Environmental persistence, bioaccumulation, and hazards of chemicals in e-cigarette e-liquids: short-listing chemicals for risk assessments.

BACKGROUND/METHODS: Increased use and sales of e-cigarettes raises concerns about the potential environmental impacts throughout their life-cycle. However, few available research studies focus on the environmental impacts and ecotoxicity of e-cigarettes. In this study, we short-list e-liquid chemicals from published literature that should be considered in future environmental impact and risk assessments. We used a combination of available laboratory bioassays-based data and predictive methods (eg, Structure-Activity Relationships) to characterise the hazards of the e-liquid chemicals (environmental persistence, bioaccumulation, and aquatic toxicity including hazardous concentration values (concentration affecting specific proportion of species)) for short-listing. RESULTS: Of the 421 unique e-liquid chemicals compiled from literature, 35 are US Environmental Protection Agency's hazardous constituents, 42 are US Food and Drug Administration's harmful or potentially harmful constituents in tobacco products and smoke, and 20 are listed as both. Per hazard characteristics, we short-listed 81 chemicals that should be considered for future environmental impact and risk assessments, including tobacco-specific compounds (eg, nicotine, N'-nitrosonornicotine), polycyclic aromatic hydrocarbons (eg, chrysene), flavours (eg, (-)caryophyllene oxide), metals (eg, lead), phthalates (eg, di(2-ethylhexyl)phthalate) and flame retardants (eg, tris(4-methylphenyl)phosphate). IMPLICATIONS: Our findings documenting various hazardous chemicals in the e-liquids underscore the importance of awareness and education when handling or disposing of e-liquids/e-cigarettes and aim to inform strategies to prevent and reduce hazards from e-cigarettes. This includes any scenario where e-liquids can come into contact with people or the environment during e-liquid storage, manufacturing, use, and disposal practices. Overall, our study characterises the environmental hazards of e-liquid chemicals and provides regulators and researchers a readily available list for future ecological and health risk assessments.

Measurement of Oxidatively Induced DNA Damage in Caenorhabditis elegans with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry.

Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of >50 µg DNA using a minimum of 250000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (<10% contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5'-cyclopurine-2'-deoxynucleosides in a toxicologically important terrestrial organism.

Agglomeration of Escherichia coli with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard Caenorhabditis elegans Toxicity Assay.

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard Caenorhabditis elegans ( C. elegans) toxicity assay containing an Escherichia coli ( E. coli) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with E. coli cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on C. elegans growth and reproduction. When the ENPs were tested in two alternate C. elegans assays that did not contain E. coli, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays.

Impacts of Silver Nanoparticles on a Natural Estuarine Plankton Community.

Potential effects of metal nanoparticles on aquatic organisms and food webs are hard to predict from the results of single-species tests under controlled laboratory conditions, and more realistic exposure experiments are rarely conducted. We tested whether silver nanoparticles (Ag NPs) had an impact on zooplankton grazing on their prey, specifically phytoplankton and bacterioplankton populations. If Ag NPs directly reduced the abundance of prey, thereby causing the overall rate of grazing by their predators to decrease, a cascading effect on a planktonic estuarine food web would be seen. Our results show that the growth rates of both phytoplankton and bacterioplankton populations were significantly reduced by Ag NPs at concentrations of ≥500 µg L(-1). At the same time, grazing rates on these populations tended to decline with exposure to Ag NPs. Therefore, Ag NPs did not cause a cascade of effects through the food web but impacted a specific trophic level. Photosynthetic efficiency of the phytoplankton was significantly reduced at Ag NPs concentrations of ≥500 µg L(-1). These effects did not occur at relatively low concentrations of Ag that are often toxic to single species of bacteria and other organisms, suggesting that the impacts of Ag NP exposure may not be apparent at environmentally relevant concentrations due to compensatory processes at the community level.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

Effects and implications of trophic transfer and accumulation of CeO2 nanoparticles in a marine mussel.

