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.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. Rapid screening assessments should be prioritized within a testing strategy . 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,roll bench 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 influence 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. In addition, coatings, either on pristine ENMs or acquired in the test media or environment, may alter toxicity.ENM amounts and forms effecting biological impacts should be understood and related to the administered dose to inform environmental risk assessment.This is the essence of dosimetry in ENM ecotoxicology. As with other exposure concerns related to hazard assessment, appropriate dose measurement depends on receptor and ENM characteristics, which are scenario dependent. For example, mammalian cells are harmed by ENMs that become internalized, yet uptake pathways depend on ENM characteristics.Then again, bacterial receptors that affect ecosystem level processes may be impacted by externally associated ENMs at the cell membrane, or even in the surrounding environment. In those cases, dosimetry relies on understanding ENM behavior in the complex media in which bacteria reside , which is scenario driven. End point observations of ENM damage will also depend on ENM processing in cells. During hazard assessments, understanding the history of biological interactions with internalized, or otherwise associated ENMs may not be feasible. Yet efforts should be made to measure and spatially associate ENM bio burden within biological receptors, and to examine the relationships of applied ENMs to apparent effective dose and to effects.Overall, it is not recommended to categorically exclude select conditions, environmental compartments, protocols, receptors, or end points, since any may be environmentally relevant.

Rather, careful experimental designs around well conceived, plausible exposure scenarios should be emphasized; also, ENM characteristics that influence biological responses under the dynamic conditions that occur in the environment and in biota should be characterized and quantified. One could imagine identifying key material environment system determinants that could be systematically varied to provide test results across relevant determinant ranges. Such ideas are not specific to ENM ecotoxicology, but could establish defensible practices for making progress in hazard assessment while the ENM industry rapidly advances.Mesocosms are “enclosed experimental systems [that] are intended to serve as miniaturized worlds for studying ecological processes.” While the distinctions between mesocosms and other experimental systems are not well delineated, mesocosms are generally larger experimental units and inherently more complex than benchtop microcosms or more simplified laboratory experiments.Mesocosms for ENM ecotoxicology are intended to increase the complexity of experimental systems, such that more realistic ENM physical compartmentalization, speciation,and uptake into biota can be achieved alongside biotic effects.Also, the intent is to realistically characterize ENM fates and interactions with environmental system components, and to reveal fluxes among compartments of the ecosystems responsive to internal system influences that are unconstrained by investigator interventions.Mesocosms have been used for testing relative biotic effects of ENM variants,and discerning ENM effects separately from effects of dissolution products .Mesocosm testing may occur following individual organism and microcosm studies . For example, to study how ENMs impact crops, one could first establish the potential for hydroponic plant population impacts,use soil microcosms to understand ENM bio availability via observing soil microbial community shifts,and then scale up to greenhouse mesocosms of soil grown crops. This sequence could provide an understanding of plant−microbe interactions,ENM transformation and uptake in plants,commercial greenhouse supplies and effects on food nutritional quality.

Still, there are relatively few published studies using mesocosms to assess ENM ecological hazards,and the design and operating variables of existing mesocosm studies are wide ranging.By contrast, wastewater associated ENMs,and their transformations,effects, and fates in wastewater treatment plants ,along with the potential for ENMs to impact WWTP processes,have been more extensively studied. Since sewage contains ENMs, WWTPs are inherent forms of mesocosms.Studies at entire WWTP scales elucidate ENM fates during wastewater treatment, including significant association with biological treatment biomass that becomes bio solids.However, only 50% of bio solids produced in the U.S. are land applied, and these bio solids are used on less than 1% of agricultural land in the U.S. . Bio solids are land applied even less in the European Union.Thus, knowledge of ENM fates in WWTPs and how final residues are disposed regionally are needed to develop plausible exposure scenarios. Concerns with mesocosms include factors that can be difficult to control and that mesocosms may respond to artifacts including “wall” or “bottle” effects.Further, mesocosms can conflate direct and indirect toxicant effects, typically do not have a full complement of control conditions, and deliver inconclusive results . Biological communities in mesocosms also lack realistic ecological interconnections, interactions, and energy flows. Nevertheless, outcomes can be improved by using carefully designed mesocosms and associated experiments.For example, combined with analyzing mesocosm samples, performing practical “functional assays” such as for heteroaggregation,allows for anticipating phenomena and later interpreting ENM transformation and compartmentalization in mesocosms.Similarly, batch physical association experiments if conducted using realistic components, and over time frames that allow for quantifiable mass transfer can assess ENM biomass association and readily suggest ENM fates in WWTPs.Still, hydrodynamic conditions are different in simplified tests versus mesocosms, which are different from those in the natural environment. Hydrodynamic conditions will impact ENM fate and transport and thus exposure concentrations at receptor boundaries. The inability to capturereal environmental hydrodynamic conditions in any experimental scale is a general shortcoming for both ecotoxicology and transport studies.Although mesocosms do not fully simulate real environments,mesocosms are useful and should be employed, albeit judiciously due to their resource intensity, within a strategy . Recommendations regarding using mesocosms for assessing ENM environmental hazards are provided in Table 2.Mesocosm studies must be designed and conducted around well conceived questions related to plausible exposure scenarios; they should use select end points, potentially including sensitive omics measurements,to answer questions or test hypotheses.Internal process and constituent characterization should be thorough and equally responsive to well conceived, realistic scenarios. Functional assays, that is, “intermediary, semi empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems”,should precede mesocosm designs and experiments, and aid interpreting mesocosm results . Mesocosm artifacts are avoidable by following best practices for design and operation, although possible interferences of particulate material testing with assays must be evaluated.As for other hazard assessments, ENMs should be tested across the product life cycle, within a motivating exposure scenario. Similarly, suitable material controls should be used to test hypotheses regarding ENM specific effects .

