Issues regarding environmental detection, uncertain concentrations, and unknown toxicity are not unique to ENMs; they are also raised for other emerging contaminants.However, because the ENM industry is rapidly evolving and scientists seek to assist in advancing environmentally safe nanotechnology, environmental relevance in ENM hazard assessment should be prioritized. To accomplish this, representatives from academia, industry, and government regulation working in ecotoxicology, exposure modeling, and social science Table S1 addressed four questions within this critical review: What exposure conditions are used in assessing ENM ecotoxicity potential for model organisms? What exposure and design considerations drive mesocosm experiments for assessments of ENM environmental hazards? What is the state of knowledge regarding ENM environmental exposure conditions, via measurements or modeling simulations? How should concepts such as exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? The main objective was to provide context and guidance to the meaning of environmental relevance in ENM environmental hazard assessment. This critical review addresses the four motivating questions and expands on detailed topics that emerged during the project . For each question, there are findings and recommendations. These serve to crystallize what is meant by environmental relevance in ENM ecotoxicology, and further coalesce ENM environmental exposure and hazard assessment endeavors.Many systems, approaches, and conditions have been used to assess ENM ecotoxicity.The applicability and challenges of standardized testing protocols under aquatic conditions have been reviewed and vetted in workshops.Also, the OECD reviewed and vetted its guidelines for testing ENMs in an expert meeting.Readers are referred to those reports for deliberations of ENM standardized testing. Research studies include laboratory-specific low- and high-throughput dose−response evaluations using select media with ENM compositional and receptor variants for assessing uptake and effect mechanism.Investigations include mechanistic gene transcriptional,DNA damage,dutch bucket for tomatoes metabolomics profiling,and transgenerational experiments.
Bottle-scale microcosms partially simulate limited levels of environmental complexity,for example, in using natural soils or sediments with or without plants and associated rhizosphere influences,or using seawater or marine sediments and associated receptors based on expected ENM compartmentalization.Nonstandard microcosms assess ecological end points, for example, microbial community composition and function related to C and N cycling.Environmental factors are examined, including how ENMs interactively affect soil water availability and soil bacterial communities.Single-species experiments that assess ENM bio-association or bio-accumulation precede and motivate using microcosms for assessing dual species trophic transfer and potential bio-magnification.Pristine ENMs, including those with surface functionalization, capping agents, or adsorbed species or coatings,are the most frequently assessed, although released and transformed versions are increasingly studied.Results for textiles, paints, and nanocomposites suggest that released particles significantly transform and age, and exhibit different environmental behavior and effects compared to pristine ENMs.Assessing changes in form and associated behavior or activity across the material’s life cycle are uniquely challenging. Nominal test exposure concentrations vary widely and are sometimes related to scenarios such as repeated applications,accidental spills,or ranges over a spatial gradient.High exposure concentrations may be used:for assessing bio-availability in soil in comparison to simpler media, or to accommodate analytical instrument detection limits. High concentrations are also used to establish no-effect limits, using limit tests; if an effect is not observed, the ENM is assumed to be nontoxic at lower concentrations,although effects could occur at longer exposure times. However, this approach is problematic if agglomeration and sedimentation of ENMs are concentration dependent, or if effect mechanisms do not scale with concentration, which might occur if organisms adaptively respond to toxicants. Some challenges to ENM ecotoxicology are familiar to conventional chemical ecotoxicology while others are unique. With conventional chemicals, toxicity is related to the effective dose of a toxicant molecule crossing the cell membrane and disrupting essential processes. However, ENMs can exert effects as particulates and in a molecular or ionic form depending on the dissolution extent in the medium.
