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Nanoplastics in focus: Exploring interdisciplinary approaches and
future directions

Julien Gigault, Mélanie Davranche

<figure><img src="image_1.png" /></figure>

PII: S2452-0748(25)00004-7

DOI: https://doi.org/10.1016/j.impact.2025.100544

Reference: IMPACT 100544

To appear in: NANOIMPACT

Received date: 26 September 2024

Revised date: 13 January 2025

Accepted date: 20 January 2025

Please cite this article as: J. Gigault and M. Davranche, Nanoplastics in focus: Exploring interdisciplinary approaches and future directions, *NANOIMPACT* (2024), https://doi.org/10.1016/j.impact.2025.100544

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© 2025 Published by Elsevier B.V.
# Nanoplastics in Focus: Exploring Interdisciplinary Approaches and Future Directions

Julien Gigault¹*, Mélanie Davranche².

1. Takuvik Laboratory, CNRS/Université Laval, 1045 Avenue de la Médecine, G1V 0A6
Québec QC

2. Géosciences Rennes, CNRS/Université Rennes, 263 av. Général Leclerc, 35000 Rennes,

Julien.gigault@cnrs.fr

Julien.gigault@takuvik.ulaval.ca
Abstract

Nanoplastics (NPs) are gaining increasing attention due to their widespread distribution and potential environmental and biological impacts. Spanning a variety of ecosystems – from soils and rivers to oceans and polar ice – NPs interact with complex biological and geochemical processes, posing risks to organisms across multiple trophic levels. Despite their growing presence, understanding the behavior, transport, and toxicity of nanoplastics remains challenging due to their diverse physical and chemical properties as well as the heterogeneity of environmental matrices. Currently, nanoplastics are often studied alongside microplastics as a single, homogeneous group, which obscures the nuanced behavior of NPs, particularly in terms of their colloidal properties and interactions within ecosystems. This perspective aims to highlight the critical gaps in nanoplastics research, stressing the importance for field studies and advanced detection/quantification methods to better capture their behavior across environmental interfaces. We advocate for a more integrated approach that account for the dynamic interactions between nanoplastics and surrounding biological, chemical, and physical environments, especially across key ecological gradients. Furthermore, long-term and transgenerational studies are essential to assess the chronic impacts of low-concentration nanoplastics exposure. Innovative and appropriate methodologies are needed to explore NP fate, transport, and toxicity in realistic environmental conditions. By combining advanced experimental tools, field studies, and ecological modeling frameworks, this paper outlines provides a roadmap for advancing our understanding of nanoplastics and their broader ecological impacts, ultimately shaping more effective environmental monitoring and mitigation strategies.
Introduction

Nanoplastics (NPs) have emerged as a significant environmental threat due to their global distribution and potential effects on environmental systems. As they spread through ecosystems, from soil, (Junhao et al., 2021; Wahl et al., 2021) to river and estuaries (Gagné et al., 2023; Hoemann et al., 2024) and finally oceans (Ter Halle, 2017) including polar ice (Materić et al., 2022), nanoplastics interact with a wide range of biological and geochemical processes, posing risks to organisms across multiple trophic levels. However, the complexity of nanoplastics' behavior, including their transport, transformation, and toxicity, remains poorly understood due to their physical and chemical properties as well as the heterogeneity of environmental matrices.

In several studies, NPs are often examined alongside the smallest microplastics, collectively referred to as MNPs, as though they represent a single entity with uniform properties. The group is defined as plastic particles or debris less than 5 mm present in the environment (Balakrishnan et al., 2019; Dube and Okuthe, 2023; Kumar et al., 2023; Matthews et al., 2021, 2021; Wang et al., 2024; Yang et al., 2023). Nanoplastics and small microplastics (MPs) differ not only in size but also in their fundamental properties. One of the most notable distinctions is that while microplastics tend to float, NPs behave like colloids (Julien Gigault et al., 2018; Gigault et al., 2021). These differences significantly drive their environmental behavior, transport pathways, and ecological impacts. As a result, assuming MPs and NPs as a single, homogeneous group introduces substantial biases. A key reason for this grouping is the ongoing difficulty in detecting and, more importantly, quantifying nanoplastics in organic matrices and environmental samples.

To accurately assess their behavior and distribution, NPs must be considered as a distinct group with unique characteristics and require specific detection and analytical methods.

