Bioaccumulation and toxicity of CuO nanoparticles by a freshwater invertebrate after waterborne and dietborne exposures.

The incidental ingestion of engineered nanoparticles (NPs) can be an important route of uptake for aquatic organisms. Yet, knowledge of dietary bioavailability and toxicity of NPs is scarce. Here we used isotopically modified copper oxide ((65)CuO) NPs to characterize the processes governing their bioaccumulation in a freshwater snail after waterborne and dietborne exposures. Lymnaea stagnalis efficiently accumulated (65)Cu after aqueous and dietary exposures to (65)CuO NPs. Cu assimilation efficiency and feeding rates averaged 83% and 0.61 g g(-1) d(-1) at low exposure concentrations (<100 nmol g(-1)), and declined by nearly 50% above this concentration. We estimated that 80-90% of the bioaccumulated (65)Cu concentration in L. stagnalis originated from the (65)CuO NPs, suggesting that dissolution had a negligible influence on Cu uptake from the NPs under our experimental conditions. The physiological loss of (65)Cu incorporated into tissues after exposures to (65)CuO NPs was rapid over the first days of depuration and not detectable thereafter. As a result, large Cu body concentrations are expected in L. stagnalis after exposure to CuO NPs. To the degree that there is a link between bioaccumulation and toxicity, dietborne exposures to CuO NPs are likely to elicit adverse effects more readily than waterborne exposures.

Stable isotope tracer to determine uptake and efflux dynamics of ZnO Nano- and bulk particles and dissolved Zn to an estuarine snail.

Zinc oxide nanoparticles (ZnO NPs) are among the most commercialized engineered nanomaterials. Their biological impact in aquatic organisms has been associated with dissolution, but there is also evidence of nanospecific effects. In this study the waterborne uptake and efflux kinetics of isotopically labeled (68)ZnO NPs (7.8 ± 1.2 nm), in comparison to aqueous (68)Zn and (68)ZnO bulk particles (up to 2 µm), were determined for the estuarine snail Peringia ulvae following a 7 d exposure (nominally 20 µg (68)Zn L(-1)) and 28 d depuration. Detection of the (68)Zn label was achieved by high precision multiple-collector ICP-MS (MC-ICP-MS). Previous characterization in artificial estuarine water revealed that the NPs underwent initial aggregation and solubilized up to 60% within 1-2 days. Bulk and aqueous forms were significantly more bioavailable than (68)ZnO NPs (p < 0.05), but after correcting for dissolution, aqueous (0.074 L(-1) g(-1) d(-1)) and NP (0.070 L(-1) g(-1) d(-1)) uptake rate constants were highly comparable. The rate constant of loss for (68)Zn aqueous (0.012 ± 0.005 d(-1)) and (68)ZnO NPs (0.012 ± 0.007 d(-1)) were identical. These results strongly suggest that in this exposure scenario the bioaccumulation of Zn from ZnO NPs is primarily dependent upon solubility.

Isotopically modified nanoparticles for enhanced detection in bioaccumulation studies.

This work presents results on synthesis of isotopically enriched (99% (65)Cu) copper oxide nanoparticles and its application in ecotoxicological studies. (65)CuO nanoparticles were synthesized as spheres (7 nm) and rods (7 × 40 nm). Significant differences were observed between the reactivity and dissolution of spherical and rod shaped nanoparticles. The extreme sensitivity of the stable isotope tracing technique developed in this study allowed determining Cu uptake at exposure concentrations equivalent to background Cu concentrations in freshwater systems (0.2-30 µg/L). Without a tracer, detection of newly accumulated Cu was impossible, even at exposure concentrations surpassing some of the most contaminated water systems (>1 mg/L).

Bioaccumulation dynamics and modeling in an estuarine invertebrate following aqueous exposure to nanosized and dissolved silver.