Bivalves are hypothesized to be key organisms in the fate and transport of engineered nanomaterials (ENMs) in aquatic environments due to their ability to filter and concentrate particles from water, but how different exposure pathways influence their interactions with ENMs is not well understood. In a five-week experiment, we tested how interactions between CeO2 ENMs and a marine mussel, Mytilus galloprovincialis, are affected through two exposure methods, direct and through sorption to phytoplankton. We found that phytoplankton sorbed ENMs in <1 h. The exposure methods used did not result in significantly different mussel tissue or pseudofeces Ce concentrations. Approximately 99% of CeO2 was captured and excreted in pseudofeces and average pseudofeces mass doubled in response to CeO2 exposure. Final mean dry tissue Ce concentration (±SE) for treatments exposed to 3 mg L(-1) CeO2 directly was 33 ± 9 µg g(-1) Ce, and 0 ± 0, 19 ± 4, 21 ± 3, and 28 ± 5 µg g(-1) for treatments exposed to 0, 1, 2, and 3 mg L(-1) CeO2 sorbed to phytoplankton. Clearance rates increased with CeO2 concentration but decreased over time in groups exposed to CeO2 directly, indicating stress. These results show the feedback between ENM toxicity and transport and the likelihood of biological mediation in the fate and transport of ENMs in aquatic environments.

No Butts on the Beach: Aquatic Toxicity of Cigarette Butt Leachate Chemicals.

OBJECTIVES: Toxic pollutants leaching from littered cigarette butts (CB) raise environmental impact concerns. The US Food and Drug Administration (FDA) is required to assess the environmental impacts of its tobacco regulatory actions per the US National Environmental Policy Act (NEPA). METHODS: We determined the chemical constituents in CB leachate through analyses of 109 field-collected CB and literature compilation and characterized their ecotoxicity to aquatic organisms. RESULTS: One-third of the 98 identified CB leachate chemicals were very toxic and 10% were toxic to aquatic organisms due to acute and chronic toxicity. Polycyclic aromatic hydrocarbons, metals, phthalates, nicotine and volatile organic compounds were the most hazardous CB leachate chemicals for aquatic organisms. Of the 98 CB leachate chemicals, 25 are included in FDA's list of harmful or potentially harmful constituents in tobacco products and tobacco smoke. CONCLUSIONS: Our study quantifies CB leachate constituents, characterizes their ecological hazard and identifies chemicals of concern. Thus, it aids in evaluating the environmental impacts of tobacco products per NEPA requirements. These results provide important information for strategies to prevent and reduce CB litter (eg, awareness programs, litter laws enforcement), thereby reducing environmental hazards from CB toxicants.

Combining secondary ion mass spectrometry image depth profiling and single particle inductively coupled plasma mass spectrometry to investigate the uptake and biodistribution of gold nanoparticles in Caenorhabditis elegans.

Analytical techniques capable of determining the spatial distribution and quantity (mass and/or particle number) of engineered nanomaterials in organisms are essential for characterizing nano-bio interactions and for nanomaterial risk assessments. Here, we combine the use of dynamic secondary ion mass spectrometry (dynamic SIMS) and single particle inductively coupled mass spectrometry (spICP-MS) techniques to determine the biodistribution and quantity of gold nanoparticles (AuNPs) ingested by Caenorhabditis elegans. We report the application of SIMS in image depth profiling mode for visualizing, identifying, and characterizing the biodistribution of AuNPs ingested by nematodes in both the lateral and z (depth) dimensions. In parallel, conventional- and sp-ICP-MS quantified the mean number of AuNPs within the nematode, ranging from 2 to 36 NPs depending on the size of AuNP. The complementary data from both SIMS image depth profiling and spICP-MS provides a complete view of the uptake, translocation, and size distribution of ingested NPs within Caenorhabditis elegans.

Impacts of metal oxide nanoparticles on marine phytoplankton.

