Optical Interaction Patterns in a Microplastics and Nanoplastics Urine Test: Cross-Donor Consistency of Aged Decentralized Human Urine Samples

Abstract:

Human urine represents a promising non-invasive matrix for scalable longitudinal monitoring of environmental microplastic and nanoplastic (MNP) exposure. However, biological matrices introduce substantial variability related to donor chemistry, hydration status, baseline coloration, biochemical composition, and sample aging. These challenges are amplified in decentralized workflows involving real-world shipping and delayed processing.

This technical note describes preliminary observations demonstrating cross-donor consistency of concentration-dependent optical interaction morphology in aged decentralized human urine samples evaluated using the EcoExposure™ optical interaction assay. Three independently collected urine samples that underwent approximately 7–10+ days of decentralized storage and shipping prior to processing nevertheless demonstrated recognizable and internally consistent organizational states after standardized assay processing.

Importantly, the observed interaction behavior remained conceptually consistent with prior experiments in filtered water, saltwater, and mixed microplastic/nanoplastic systems. These findings support the possibility that transferable interaction-state dynamics may persist across multiple matrices despite substantial biochemical and operational variability.

This paper is also available at:

https://doi.org/10.5281/zenodo.20112394

Background

Microplastics and nanoplastics have been identified in a growing number of biological matrices including blood, urine, and tissue. Scalable approaches for decentralized exposure monitoring remain limited by concerns regarding matrix variability, contamination, storage conditions, and operational robustness.

Urine is particularly challenging because baseline appearance and composition vary substantially between individuals. Additional changes occur after collection, including oxidation of urobilinogen to urobilin, mild bacterial metabolism with ammonia generation, minor evaporation, and progressive baseline darkening. These effects are expected to become more pronounced during decentralized shipping and delayed processing workflows.

Despite these challenges, preliminary observations from the EcoExposure™ urine test, an optical interaction assay. suggest that concentration-associated organizational behavior may remain interpretable even in stressed biological matrices.

Experimental Overview

Three decentralized participant-collected urine samples were evaluated. All samples:

• underwent approximately 7–10+ days of decentralized storage and transport,

• were stored in steel collection containers,

• and were processed after delayed retrieval.

Samples were diluted using a standardized protocol and the plant-based interaction reagent was added, followed by standard optical imaging at 15- and 30-minute timepoints.

The experiments utilized heterogeneous fragmented multi-polymer microplastics (rather than idealized monodisperse polystyrene spherical particles) at a range of prespecified concentrations designed to be realistic and biologically relevant (≤100 MP/L).

Cross-Donor Consistency

Despite donor-to-donor biochemical variability, expected oxidation-associated darkening, prolonged storage, decentralized handling, and aged-sample conditions, the assay demonstrated notable preservation of concentration-associated interaction morphology across all three donors. Representative organizational states remained recognizable despite substantial baseline matrix variability. These observations suggest that donor-specific matrix differences may modulate reaction kinetics without abolishing the broader interaction-state structure.

Figure 1. Cross-donor comparison of 100 microplastic particles/L conditions in aged decentralized urine samples at 30 minutes.Representative photographs from three independent donor urine samples evaluated approximately 30 minutes after assay initiation under the 100 microplastic particles/L condition. Samples had undergone decentralized storage and transport prior to processing. Despite baseline donor-to-donor differences in urine coloration and expected aging-associated biochemical changes, recognizable optical interaction organization remained observable across all samples. These findings further support that baseline urine color variation alone does not abolish interpretable optical interaction analysis following standardized dilution and grayscale-compatible imaging workflows.

Consistency with Prior Matrix Observations (e.g. saltwater, tap / environmental samples)

One of the most important findings was that the interaction morphology observed in aged urine samples remained conceptually consistent with prior experiments performed in filtered water, saltwater, filtered urine, raw urine, and mixed microplastic/nanoplastic systems.

The recurrence of these organizational states across chemically distinct matrices suggests that the observed behavior may reflect transferable interaction-state dynamics rather than purely matrix-specific artifacts.

Multi-Polymer Fragmented Particle Relevance

The experiments did not rely on idealized monodisperse bead systems. Instead, heterogeneous fragmented particles with irregular morphologies, mixed polymer compositions, and broad size distributions were used. This improves translational relevance because real-world environmental and biological exposures are highly heterogeneous. The persistence of recognizable interaction regimes despite this particle heterogeneity further supports the robustness of the interaction-based analytical framework.

Operational Stress-Test Significance

The evaluated samples represented intentionally stressful operational conditions relative to intended product workflows. In planned decentralized observational studies and future consumer use, participants will collect urine, perform dilution (by adding the diluent provided), add reagent, and capture images in real-time or within minutes (perhaps hours) of collection. By contrast, these samples experienced prolonged storage 7-10 days+ before analysis), decentralized shipping, delayed retrieval, biochemical drift, and oxidation-associated changes prior to processing.

The preservation of interpretable interaction morphology under these stressed conditions provides preliminary support for the robustness of decentralized workflows.

Table 1. Comparison of Intended Use Conditions vs. Extreme Decentralized Stress-Test Conditions

Intended / Proposed Use Condition	Tested Extreme AgedUrine Sample	Notes / Implications for Robustness
Decentralized at-home or research-office workflow with assay performed within minutes to hours of urine collection	Approximately 7–10+ days of decentralized storage, shipping, and delayed retrieval in steel containers	Represents an extreme operational stress-test condition relative to intended product use. Planned observational and decentralized workflows are expected to use fresh urine processed shortly after collection.
Relatively stable fresh urine biochemical environment	Oxidation of urobilinogen to urobilin, mild bacterial metabolism with ammonia generation, possible minor evaporation, baseline pigment darkening, and occasional sediment/debris formation	Despite expected aging-associated biochemical drift, concentration-dependent optical interaction patterns remained distinguishable and internally consistent across donors following standardized dilution.
Minimal storage-associated matrix alteration expected during routine use	Extended storage interval prior to assay processing	Standardized dilution with urine-mimic saline, grayscale-compatible imaging, and interaction-based optical analysis appeared to mitigate sensitivity to baseline color variation and moderate matrix aging effects.
Controlled same-day assay workflow anticipated in routine deployment	Heterogeneous donor-to-donor biochemical variability amplified by aging and decentralized handling	Cross-donor consistency was still preserved across concentration regimes, supporting preliminary robustness of the assay framework under real-world decentralized conditions.

Interpretation

The combined findings suggest that baseline urine color variability, oxidation-associated darkening, mild biochemical drift, and donor-specific matrix differences did not abolish interpretable interaction-state behavior under the assay conditions evaluated. Instead, concentration-associated organizational states remained recognizable across donors and conceptually aligned with prior non-biological matrix experiments. These observations support continued exploration of interaction-based multi-matrix optical frameworks for scalable environmental exposure monitoring.

Limitations

This technical note represents preliminary exploratory observations and not formal analytical validation. Limitations include small donor count, decentralized uncontrolled storage conditions, absence of formal stability controls, qualitative morphology interpretation, and exploratory interaction-state analysis. Additional controlled longitudinal studies remain necessary.

Conclusion

Preliminary observations from aged decentralized urine samples demonstrated notable cross-donor consistency of concentration-associated optical interaction morphology despite substantial biological and operational variability. Importantly, these organizational states remained conceptually consistent with prior filtered-water, saltwater, and mixed microplastic/nanoplastic experiments, suggesting the possibility of transferable interaction-state dynamics across multiple matrices. These findings support continued development of decentralized multi-matrix optical interaction assays for scalable environmental exposure monitoring of microplastics and nanoplastics.