Predicting the environmental impact of engineered nanomaterials (ENMs) is increasingly important owing to the prevalence of emerging nanotechnologies. We derived waterborne uptake and efflux rate constants for the estuarine snail, Peringia ulvae, exposed to dissolved Ag (AgNO(3)) and silver nanoparticles (Ag NPs), using biodynamic modeling. Uptake rates demonstrated that dissolved Ag is twice as bioavailable as Ag in nanoparticle form. Biphasic loss dynamics revealed the faster elimination of Ag from Ag NPs at the start of depuration, but similar slow efflux rate constants. The integration of biodynamic parameters into our model accurately predicted Ag tissue burdens during chronic exposure with 85% of predicted values within a factor of 2 of observed values. Zeta potentials for the Ag NPs were lower in estuarine waters than in waters of less salinity; and uptake rates in P. ulvae were slower than reported for the freshwater snail Lymnaea stagnalis in similar experiments. This suggests aggregation of Ag NPs occurs in estuarine waters and reduces, but does not eliminate, bioavailability of Ag from the Ag NPs. Biodynamic modeling provides an effective methodology to determine bioavailable metal concentrations (originating from metal and metal-oxide nanoparticles) in the environment and may aid future ENM risk assessment.

Silver bioaccumulation dynamics in a freshwater invertebrate after aqueous and dietary exposures to nanosized and ionic Ag.

We compared silver (Ag) bioavailability and toxicity to a freshwater gastropod after exposure to ionic silver (Ag(+)) and to Ag nanoparticles (Ag NPs) capped with citrate or with humic acid. Silver form, exposure route, and capping agent influence Ag bioaccumulation dynamics in Lymnaea stagnalis. Snails efficiently accumulated Ag from all forms after either aqueous or dietary exposure. For both exposure routes, uptake rates were faster for Ag(+) than for Ag NPs. Snails efficiently assimilated Ag from Ag NPs mixed with diatoms (assimilation efficiency (AE) ranged from 49 to 58%) and from diatoms pre-exposed to Ag(+) (AE of 73%). In the diet, Ag NPs damaged digestion. Snails ate less and inefficiently processed the ingested food, which adversely impacted their growth. Loss rates of Ag were faster after waterborne exposure to Ag NPs than after exposure to dissolved Ag(+). Once Ag was taken up from diet, whether from Ag(+) or Ag NPs, Ag was lost extremely slowly. Large Ag body concentrations are thus expected in L. stagnalis after dietborne exposures, especially to citrate-capped Ag NPs. Ingestion of Ag associated with particulate materials appears as the most important vector of uptake. Nanosilver exposure from food might trigger important environmental risks.

Cellular internalization of silver nanoparticles in gut epithelia of the estuarine polychaete Nereis diversicolor.

Silver nanoparticles (AgNPs) are widely used which may result in environmental impacts, notably within aquatic ecosystems. As estuarine sediments are sinks for numerous pollutants, but also habitat and food for deposit feeders such as Nereis diversicolor, ingested sediments must be investigated as an important route of uptake for NPs. N. diversicolor were fed sediment spiked with either citrate capped AgNPs (30 ± 5 nm) or aqueous Ag for 10 days. Postexposure AgNPs were observed in the lumen of exposed animals, and three lines of evidence indicated direct internalization of AgNPs into the gut epithelium. With TEM, electron-dense particles resembling AgNPs were observed associated with the apical plasma membrane, in endocytotic pits and in endosomes. Energy dispersive X-ray analysis (EDX) confirmed the presence of Ag in these particles, which were absent in controls. Subcellular fractionation revealed that Ag accumulated from AgNPs was predominantly associated with inorganic granules, organelles, and the heat denatured proteins; whereas dissolved Ag was localized to the metallothionein fraction. Collectively, these results indicate separate routes of cellular internalization and differing in vivo fates of Ag delivered in dissolved and NP form. For AgNPs an endocytotic pathway appears to be a key route of cellular uptake.

Aquatic insect ecophysiological traits reveal phylogenetically based differences in dissolved cadmium susceptibility.

