TABLE 1:

Challenges and potential solutions for the eco-exposome assessment

Challenge	Description	Possible solutions
Comprehensiveness of chemical exposure assessment	In many cases, the identity of internal xenobiotic chemicals will be only partially known, hampering a comprehensive assessment because of methodological limitations such as lack of analytical standards, insensitivity of detection as a result of matrix effects, and masking of exogenous chemicals by endogenous chemicals (see “Endogenous vs. exogenous chemicals”).	Suspect screening may reveal the identity of more compounds of toxicological relevance. Matrix or masking effects from ions or macromolecules such as proteins may be avoided by using passive samplers to extract tissues or biofluids. Alternatively, proxies (e.g., external exposure assessment) may be used to infer internal exposure.
Endogenous versus exogenous compounds	Extraction of a biofluid, tissue, or whole-organism sample will yield both endogenous and exogenous compounds. The large number and often higher concentrations of endogenous compounds may mask the detection of compounds that have been taken up from the environment. This can be challenging for untargeted chemical analysis and may also influence results from bioanalytical assessment.	The use of surrogate environmental matrices or passive samplers (mimicking organisms) could help to differentiate between endogenous and exogenous compounds. By comparing sample extracts to extracts obtained from unexposed control organisms it may be possible to identify compounds of exogenous origin.
Formation of adducts	Reactive xenobiotic electrophiles can form adducts with biological macromolecules (e.g., DNA, proteins). These adducts are not captured by standard extraction protocols but are highly relevant with respect to biological responses (e.g., cancer, cytotoxicity).	Specific protocols are available to identify DNA or protein adducts and could be applied to complement standard extraction procedures.
Organ-specific assessment	It may not be feasible to analyze all relevant tissues in an organism. Furthermore, the assessment of internal concentrations in small organisms may only be possible in whole-body extracts.	Experience may indicate organs for which major differences in internal exposure can be expected, informing subsequent tissue sampling efforts. For small organisms, chemical imaging may provide information on tissue distribution
Exposure over lifetime	Albeit to a lesser degree than for external exposure, an individual sample taken for an exposome assessment represents a snapshot. Hence, exposure over the lifetime of an organism may be difficult to characterize if a limited number of “snapshot” samples are available. This would apply particularly to nonpersistent and/or hydrophilic chemicals, which may be rapidly excreted from an organism during recovery periods.	For organisms that have relatively short life cycles, fluctuations in external exposure over a relatively large time span may be less relevant than for long-lived organisms. Lipid-rich tissues may be useful for evaluating exposures to hydrophobic chemicals because these chemicals tend to remain in such tissues for a long period of time. Other chemicals with high persistence such as perfluoroalkyl substances may also be detected from snapshot samples in case of fluctuating exposures. Proper attention to sampling design and properties of targeted chemicals may increase the likelihood that limited samples adequately capture the nature of the exposure (see “Translation of the exposure event”).
Life-stage specificity	Organisms undergo complex transitions in their development, passing through windows of sensitivity with particular vulnerability to adverse toxicological effects. Exposure would only be relevant if it was present during such periods, and hence exposure and effect data may not match.	Trans-sectional sampling from populations could partially address this issue.
Species specificity	An exposome assessment inherently accounts for species differences in TK. Knowledge of TK could, however, be relevant for extrapolation between species. Current practice is the collection of data only for a limited number of species.	If the TK of a given chemical and organism are well understood, the toxicodynamics are conserved, and an appropriate TK model is available, extrapolation of chemical kinetics and effects from a tested species to untested species may be possible.
Modulation of internal exposure by diet, trophic level, the microbiome, parasites, habitat, and behavior (migration)	Diet, trophic level, the microbiome, habitat, parasites, and behavior can impact the translation of external exposures to internal concentrations. Diet and the microbiome may also impact the biotransformation of both endogenous and exogenous chemicals. Diet can be influenced by population structure, which may regulate access to food and exposure to certain chemicals.	These factors would be reflected in the assessment of internal exposure, but their individual impacts on the exposure would be unknown. For data interpretation and extrapolation among different species and environmental settings it would be useful to understand the impact of modulating factors on internal concentrations of exogenous chemicals. Controlled experiments could be conducted to study these impacts.
Endogenous chemicals changed by nonchemical stressors	Climate, nutrition, and habitat represent factors that may impact the levels of endogenous signaling chemicals and could confound identification of effects caused by exposure to exogenous chemicals.	Controlled experiments that study the impact of climate, nutrition, or habitat could help to identify changes in endogenous chemical levels unlikely to be caused by exposure to exogenous chemicals. Furthermore, tracers of diet and habitat (e.g., stable isotopes or fatty acids) may reveal changes in endogenous chemicals related to nonchemical stressors.
Translation of the exposure event	Temporal variations in exposure (e.g., peak runoff events vs. exposure to continuous discharge) may be differentially translated to internal concentrations, depending on the chemicals of interest. Particularly, peak exposure may be difficult to detect for nonpersistent compounds if samples are obtained outside of an ongoing discharge/event.	Sampling test species at different times and locations corresponding to different exposure situations would allow for a better resolution of fluctuating exposure.
Deriving population effects	The eco-exposome is based on an assessment of individual organisms. For environmental risk assessment, exposures and effects experienced at the individual level would have to be translated to the population level.	Translation of effects from individuals to populations is not a specific requirement in the eco-exposome assessment and is already part of the regulation of single chemicals. Existing models for translation to population levels could be combined with eco-exposome assessment.

TK = toxicokinetics.