Information on the toxicity of environmentally relevant concentrations of nanoparticles in marine ecosystems is needed for informed regulation of these emerging materials. We tested the effects of two types of metal oxide nanoparticles, TiO(2) and ZnO, on population growth rates of four species of marine phytoplankton representing three major coastal groups (diatoms, chlorophytes, and prymnesiophytes). These metal oxide nanoparticles (NPs) are becoming common components in many industrial, household, and cosmetic products that are released into coastal ecosystems. Titania NPs showed no measurable effect on growth rates of any species, while ZnO NPs significantly depressed growth rate of all four species. ZnO NPs aggregated rapidly in seawater, forming particles >400 nm hydrodynamic diameter within 30 min, and dissolved quickly, reaching equilibrium concentrations within 12 h. Toxicity of ZnO NPs to phytoplankton was likely due to dissolution, release, and uptake of free zinc ions, but specific nanoparticulate effects may be difficult to disentangle from effects due to free zinc ions. A modeling approach based on a Dynamic Energy Budget (DEB) framework was used to estimate sublethal effects of the two NPs on phytoplankton populations. Concentrations that were estimated to have no effect on population growth (NEC) were (one standard error in parentheses) 428 (58) µg L(-1) ZnO for the diatom Skeletonema marinoi and 223 (56) µg L(-1) for Thalassiosira pseudonana. NEC could not be estimated for the other taxa but were within the range of 500-1000 µg L(-1). Our results suggest that effects of metal oxide NPs on marine organisms is likely to vary with particle type and organism taxonomy.

Efficient electrochemical degradation of multiwall carbon nanotubes.

As the production mass of multiwall carbon nanotubes (MWCNT) increases, the potential for human and environmental exposure to MWCNTs may also increase. We have shown that exposing an aqueous suspension of pristine MWCNTs to an intense oxidative treatment in an electrochemical reactor, equipped with an efficient hydroxyl radical generating Boron Doped Diamond (BDD) anode, leads to their almost complete mineralization. Thermal optical transmittance analysis showed a total carbon mass loss of over two orders of magnitude due to the electrochemical treatment, a result consistent with measurements of the degraded MWCNT suspensions using UV-vis absorbance. Liquid chromatography data excludes substantial accumulation of the low molecular weight reaction products. Therefore, up to 99% of the initially suspended MWCNT mass is completely mineralized into gaseous products such as CO2 and volatile organic carbon. Scanning electron microscopy (SEM) images show sporadic opaque carbon clusters suggesting the remaining nanotubes are transformed into structure-less carbon during their electrochemical mineralization. Environmental toxicity of pristine and degraded MWCNTs was assessed using Caenorhabditis elegans nematodes and revealed a major reduction in the MWCNT toxicity after treatment in the electrochemical flow-by reactor.

Counting Caenorhabditis elegans: Protocol Optimization and Applications for Population Growth and Toxicity Studies in Liquid Medium.

The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. However, standardized methods for counting nematodes in liquid culture do not exist despite the wide use of nematodes and need for accurate measurements. Herein, we provide a simple and affordable counting protocol developed to maximize count accuracy and minimize variability in liquid nematode culture. Sources of variability in the counting process were identified and tested in 14 separate experiments. Three variables resulted in significant effects on nematode count: shaking of the culture, priming of pipette tips, and sampling location within a microcentrifuge tube. Between-operator variability did not have a statistically significant effect on counts, even among differently-skilled operators. The protocol was used to assess population growth rates of nematodes in two different but common liquid growth media: axenic modified Caenorhabditis elegans Habitation and Reproduction medium (mCeHR) and S-basal complete. In mCeHR, nematode populations doubled daily for 10 d. S-basal complete populations initially doubled every 12 h, but slowed within 7 d. We also detected a statistically significant difference between embryo-to-hatchling incubation period of 5 d in mCeHR compared to 4 d in S-basal complete. The developed counting method for Caenorhabditis elegans reduces variability and allows for rigorous and reliable experimentation.

Redox-active nanomaterials for nanomedicine applications.