We used a phylogenetically based comparative approach to evaluate the potential for physiological studies to reveal patterns of diversity in traits related to susceptibility to an environmental stressor, the trace metal cadmium (Cd). Physiological traits related to Cd bioaccumulation, compartmentalization, and ultimately susceptibility were measured in 21 aquatic insect species representing the orders Ephemeroptera, Plecoptera, and Trichoptera. We mapped these experimentally derived physiological traits onto a phylogeny and quantified the tendency for related species to be similar (phylogenetic signal). All traits related to Cd bioaccumulation and susceptibility exhibited statistically significant phylogenetic signal, although the signal strength varied among traits. Conventional and phylogenetically based regression models were compared, revealing great variability within orders but consistent, strong differences among insect families. Uptake and elimination rate constants were positively correlated among species, but only when effects of body size and phylogeny were incorporated in the analysis. Together, uptake and elimination rates predicted dramatic Cd bioaccumulation differences among species that agreed with field-based measurements. We discovered a potential tradeoff between the ability to eliminate Cd and the ability to detoxify it across species, particularly mayflies. The best-fit regression models were driven by phylogenetic parameters (especially differences among families) rather than functional traits, suggesting that it may eventually be possible to predict a taxon's physiological performance based on its phylogenetic position, provided adequate physiological information is available for close relatives. There appears to be great potential for evolutionary physiological approaches to augment our understanding of insect responses to environmental stressors in nature.

Influence of remediation in a mine-impacted river: metal trends over large spatial and temporal scales.

The effectiveness of mine-waste remediation at the Clark Fork River Superfund site in western Montana, USA, was examined by monitoring metal concentrations in resident biota (caddisfly, Hydropsyche spp.) and bed sediment over a 19-year period. Remediation activities began in 1990 and are ongoing. In the upper 45 km, reduced Cu and Cd concentrations at some sites were coincident with remediation events. However, for a period of three years, the decline in Cu and Cd directly below the treatment ponds was offset by high arsenic concentrations, suggesting that remediation for cations (e.g., Cu and Cd) mobilized anions such as arsenic. The impact of remediation in the middle and lower reaches was confounded by a significant positive relationship between metal bioaccumulation and stream discharge. High flows did not dilute metals but redistributed contaminants throughout the river. The majority of clean-up efforts were focused on reducing metal-rich sediments in the most contaminated upstream reach, implicitly assuming that improvements upstream will positively impact the downstream stations. We tested this assumption by correlating temporal metal trends in sediment between and among stations. The strength of that association (r value) was our indicator of spatial connectivity. Connectivity for both Cu and Cd was strong at small spatial scales. Large-scale connectivity was strongest with Cu since similar temporal reductions were observed at most monitoring stations. The most upstream station, closest to remediation, had the lowest connectivity, but the next three downstream sites were strongly correlated to trends downstream. Targeted remediation in this reach would be an effective approach to positively influencing the downstream stations.

Characterisation of carbon nanotubes in the context of toxicity studies.

Nanotechnology has the potential to revolutionise our futures, but has also prompted concerns about the possibility that nanomaterials may harm humans or the biosphere. The unique properties of nanoparticles, that give them novel size dependent functionalities, may also have the potential to cause harm. Discrepancies in existing human health and environmental studies have shown the importance of good quality, well-characterized reference nanomaterials for toxicological studies.Here we make a case for the importance of the detailed characterization of nanoparticles, using several methods, particularly to allow the recognition of impurities and the presence of chemically identical but structurally distinct phases. Methods to characterise fully, commercially available multi-wall carbon nanotubes at different scales, are presented.

Three-layered silver nanoparticles to trace dissolution and association to a green alga.

Core-shell silver nanoparticles (NPs) consisting of an inner Ag core and successive layers of Au and Ag (Ag@Au@Ag) were used to measure the simultaneous association of Ag NPs and ionic Ag by the green alga Chlamydomonas (C.) reinhardtii. Dissolution of the inner Ag core was prevented by a gold (Au) layer, while the outer Ag layer was free to dissolve. In short-term experiments, we exposed C. reinhardtii to a range of environmentally realistic Ag concentrations added as AgNO3 or as NPs. Results provide three lines of evidence for the greater cell-association of NPs compared to dissolved Ag over the concentration range tested, assuming that cell-association comprises both uptake and adsorption. First, the cell-association rate constants (kuw) for total Ag (AgNP+D), NPs (AgNP) and AuNP were similar and 2.2-fold higher than the one from AgD exposure, suggesting predominant association of the particles over the dissolved form. Second, model calculations based on Ag fluxes suggested that only 6-33% of algal burden was from AgD. Third, the significantly lower AgNP/Au ratio measured with the algae after exposure (2.1 ± 0.1) compared to the AgNP/Au ratio of the NPs in the media (2.47 ± 0.05) suggests cell-association of NPs depleted in Ag. Core-shell NPs provide an innovative tool to understand NP behavior and to directly delineate Ag accumulation from ion and NPs in aquatic systems.