The effective ENM dose which impacts the assay results may be unknown or changing because it varies with media conditions or with dynamic physicochemical interactions of receptors and toxicants.One effective dose metric may be insufficient to characterize an observed effect that could be related to both a physical interaction of the ENM particulate and the dissolved ionic form. Also, bio-availability and effective ENM doses change because ENMs can transform abiotically and biotically during assessment. While such influences on stability exist in conventional ecotoxicology, stability in a particulate exposure must also consider the ENM size distribution, and potential physical changes to the ENM such as dissolution and agglomeration. Therefore, effective and nominal doses may or may not be related. Although multiple ENMs may co-occur in product formulations, different ENMs are infrequently studied together or with cocontaminants.ENMs can conditionally sorb and modulate the toxicity and uptake of other contaminants and vice versa.ENMs can acquire coatings such as natural organic matter ,and age with varying pH and sunlight.Assessment outcomes are affected by media chemistry, physical characteristics, and additives . The exposure concentration at the receptor and consequent effects depend on ENM dissolution perhaps assisted by biotic ligands,and speciation,shed ions,surface associations, and heteroaggregation with colloids and particulate matter,r agglomeration and settling.All of these vary with ENM types and their varying properties, and ambient conditions.Changes in ENM properties may change bioavailability and toxicity.Various test durations and end points have been studied, from acute responses measured by standard test protocols to short-term microbial biodiversity and community composition effects or plant genotoxicity and nutrient composition changes.Multiple end points, toxicant characterization methods, and experimental controls increase assessment comprehensiveness and reduce artifacts and misinterpretations.Experimental controls based on coatings and dispersants enable determining if the apparent toxicity is attributable to the ENM itself or to ligand or surface groups. Yet whether and how experimental controls are used varies widely, for example metal salts that allow interpreting biological responses relative to ENM dissolution products versus intact ENMs.The appropriate analytical method for quantifying, locating and characterizing organism-associated ENMs depends on ENM chemistry and amounts or concentrations.
For ENMs exhibiting fluorescence or for ENMs containing heavy elements, high resolution microscopy can assess biological uptake and compartmentalization.X-ray synchrotron methods can sensitively locate bio-accumulated metal oxide ENMs and their transformation products in biota.No single analytical tool is suitable for all ENMs; however, the general lack of methodologies that can be routinely implemented to quantify ENM exposure in complex matrices continues to be a major challenge to the fundamental understanding of ecological effects. Alternative testing strategies can simultaneously assess many material types, controls, and concentrations,which is useful for ENMs not available insufficient quantities for microcosms. However, the potential for interferences in screening assay performance, depending on the specific ENM properties and toxicity test conditions,are increasingly recognized, and therefore should be controlled or accounted for in study designs.To date, ENM ecological hazard assessments have not adequately explored numerous well-conceived and plausible exposure scenarios that are founded in theory, hypothesis,blueberry grow pot mechanism and occurrence probability; yet scenarios increase certainty and predictability when addressing nanotechnology related material safety. For instance, hazard assessments have been mainly skewed toward as-produced ENMs without full consideration of potential aging, since aging cannot be fully standardized in a realistic context. However, such studies can and should further be conducted. Biological uptake, and the compartmentalization and speciation of ENMs, are still infrequently studied, limiting possibilities for attributing ENM exposure to effects at biological receptors.Varying degrees of rigor have been applied in designing and incorporating controls that are relevant to experimental questions or hypotheses. Varying degrees of attention are paid to experimental artifacts. While great advancements have been made in ENM ecotoxicology, improvements are needed to increase the environmental relevance of future research.To understand hazards for plausible exposure initiation scenarios, assessment conditions will need to depart from standardized testing protocols.It may be helpful to link or compare data obtained for ENMs under plausible scenarios to those obtained with standard methods using standardized media to facilitate interpreting complex multivariate experiments or comparing results among multiple laboratories. Herein, several recommendations regard exposure conditions that should be used in assessing ENM ecotoxicity .Soil ecotoxicity studies should specify the soil taxonomy and characteristics such as pH, clay content, and organic matter content, using standard methods.Similarly, characterization of sediments should be provided. Natural soils and sediments are preferred, because artificial media do not harbor natural soil communities, and do not reflect chemical and physical characteristics that influence ENM effects and bio-availability. Water characteristics determine ENM agglomeration, dissolution, and other behaviors affecting aquatic system compartmentalization and hence need definition. When adequately documented, media characteristics can be used retrospectively to interpret conditional ENM bio-availability or effect mechanisms.ENMs in testing should represent the particulate material form relevant to the environmental scenario . ENMs should be fully characterized and their history should be adequately described to allow comparing between studies. For toxic coatings,coating identity and degree of coverage should be related to observed effects. Impurities in ENMs introduced during product synthesis and handling should be characterized, since they can sometimes account for apparent ENM ecotoxicity.Nominal concentrations should scale according to exposure scenarios, or to specific objectives such as mechanistic research or quantifying biotic uptake.