Research on the eco-toxicological effects of nanoplastics has primarily focused on their interactions with individual organisms or specific environmental conditions. However, this reductionist approach often overlooks the complex biological and physic-chemical gradients that nanoplastics encounter in real-world ecosystems. For example, biological processes like migration and developmental stages can significantly influence how NPs affect organisms, while environmental factors such as salinity and organic matter content can modify the surface properties and reactivity of these particles. This creates a crucial gap in our understanding of how nanoplastics behave across diverse ecological contexts.
Addressing the impact of nanoplastics therefore requires an integrated, interdisciplinary approach that takes into account the diverse physical, chemical, and biological environments in which these particles exist. By considering the dynamic interactions between nanoplastics, organisms, and their surroundings environment, we can begin to unravel the full scope of their environmental impact.

In this paper, we propose some directions and perspective to more accurately assess the presence, the fate and toxicity of nanoplastics by leveraging novel and underexplored approaches and advanced experimental techniques, such as microfluidic systems and non-targeted analysis. Our goal is to highlight the need for innovative methodologies that can capture the complexity of nanoplastic interactions and offer a more holistic view of their behavior in ecosystems. Developing tools and frameworks capable of integrating biological, chemical, and physical factors, is the main way to advance our understanding of nanoplastics environmental fate, thereby informing more effective strategies for monitoring and mitigating their impact on ecosystems.

## The complexity of nanoplastics' toxicity assessment

Figure 1 illustrates, the pathways to determine whether NPs can alter living organisms. Based on available literature, the ecotoxicological impact of nanoplastics is often assessed on specific organisms at particular growth stages or ecological roles. These experiments typically simulate an environment with highly specific conditions, including the presence of NPs with particular compositions, shape and surface properties far from that observed in nature. However, He et al. (2023) highlighted that NP of irregular shapes can cause stronger toxicity in earthworms than spherical nanoplastics. Moreover, many studies focus on high NP concentrations not relevant regarding the environment to specifically observed toxic effect. These conditions are represented by a black dot in Figure 1.

From these unique conditions, the authors often extrapolate a (eco)toxic effect to all nanoplastics. However, these studies fail to consider both the biological gradients (such as trophic levels, juvenile to adult stages, transgenerational effects), the physic-chemical gradients of the environment (green circle) or nanoplastics (red circle) as well as the influence of
prolonged exposure (long-term). Consequently, defining the toxicity of nanoplastics based on
such limited conditions is highly uncertain.

As a future perspective, it is crucial to adopt an integrated and interdisciplinary approach to address the impact of nanoplastics. This requires considering the gradients, which are illustrated by trapezoidal shapes for each condition. It all begins with identifying potential sources of nanoplastics. Based on these sources, it is necessary to determine which organisms are most exposed and their specific characteristics (e.g., juveniles vs. adults, resident vs. migratory, trophic levels).

Within these gradients, nanoplastics can be categorized as either primary NPs, which are pre-manufactured polymer nanoparticles, or secondary NPs, which result from the degradation of macro- and microplastics in the environment. While primary NPs are more uniform in their physical and chemical properties, they are far less abundant by mass compared to secondary NPs. The pathways illustrated in Figure 1 apply to both anthropogenic and natural nanoparticles.

To identify relevant sources, it is essential to conduct field studies. These studies must focus on selecting sites capable of providing detectable quantities of nanoplastics across various environmental compartments, while also reflecting concentrations representative (ex. from 0.4 to 2 ng g⁻¹ of particles in water and up to 25 µg g⁻¹ of particles in soil) of those typically found in the environment. For continental surfaces, several key sources have been identified, including landfills, soils treated with organic amendments derived from household waste, and agricultural soils covered with plastic mulch (He et al., 2019; Khalid et al., 2023; Wahl et al., 2021, 2024b).

In estuarine and oceanic systems, the sea surface and sediment surfaces, where biological production is highest, are key interfaces to focus on. Organic matter quickly forms hetero-associations with nanoplastics (NPs), keeping them suspended in the water column or causing them to flocculate and settle on the ocean floor (Pradel et al., 2021a). These specific sites should facilitate the determination of relevant nanoplastics concentrations across different environmental compartments, as well as the pathways through which NPs reach biological targets. At these sites, it is critical to cross-reference faunal and floral biodiversity data with phenotypic (e.g., life history traits, performance traits) and physiological impacts (e.g., metabolism, energetic and redox status, endocrine and inflammatory responses). This approach must be complemented by nanoplastics characterization and quantification inside organisms within the trophic chain and the ecosystem to provide insights into ecotoxico-dynamic processes
and enhance our understanding of the biological dispersion of NPs. To date, relatively few studies have quantified the translocation factor of nanoplastics in barley under experimental conditions (Luo et al., 2022; Ryzhenko et al., 2024; Shi et al., 2024). While experimental studies provide valuable insights, they represent only part of the puzzle. To fully understand the long-term and transgenerational effects of nanoplastics exposure, field studies must be complemented by controlled mesocosm experiments that address confounding factors such as soil composition, trophic interactions, and species migration. Long-term studies, combined with fieldwork, are essential to elucidate the effects of chronic low-concentration NP exposure, and transgenerational research is particularly critical, as such exposure may impact multiple generations or having an impact only after several generations. The sources of NPs will also determine transportation and transformation pathways of NPs across highly reactive environmental interfaces (e.g. such as estuaries or the Arctic Sea ice), characterized by physico-chemical gradients (red trapezoid in green circle) that influence the molecular properties of NPs (blue trapezoid in red circle).