Cadmium biodynamics in the oligochaete Lumbriculus variegatus and its implications for trophic transfer.

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake (k(u)) and efflux (k(e)) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies.

Determining metal assimilation efficiency in aquatic invertebrates using enriched stable metal isotope tracers.

We employ a novel approach that combines pulse-chase feeding and multi-labelled stable isotopes to determine gut passage time (GPT), gut retention time (GRT), food ingestion rate (IR) and assimilation efficiency (AE) of three trace elements for a freshwater gastropod. Lettuce isotopically enriched in (53)Cr, (65)Cu and (106)Cd was fed for 2h to Lymnaea stagnalis. The release of tracers in feces and water was monitored for 48 h, during which unlabelled lettuce was provided ad libidum. The first defecation of (53)Cr occurred after 5h of depuration (GPT), whereas 90% of the ingested (53)Cr was recovered in the feces after 22.5h of depuration (GRT). (53)Chromium was not significantly accumulated in the soft tissues upon exposure. In contrast, (65)Cu and (106)Cd assimilation was detectable for most experimental snails, i.e., (65/63)Cu and (106/114)Cd ratios in exposed snails were higher than those for controls. Food IR during the labelled feeding phase was 0.16+/-0.07 g g(-1)d(-1). IR was inferred from the amount of (53)Cr egested in the feces during depuration and the concentration of (53)Cr in the labelled lettuce. Assimilation efficiencies (+/-95% CI) determined using mass balance calculations were 84+/-4% for Cu and 85+/-3% for Cd. The ratio method yields similar AE estimates. Expanding the application of this novel stable isotope tracer technique to other metals in a wide variety of species will provide unique opportunities to evaluate the interplay between digestive processes and dietary influx of metals. Understanding the biological processes that modulate dietborne metal uptake is crucial to assess the toxicity of dietborne metals.

Biological uptake of polychlorinated biphenyls by Macoma balthica from sediment amended with activated carbon.

This work characterizes the efficacy of activated carbon amendment in reducing polychlorinated biphenyl (PCB) bioavailability to clams (Macoma balthica) from field-contaminated sediment (Hunters Point Naval Shipyard, San Francisco Bay, CA, USA). Test methods were developed for the use of clams to investigate the effects of sediment amendment on biological uptake. Sediment was mixed with activated carbon for one month. Bioaccumulation tests (28 d) were employed to assess the relationships between carbon dose and carbon particle size on observed reductions in clam biological uptake of PCBs. Extraction and cleanup protocols were developed for the clam tissue. Efficacy of activated carbon treatment was found to increase with both increasing carbon dose and decreasing carbon particle size. Average reductions in bioaccumulation of 22, 64, and 84% relative to untreated Hunters Point sediment were observed for carbon amendments of 0.34, 1.7, and 3.4%, respectively. Average bioaccumulation reductions of 41, 73, and 89% were observed for amendments (dose = 1.7% dry wt) with carbon particles of 180 to 250, 75 to 180, and 25 to 75 microm, respectively, in diameter, indicating kinetic phenomena in these tests. Additionally, a biodynamic model quantifying clam PCB uptake from water and sediment as well as loss through elimination provided a good fit of experimental data. Model predictions suggest that the sediment ingestion route contributed 80 to 95% of the PCB burdens in the clams.

Modernizing Water Quality Criteria in the United States: A Need to Expand the Definition of Acceptable Data.

The development of water quality criteria (WQC) for the protection of aquatic life is a fundamental component of the Clean Water Act-the primary US legislation responsible for protecting aquatic ecosystems from pollution. Water quality criteria define acceptable levels of contamination in the environment and thus play an important role in society. Rules for how science is used to develop WQC were created in 1985. Most rely on only data and knowledge obtained through a single methodology, the single-species laboratory toxicity test. Since 1985, understanding of the fate and effects of environmental contaminants has advanced markedly from multiple perspectives and disciplines. However, many of these advances are routinely discarded in WQC development because they do not adhere to data limits imposed by the 1985 guidelines. The present Focus article outlines how multiple lines of inquiry have played important roles in improving understanding of the ecological implications of environmental contaminants. The authors focus on gains in understanding that would not have been possible through traditional toxicity bioassays alone and argue that more robust scientific understanding can be used to modernize WQC development. In particular, the present article highlights ways to increase the relevance of toxicity testing (at different spatiotemporal scales) and incorporate all relevant lines of evidence into WQC modernization. Environ Toxicol Chem 2017;36:285-291. © 2017 SETAC.