The recommendations made regarding exposure conditions in assessing ENM hazard potentials for model organisms should be followed for mesocosm studies . Additionally, mesocosm designs should incorporate exposure durations, which should be sufficiently long to address population growth, reproduction, bio accumulation, trophic transfer, and possibly transgenerational effects. Sufficient measurements of ENM concentrations and time dependent properties must be made for clear interpretations. Key to successfully interpreting mesocosm studies is using validated methods for measuring ENMs in complex media. Measurements should include the size distribution, concentration and chemical composition of ENMs in the test system, including biological tissues,over time.In some cases, transformation products are inventoried thoroughly during long term field relevant exposures.Detection schemes require sample preparation to assess in situ exposures before quantitative analyses, or drying and embedding before visual confirmation by electron microscopy. The potential for artifact introduction should be recognized. Recovery methods continually develop, such as cloud point extraction for concentrating ENMs from aqueous matrices.Depending on the exposure scenario, in situ aging may be a study objective. However, it is important to define what “aging” really means and the specific application domain, since “aging” is a wide ranging term and can be used in different contexts, making comparisons impossible. At least, studies should be undertaken over sufficiently long time frames , which may include repeated ENM applications,such that appropriate aging, that is, time dependent transformation under realistic conditions, could occur. Alternatively, preaged ENMs could be used. However, preaging protocols are not yet standardized and, while some convention could allow for comparing across studies, the appropriate aging protocol would depend on the envisioned exposure scenario. Cocontaminants should be considered and potentially introduced into mesocosms, since some ENMs sorb, concentrate, and increase exposure to other contaminants.Select end points should account for ENMs as chemosensitizers.Also, mesocosm study designs should anticipate and plan for measuring secondary effects . In summary, while few mesocosms have been used in assessing ENM ecotoxicity and are also rare for conventional chemical testing, such systems potentially offer greater realism. Still, mesocosm exposure and design considerations should derive from immediate environmental applicability. The value of mesocosms to ENM ecotoxicology can increase by following recommendations including: addressing context dependent questions while using relevant end points; considering and minimizing artifacts; using realistic exposure durations; quantifying ENMs and their products; and considering ENM aging, cocontaminants, and secondary biological effects . Further, it should be acknowledged that mesocosms do not fully recreate natural environmental complexity. For example, aquatic mesocosms do not recreate actual environmental hydrodynamic, or temperature cycling, conditions. Hydrodynamics can significantly impact ENM aggregation or heteroaggregation, and fate and transport . Therefore, potential impacts on the resulting concentrations at the receptor boundaries should be considered.ENM environmental exposure conditions herein refer to where, how much, and in what forms ENMs may occur in the environment. These are central issues for ecotoxicology of ENMs because they suggest test exposure scenarios in which ENMs could impact biological receptors within environmental compartments influenced by various factors . These issues also influence outcomes of key regulatory concern: persistence, bio accumulation, and toxicity.Discharges underpin exposure scenarios,are initiated by situational contaminant releases , and are referred to as source terms. Mass balance based multimedia simulations mathematically account for released contaminants as they are transported and exchanged between environmental media, where contaminants may be transformed and may ultimately concentrate, potentially with altered compositions and structures . Far field exposure modeling approaches vary by question, the modeling purpose , the required spatial resolution , the temporal conditions , and the predictive accuracy required.Material Flow Analysis , which is a type of life cycle inventory analysis, has been advanced to track ENM flows through various use patterns into volumes released into broad environmental compartments,scaled to regional ENM concentrations that release via WWTPs to water, air, landfills, and soil.Such models estimate exposure concentrations in part via engineering assumptions and in part via heuristics.Also, such material flow analysis models depend on the underlying data which are not readily available, making it difficult to validate model results and potentially leading to inaccurate estimates.