By considering the unique physico-chemical properties of nanoplastics, the gradients they encounter in their environments, and the distribution of organisms within these environments, we should better understand their fate and potential toxicity. For example, investigating the presence of nanoplastics in estuarine systems cannot be effectively done without considering the interplay between nanoplastic degradation pathways, salinity gradients, and the associated diversity of aquatic organisms inhabiting and moving through these environments. In a recent paper we published, we combined these physical and chemical transportation and transformation pathways with estuarine-specific benthic organisms using environmentally-relevant nanoplastics models passing through a salinity gradient (Arini et al., 2022). This approach will be crucial in the coming years to address this emerging issue.
<figure><img src="image_2.png" /><figcaption>Figure 1: Illustration of the main pathways for determining the toxic behavior of nanoplasics in the environment. While the black dot represents the traditional approach in the literature, we propose a new approach that involves integrating and interconnecting various physical, chemical, and biological gradients at each level (from ecosystem to molecular scales) to assess the full spectrum of potential effects of nanoplasics.</figcaption></figure>

A revolution needed in Experimentation of nanoplasics’ fate investigation

The transport of nanoplasics is a critical factor in understanding their migration from source environments. One of the main challenges in studying environmental matrices, such as soil and water, is the limited accessibility to the dynamic processes that influence the fate of nanoplasics and associated elements. While the migration of nanoplasics is primarily driven by macro-scale water flow and transport mechanisms, it is well-established that molecular-, nano-, and micro-scale interactions play a crucial role in shaping the environmental fate of nanoplasics and associated metals. The transport behavior of nanoplasics is determined by their intrinsic properties, such as composition, size, shape, surface characteristics, and the elements they carry, yet comprehensive data on these features are still lacking.

The current scarcity of quantitative data on both the migration of nanoplasics across
environmental compartments and the factors controlling this migration impedes the development
of reliable models for predicting their environmental fate. This knowledge gap is further
compounded by our limited ability to observe and understand the complex, dynamic processes
governing nanoplastic interactions with surrounding elements. In particular, understanding the
behavior of nanoplasics at the soil-water interface is crucial for assessing their potential for
mobilization, transformation and transport under environmental conditions. While macro-scale
processes remain important, it is essential to also investigate the molecular- and micro-scale
dynamics. This is particularly critical when considering the potential microbiological
transformation of plastics from the microscale and nanoscale down to oligomers and monomers.
To the best of our knowledge, there is no representative experimental investigation of such
degradation processes for nanoplastics. Furthermore, the kinetics of nanoplastics
photodegradation into oligomers remains unclear, primarily due to the lack of environmentally
relevant nanoplastics models.

Historically, most studies have focused on spherical polystyrene (PS) nanoplastics, neglecting
the complexity introduced by polymorphic nanoplastics. Pradel et al. highlights the need for
further exploration of these dynamics (Pradel et al., 2023). It was demonstrated that polymorphic
nanoplastics in presence of natural organic matter exhibit greater retention in porous media
compared to spherical nanoplastics, likely due to their heterogeneous shape rather than particle
concentration (Dong et al., 2020; Pradel et al., 2020). Similar observations were made for
nanoplastics and microplastics in polar ice, where spherical and polymorphic particles exhibit
contrasting behaviors when expelled from the ice with the brine (Pradel et al., 2021b). These
findings emphasize the need for more advanced approaches to study nanoplastics transport under
conditions that more accurately reflect natural environments.

Nanoplastics, once introduced into the environment, exhibit behaviors like other reactive
nanoparticles, quickly forming hetero associations with both natural and anthropogenic materials
present in their surroundings. Their surface properties make them highly prone to adsorption
onto or incorporation into environmental particles, organic matter, and living organisms.
Extensive literature demonstrates that both terrestrial and marine organic matter (Pradel et al.,
2023), such as humic substances, dissolved organic carbon, and other organic particles, readily
interact with nanoplastics.