Comparison of Selenium bioaccumulation in the clams Corbicula fluminea and Potamocorbula amurensis: a bioenergetic modeling approach.

Selenium uptake from food (assimilation efficiency) and dissolved phase (influx rate) as well as loss kinetics (efflux rate) were compared between two bivalves, Corbicula fluminea and Potamocorbula amurensis. The effects of salinity and temperature on these kinetic parameters for both clam species also were evaluated. The Asiatic clam, C. fluminea, more efficiently assimilated Se associated with algae (66-87%) than Se associated with oxic sediments (20-37%). However, no consistent difference was found between Se assimilation efficiencies from both food types (19-60%) for P. amurensis. The temperature and salinity had a minor influence on the Se assimilation from ingested food. However, the effects of temperature and salinity were more evident in the uptake from dissolved sources. The influx rate of Se(IV) increased by threefold with the increase of temperature from 5 to 21 degrees C for C. fluminea. The increase of salinity from 4 to 20 psu decreased the uptake rate constant (ku) of Se in P. amurensis from 0.011 to 0.005 L/g/h, whereas salinity change (0-8 psu) had a negligible effect on the Se influx rate of C. fluminea. The Se influx rate of P. amurensis decreased by half with the 3.5-fold increase in tissue dry weight. The rate constant of loss was greater for P. amurensis (0.029/d at 8 psu) than for C. fluminea (0.014/d at 0 psu and 0.01/d at 8 psu). A bioenergetic model suggests that dietary uptake is the dominant pathway for Se bioaccumulation in the two clams in San Francisco Bay and that interspecies differences in Se bioaccumulation can be explained by differences in food ingestion rates.

Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica.

The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, k((e), ranged from 0.20 to 0.24/d (t 1/2 approximately 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 +/- 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 +/- 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 +/- 7 ng/g) than in those from the contaminated site (42 +/- 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects.

Opportunities and challenges of integrating ecological restoration into assessment and management of contaminated ecosystems.

Ecosystem restoration planning near the beginning of the site assessment and management process ("early integration") involves consideration of restoration goals from the outset in developing solutions for contaminated ecosystems. There are limitations to integration that stem from institutional barriers, few successful precedents, and limited availability of guidance. Challenges occur in integrating expertise from various disciplines and multiple, sometimes divergent interests and goals. The more complex process can result in timing, capacity, communication, and collaboration challenges. On the other hand, integrating the 2 approaches presents new and creative opportunities. For example, integration allows early planning for expanding ecosystem services on or near contaminated lands or waters that might otherwise have been unaddressed by remediation alone. Integrated plans can explicitly pursue ecosystem services that have market value, which can add to funds for long-term monitoring and management. Early integration presents opportunities for improved and productive collaboration and coordination between ecosystem restoration and contaminant assessment and management. Examples exist where early integration facilitates liability resolution and generates positive public relations. Restoration planning and implementation before the completion of the contaminated site assessment, remediation, or management process ("early restoration") can facilitate coordination with offsite restoration options and a regional approach to restoration of contaminated environments. Integration of performance monitoring, for both remedial and restoration actions, can save resources and expand the interpretive power of results. Early integration may aid experimentation, which may be more feasible on contaminated lands than in many other situations. The potential application of concepts and tools from adaptive management is discussed as a way of avoiding pitfalls and achieving benefits in early integration. In any case, there will be challenges with early integration of restoration concepts for contaminated ecosystems, but the benefits are likely to outweigh them.

Delineating copper accumulation pathways for the freshwater bivalve Corbicula using stable copper isotopes.