Nanomedicine utilizes the remarkable properties of nanomaterials for the diagnosis, treatment, and prevention of disease. Many of these nanomaterials have been shown to have robust antioxidative properties, potentially functioning as strong scavengers of reactive oxygen species. Conversely, several nanomaterials have also been shown to promote the generation of reactive oxygen species, which may precipitate the onset of oxidative stress, a state that is thought to contribute to the development of a variety of adverse conditions. As such, the impacts of nanomaterials on biological entities are often associated with and influenced by their specific redox properties. In this review, we overview several classes of nanomaterials that have been or projected to be used across a wide range of biomedical applications, with discussion focusing on their unique redox properties. Nanomaterials examined include iron, cerium, and titanium metal oxide nanoparticles, gold, silver, and selenium nanoparticles, and various nanoscale carbon allotropes such as graphene, carbon nanotubes, fullerenes, and their derivatives/variations. Principal topics of discussion include the chemical mechanisms by which the nanomaterials directly interact with biological entities and the biological cascades that are thus indirectly impacted. Selected case studies highlighting the redox properties of nanomaterials and how they affect biological responses are used to exemplify the biologically-relevant redox mechanisms for each of the described nanomaterials.

Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis.

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

Deposition of carbon nanotubes by a marine suspension feeder revealed by chemical and isotopic tracers.

Carbon nanotubes (CNTs) are one of the few truly novel nanomaterials and are being incorporated into a wide range of products, which will lead to environmental release and potential ecological impacts. We examined the toxicity of CNTs to marine mussels and the effect of mussels on CNT fate and transport by exposing mussels to 1, 2, or 3mg CNTsl(-1) for four weeks and measuring mussel clearance rate, shell growth, and CNT accumulation in tissues and deposition in biodeposits. We used metal impurities and carbon stable isotope ratios of the CNTs as tracers of CNT accumulation. Mussels decreased clearance rate of phytoplankton by 24% compared with control animals when exposed to CNTs. However, mussel growth rate was unaffected by CNT concentrations up to 3mgl(-1). Based on metal concentrations and carbon stable isotope values, mussels deposited most CNTs in biodeposits, which contained >110mg CNTsg(-1) dry weight, and accumulated about 1mg CNTsg(-1) dry weight of tissue. We conclude that extremely high concentrations of CNTs are needed to illicit a toxic response in mussels but the ability of mussels to concentrate and deposit CNTs in feces and pseudofeces may impact infaunal organisms living in and around mussel beds.

Accumulation and Toxicity of Copper Oxide Engineered Nanoparticles in a Marine Mussel.

Cu is an essential trace element but can be highly toxic to aquatic organisms at elevated concentrations. Greater use of CuO engineered nanoparticles (ENPs) may lead to increased concentrations of CuO ENPs in aquatic environments causing potential ecological injury. We examined the toxicity of CuO ENPs to marine mussels and the influence of mussels on the fate and transport of CuO ENPs. We exposed marine mussels to 1, 2, or 3 mg L-1 CuO ENPs for four weeks, and measured clearance rate, rejection, excretion and accumulation of Cu, and mussel shell growth. Mussel clearance rate was 48% less, and growth was 68% less, in mussels exposed to 3 mg L-1 than in control animals. Previous studies show 100% mortality at 1 mg Cu L-1, suggesting that CuO ENPs are much less toxic than ionic Cu, probably due to the slow dissolution rate of the ENPs. Mussels rejected and excreted CuO ENPs in biodeposits containing as much as 110 mg Cu g-1, suggesting the potential for magnification in sediments. Mussels exposed to 3 mg L-1 CuO ENPs accumulated 79.14 ± 12.46 µg Cu g-1 dry weight, which was 60 times more Cu than in control animals. Our results suggest that mussels have the potential to influence the fate and transport of CuO ENPs and potentially cause magnification of CuO ENPs in mussel bed communities, creating a significant source of Cu to marine benthos.

Accumulation and toxicity of metal oxide nanoparticles in a soft-sediment estuarine amphipod.