Dose verification, including size distribution and ENM concentration, is also desirable, although heretofore challenging in soil and sediment exposures. ENM physicochemical changes during release and in the environment should be studied to uncover properties of ENMs that reach biological receptors. All potential forms of ENMs, including transformation products and residual reagents used in synthesis, should be accounted for, such that all toxicants can be related to biological responses.Study designs should anticipate dispersing agent effects and the nature of transformed ENMs plus cocontaminants, by including controls to account for effects, fate, and kinetics of ENMs in the test medium.The ENM physicochemical states should be understood before conducting hazard assessments.This is important because some ENMs agglomerate or dissolve or otherwise change in laboratory media, resulting in nonuniform exposures and uncertain bio-availability. Such unevenness precludes relating measured effects to the applied dose. Spatial bio-association, bio-accumulation,and intraorganism compartmentalization should be assessed to locate ENMs and their components.Important advances have been made toward characterizing the physicochemical factors influencing ENM behavior in environmental and test media, and toward utilizing that information to develop standardized methods for conducting ENM ecotoxicity testing.However, aquatic or terrestrial species and even different species belonging to the same order respond differently to ENMs using the same tests. Thus, test species and end points should be carefully chosen to enhance the relevance of ENM ecotoxicity testing. Some complex interrelationships and dependencies between species comprising ecosystems have been described.However, focused research could rationally identify species for routine evaluations; likewise, the scientific rationale behind test species should be reported. Ecosystems are more complex than conditions of routine ENM ecotoxicity evaluations. Thus, research should define an optimal suite of test species and end points to determine the ecosystem response to a given ENM. In general, biological receptors should be chosen for expected exposures stemming from realistic exposure scenarios. For example, relatively insoluble ENMs may, depending on their density, size and agglomeration state, rapidly settle out of suspension and associate with aquatic sediments. In that case, initial hazard assessment could focus on benthic, rather than pelagic, receptor organisms.Conversely, for ENMs that rapidly dissolve under environmental exposure conditions, conventional ecotoxicological exposure scenarios may be applied and receptors chosen to assess dissolution product toxicity. However, ENM dissolution rates vary, and pelagic organisms can be more sensitive than benthic organisms.Thus, both ENM compartmentalization and form must be accounted for when choosing receptors.Multiple effects measurements should be applied to answer research questions.Mechanistically understanding overt toxicity is needed, which may require measuring more omics end points and choosing variables for developing mathematical models to predict toxicity at untested concentrations or conditions.Omics technologies can also identify potential modes of action that are conserved among different species. However, different scientific communities will have varying preferences in defining needs for omics-level investigations.Effects interpretation requires understanding the effective toxicant dose or other basis of impacts.For ENMs, the mass concentration basis of dosing may relate only partially to the effective applied dose, since biological effects often originate from surface interactions with receptors.Furthermore, ENMs are more complex than conventional chemicals because ENM shape, aggregation state and surface area may in- fluence toxicity.Thus, surface area applied has been suggested as a supplemental dosing metric.However, ENM surface area in suspension/solid media is not a straightforward assessment given that ENMs may aggregate with a size distribution that is affected by the medium in which they are dispersed.