Delineation of metal uptake routes in aquatic invertebrates is critical for characterizing bioaccumulation dynamics and assessing risks associated with metal exposure. Here we demonstrate that Cu stable isotopic ratios can be manipulated in both exposure media and algae to determine the efflux rate constant (ke) and to estimate Cu assimilation efficiency (AE) from ingested food in a freshwater bivalve (Corbicula fluminea). The Cu AE in Corbicula fed 65Cu-spiked Cryptomonas ozolini was 38%. Copper uptake routes had no significant influence on efflux; ke of 0.004 per day characterized the slowest component of efflux following short-term exposures to 65Cu in water or in both food and water. Incorporation of the physiological parameters for dietary and dissolved uptake as well as rate constants of loss into a bioaccumulation model allowed for assessing the relative contribution of water and food as Cu sources. At [65Cu2+] of 6.7 microg/L, Corbicula accumulated twice as much Cu from diet as from water. In most freshwater systems, the dietary pathway is likely to act as the major Cu uptake route for Corbicula. Extrapolation of our laboratory results to the San Francisco Bay-Delta (California, USA) indicated that our biodynamic model and the laboratory-derived parameters for dietary 65Cu uptake provided a realistic representation of the processes involved in Cu accumulation by the bivalve Corbicula.

Influence of hardness on the bioavailability of silver to a freshwater snail after waterborne exposure to silver nitrate and silver nanoparticles.

The release of Ag nanoparticles (AgNPs) into the aquatic environment is likely, but the influence of water chemistry on their impacts and fate remains unclear. Here, we characterize the bioavailability of Ag from AgNO(3) and from AgNPs capped with polyvinylpyrrolidone (PVP AgNP) and thiolated polyethylene glycol (PEG AgNP) in the freshwater snail, Lymnaea stagnalis, after short waterborne exposures. Results showed that water hardness, AgNP capping agents, and metal speciation affected the uptake rate of Ag from AgNPs. Comparison of the results from organisms of similar weight showed that water hardness affected the uptake of Ag from AgNPs, but not that from AgNO(3). Transformation (dissolution and aggregation) of the AgNPs was also influenced by water hardness and the capping agent. Bioavailability of Ag from AgNPs was, in turn, correlated to these physical changes. Water hardness increased the aggregation of AgNPs, especially for PEG AgNPs, reducing the bioavailability of Ag from PEG AgNPs to a greater degree than from PVP AgNPs. Higher dissolved Ag concentrations were measured for the PVP AgNPs (15%) compared to PEG AgNPs (3%) in moderately hard water, enhancing Ag bioavailability of the former. Multiple drivers of bioavailability yielded differences in Ag influx between very hard and deionized water where the uptake rate constants (k(uw), l g(-1) d(-1) ± SE) varied from 3.1 ± 0.7 to 0.2 ± 0.01 for PEG AgNPs and from 2.3 ± 0.02 to 1.3 ± 0.01 for PVP AgNPs. Modeling bioavailability of Ag from NPs revealed that Ag influx into L. stagnalis comprised uptake from the NPs themselves and from newly dissolved Ag.

Inhibition of potential uptake pathways for silver nanoparticles in the estuarine snail Peringia ulvae.

Mechanisms involved in the uptake of Ag NPs, and NPs in general, have been long debated within nano-ecotoxicology. In vitro studies provide evidence of the different available uptake pathways, but in vivo demonstrations are lacking. In this study, pharmacological inhibitors were employed to block specific uptake pathways that have been implicated in the transport of metal NPs and aqueous metal forms; phenamil (inhibits Na(+) channel), bafilomycin A1 (H(+) proton pump), amantadine (clathrin-mediated endocytosis), nystatin (caveolae-mediated endocytosis) and phenylarsine oxide (PAO, macropinocytosis). Peringia ulvae (snails) were exposed to 150 µg Ag L(-1) added as citrate capped Ag NPs or aqueous Ag (AgNO3) in combination with inhibitor treatment (determined by preliminary studies). Reductions in accumulated tissue burdens caused by the inhibitors were compared to control exposures (i.e. no inhibition) after 6 and 24 h. No inhibitor treatment completely eliminated the uptake of Ag in either aqueous or NP form, but all inhibitor treatments, except phenamil, significantly reduced the uptake of Ag presented as Ag NPs. Clathrin- and caveolae-mediated endocytosis appear to be mechanisms exploited by Ag NPs, with the latter pathway only active at 24 h. Inhibition of the H(+) proton pump showed that a portion of Ag NP uptake is achieved as aqueous Ag and is explained by the dissolution of the particles (∼25% in 24 h). This in vivo study demonstrates that uptake of Ag from Ag NPs is achieved by multiple pathways and that these pathways are simultaneously active.