Estuarine and marine sediments are a probable end point for many engineered nanoparticles (ENPs) due to enhanced aggregation and sedimentation in marine waters, as well as uptake and deposition by suspension-feeding organisms on the seafloor. Benthic infaunal organisms living in sediments encounter relatively high concentrations of pollutants and may also suffer toxic effects of ENPs. We tested whether three heavily used metal oxide ENPs, zinc oxide (ZnO), copper oxide (CuO), and nickel oxide (NiO) were toxic to an estuarine amphipod, Leptocheirus plumulosus. We used results from 10-day laboratory bioassays to estimate potential demographic impacts of ENP exposure. We also evaluated fate and transport pathways of the ENPs in the experiments to elucidate routes of uptake and exposure. Dissolved Zn was found in sediment pore water and overlying water samples at 10 fold the concentrations of Cu or Ni, a pattern indicative of the relatively high dissolution rate of ZnO ENPs compared with CuO and NiO ENPs. Accumulation of metals in amphipod tissues increased with exposure concentrations for all three ENPs, suggesting possible exposure pathways to higher taxa. Amphipods accumulated ≤600 µg Zn and Cu g(-1) and 1000 µg Ni g(-1). Amphipod mortality increased with ZnO and CuO concentrations, but showed no significant increase with NiO to concentrations as high as 2000 µg g(-1). The median lethal concentration in sediment (LC50) of ZnO was 763 µg g(-1) and 868 µg g(-1) for CuO ENPs. Our results indicate that ZnO and CuO ENPs, but not NiO ENPs, are toxic to L. plumulosus and that ZnO toxicity primarily results from Zn ion exposure while CuO toxicity is due to nanoparticle exposure.

Impact of engineered zinc oxide nanoparticles on the individual performance of Mytilus galloprovincialis.

The increased use of engineered nanoparticles (ENPs) in consumer products raises the concern of environmental release and subsequent impacts in natural communities. We tested for physiological and demographic impacts of ZnO, a prevalent metal oxide ENP, on the mussel Mytilus galloprovincialis. We exposed mussels of two size classes, <4.5 and ≥ 4.5 cm shell length, to 0.1-2 mg l(-1) ZnO ENPs in seawater for 12 wk, and measured the effect on mussel respiration, accumulation of Zn, growth, and survival. After 12 wk of exposure to ZnO ENPs, respiration rates of mussels increased with ZnO concentration. Mussels had up to three fold more Zn in tissues than control groups after 12 wk of exposure, but patterns of Zn accumulation varied with mussel size and Zn concentrations. Small mussels accumulated Zn 10 times faster than large mussels at 0.5 mg l(-1), while large mussels accumulated Zn four times faster than small mussels at 2 mg l(-1). Mussels exposed to 2 mg l(-1) ZnO grew 40% less than mussels in our control group for both size classes. Survival significantly decreased only in groups exposed to the highest ZnO concentration (2 mg l(-1)) and was lower for small mussels than large. Our results indicate that ZnO ENPs are toxic to mussels but at levels unlikely to be reached in natural marine waters.

Uptake, accumulation, and biotransformation of metal oxide nanoparticles by a marine suspension-feeder.

A growing body of evidence indicates that some engineered nanoparticles (ENPs) are toxic to organisms that perform important ecosystem services in terrestrial and aquatic ecosystems. However, toxicity can be influenced by the biotransformation of contaminants, including ENPs, as it may alter the fate and transport of these substances. In turn, fate and transport can influence their bioavailability. To understand how biotransformation influences the fate and transport of ENPs in marine ecosystems, we exposed suspension-feeding mussels, Mytilus galloprovincialis, to two common nano-metal oxides, CeO(2) and ZnO, over a range of concentrations from 1mg L(-1) to 10mg L(-1), in a laboratory experiment. Mussels exposed to 10mg L(-1) accumulated 62µg g(-1) of Ce and 880µg g(-1) of Zn on a dry tissue basis but rejected 21,000µg g(-1) for Ce and 63,000µg g(-1) for Zn in pseudofeces. Scanning electron microscope evidence indicates CeO(2) remained as ENPs but ZnO did not after being rejected by the mussels. Mussels filtered most of the CeO(2) from the aqueous media, while a significant fraction of Zn remained in solution. Differences in ENP solubility affect ENP uptake, excretion, and accumulation in mussels. Our study highlights the potential role of marine suspension feeders in biotransformation of ENPs.