But, micro and nano-processes that explain nanoplastics reactions in environment systems with
chemical species, organic matter, or microorganisms can only be observed over relatively short
timescales (e.g., milliseconds to seconds). Given the significant spatial and temporal variability
of these processes, it is very difficult, if not impossible, to observe and characterize them directly
in the environment. Therefore, representative laboratory experiments using micro- and
mesocosms that mimic natural conditions with different levels of complexity are required (Arnott
et al., 2021; Long et al., 2015).
However, while these systems are useful for studying large-scale behaviors in algae populations or larger objects (Long et al., 2015), they are oversized for biogeochemical studies involving small particles and living species in the environment (Arnott et al., 2021). In case of sinking particles investigation in marine system, a recent study showed that the mass loss due to the degradation dynamics of sinking particles is directly related to particle size, and the presence of flow around the particle, characterized by the Reynolds number (Re) and Sherwood (Sh) function (Alcolombri et al., 2021). For final particle radii ranging from 1 mm to 1 µm, with typical sinking speeds of 100 m d⁻¹ down to 0.1 mm d⁻¹, and the kinematic viscosity of water at room temperature, the particle’ Reynolds number ranges from 1 to 0.001 (Alcolombri et al., 2021; Julien Gigault et al., 2018; Venel et al., 2021). However, in classical and large-scale experimental designs (micro- and mesocosms), the Re is generally larger than 1,000 and can go up to 100,000.

To meet these dynamic conditions and understand the interactions at the nanoplastics and microorganism interface, the use of microfluidic tools allows investigating these interactions under realistic conditions (e.g., Reynolds number <1). Recent advancements have enabled the use of microfluidic devices to investigate nanoplastics behavior at the pore scale, addressing some of these complexities. Unlike traditional porous media column devices with opaque porous media that limit real-time observation, microfluidic systems offer a unique opportunity to visualize and monitor the aggregation, sorption, and interaction of nanoplastics with other elements under flowing conditions, making them much more representative of natural environmental processes. In recent years, thanks to new microfluidic technology, it has been possible to reproduce porous media (Pradel et al., 2024) or salinity gradient of estuarine systems (J. Gigault et al., 2018; Venel et al., 2021). With this approach, unlike batch experiments that show rapid and extensive polystyrene nanoplastics aggregation in a saline environment, it has been demonstrated that under flow conditions, polystyrene nanoplastics can hetero-aggregate in such a way that their stability is significantly increased over long periods (Venel et al., 2021).

While large-scale experiments do not allow for the coupling of measurement methods capable of detecting chemical phenomena on nanoplastics surfaces as small as a few nanometers (i.e., nm²) and over very short timescales (i.e., ns to ms). Microfluidic devices allow for in situ studies of nanoplastics reactivity with metals and flocculants, using advanced coupled or in-situ techniques such as Static Light Scattering (SLS) for size determination, synchrotron-based tomography and
high-speed microscopy for aggregation analysis, nano-resolved microscopy with the FlowCam
Nano for shape characterization, and X-ray Absorption Spectroscopy (XAS) for metal-binding
studies (Chaussavoine et al., 2020).

Microfluidic devices open new avenues for studying key processes such as sorption and aggregation under controlled flow conditions, bridging the gap between laboratory-scale studies and real-world environmental dynamics. Their ability to closely replicate environmental flow regimes makes microfluidics a powerful tool for unraveling the complexities of nanoplasics transport, providing more accurate insights into their behavior in natural systems. In parallel, the development of modeling frameworks to simulate nanoplasics-metal interactions will complement these experimental approaches, enabling the prediction of transport and retention patterns across multiple spatial scales. Together, these advancements hold great promise for enhancing our understanding of the fate of nanoplasics in the environment and offer critical insights into their broader ecological impact.

Challenges in Non-Targeted Analysis of Nanoplastics

Many publications explicitly use the term "nanoplastics" in their titles, implying that the findings presented apply broadly to all types of plastics, regardless of the matrix in which they are found. This term covers a wide range of polymers and hybrid materials, which may include various metallic additives, in either ionic or nanoparticle form, as well as organic compounds like phthalates and UV filters. In earlier work (Gigault et al., 2021; Gigault et al., 2018), we already initiated the discussion on classifying microplastics and nanoplastics based on size and shape. However, it is crucial to address the need to disentangle the presence, sources, behaviors, and impacts of nanoplastics, given the significant heterogeneity captured by the term "plastic." In analytical science, each analyte requires a specific approach tailored to a simple, well-defined matrix, necessitating customized sampling strategies and sample pre-treatment.

The first challenge is to develop new standards and models for nanoplastics, which are essential
for advancing quantitative methods and calibration curves in environmental matrices, as well as
for building databases that support both targeted and non-targeted analysis. