The recovery of REEs in the SLM configuration, which involved the impregnation of the solvent in a hydrophobic membrane, was slower than the LEM process. However, the SLM process was notably more selective for the heavy (and higher value) REEs, while the conventional extraction and LEM processes were more selective for the light REEs. A flux-based model of the extraction processes suggested that recovery rates were limited by REE affinity for the solvent chelator in the SLM, while the rates of REEs separation via LEM were limited by diffusive mass transfer across the liquid membrane. Altogether, these results help to identify specific steps in the recovery process that future work should target in the development of scalable liquid membrane separations for REE recovery.

Tailoring the Core-Satellite Nanoassembly Architectures by Tuning Internanoparticle Electrostatic Interactions.

The use of plasmonic nanoplatforms has received increasing interest in a wide variety of fields ranging from theranostics to environmental sensing to plant biology. In particular, the development of plasmonic nanoparticles into ordered nanoclusters has been of special interest due to the new chemical functionalities and optical responses that they can introduce. However, achieving predetermined nanocluster architectures from bottom-up approaches in the colloidal solution state still remains a great challenge. Herein, we report a one-pot assembly approach that provides flexibility in precise control of core-satellite nanocluster architectures in the colloidal solution state. We found that the pH of the assembly medium plays a vital role in the hierarchy of the nanoclusters. The architecture along with the size of the satellite gold nanoparticles determines the optical responses of nanoclusters. Using electron microscopy and optical spectroscopy, we introduce a set of design rules for the synthesis of distinct architectures of silica-core gold satellites nanoclusters in the colloidal solution state. Our findings provide insight into advancing the colloidal solution state nanoclusters formation with predictable architectures and optical properties.

Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment.

Despite the rapid rise in diversity and quantities of engineered nanomaterials produced, the impacts of these emerging contaminants on the structure and function of ecosystems have received little attention from ecologists. Moreover, little is known about how manufactured nanomaterials may interact with nutrient pollution in altering ecosystem productivity, despite the recognition that eutrophication is the primary water quality issue in freshwater ecosystems worldwide. In this study, we asked two main questions: (1) To what extent do manufactured nanoparticles affect the biomass and productivity of primary producers in wetland ecosystems? (2) How are these impacts mediated by nutrient pollution? To address these questions, we examined the impacts of a citrate-coated gold nanoparticle (AuNPs) and of a commercial pesticide containing Cu(OH)2 nanoparticles (CuNPs) on aquatic primary producers under both ambient and enriched nutrient conditions. Wetland mesocosms were exposed repeatedly with low concentrations of nanoparticles and nutrients over the course of a 9-month experiment in an effort to replicate realistic field exposure scenarios. In the absence of nutrient enrichment, there were no persistent effects of AuNPs or CuNPs on primary producers or ecosystem productivity. However, when combined with nutrient enrichment, both NPs intensified eutrophication. When either of these NPs were added in combination with nutrients, algal blooms persisted for >50 d longer than in the nutrient-only treatment. In the AuNP treatment, this shift from clear waters to turbid waters led to large declines in both macrophyte growth and rates of ecosystem gross primary productivity (average reduction of 52% ± 6% and 92% ± 5%, respectively) during the summer. Our results suggest that nutrient status greatly influences the ecosystem-scale impact of two emerging contaminants and that synthetic chemicals may be playing an under-appreciated role in the global trends of increasing eutrophication. We provide evidence here that chronic exposure to Au and Cu(OH)2 nanoparticles at low concentrations can intensify eutrophication of wetlands and promote the occurrence of algal blooms.

Incorporation of Cellulose Nanocrystals (CNCs) into the Polyamide Layer of Thin-Film Composite (TFC) Nanofiltration Membranes for Enhanced Separation Performance and Antifouling Properties.

To achieve greater separation performance and antifouling properties in a thin-film composite (TFC) nanofiltration membrane, cellulose nanocrystals (CNCs) were incorporated into the polyamide layer of a TFC membrane for the first time. The results of Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy (XPS) confirmed the successful formation of the CNC-polyamide composite layer. Surface characterization results revealed differences in the morphologies of the CNC-TFC membranes compared with a control membrane (CNC-TFC-0). Streaming potential measurements and molecular weight cutoff (MWCO) characterizations showed that the CNC-TFC membranes exhibited a greater negative surface charge and a smaller MWCO as the CNC content increased. The CNC-TFC membranes showed enhanced hydrophilicity and increased permeability. With the incorporation of only 0.020 wt % CNCs, the permeability of the CNC-TFC membrane increased by 60.0% over that of the polyamide TFC without CNC. Rejection of Na2SO4 and MgSO4 by the CNC-TFC membranes was similar to that observed for the CNC-TFC-0 membrane, at values of approximately 98.7% and 98.8%, respectively, indicating that divalent salt rejection was not sacrificed. The monovalent ion rejection tended to increase as the CNC content increased. In addition, the CNC-TFC membranes exhibited enhanced antifouling properties due to their increased hydrophilicity and more negatively charged surfaces.

Stress Responses of Aquatic Plants to Silver Nanoparticles.

Silver nanoparticles (AgNPs) are increasingly used in consumer products, biotechnology, and medicine, and are released into aquatic ecosystems through wastewater discharge. This study investigated the phytotoxicity of AgNPs to aquatic plants, Egeria densa and Juncus effusus by measuring physiologic and enzymatic responses to AgNP exposure under three release scenarios: two chronic (8.7 mg, weekly) exposures to either zerovalent AgNPs or sulfidized silver nanoparticles; and a pulsed (450 mg, one-time) exposure to zerovalent AgNPs. Plant enzymatic and biochemical stress responses were assessed using superoxide dismutase (SOD) and peroxidase (POD) activity, malondialdehyde (MDA) concentrations and chlorophyll content as markers of defense and phytotoxicity, respectively. The high initial pulse treatment resulted in rapid changes in physiological characteristics and silver concentration in plant tissue at the beginning of each AgNPs exposure (6 h, 36 h, and 9 days), while continuous AgNP and sulfidized AgNP chronic treatments gave delayed responses. Both E. densa and J. effusus enhanced their tolerance to AgNPs toxicity by increasing POD and SOD activities to scavenge free radicals but at different growth phases. Chlorophyll did not change. After AgNPs exposure, MDA, an index of membrane damage, was higher in submerged E. densa than emergent J. effusus, which suggested that engineered nanoparticles exerted more stress to submerged macrophytes.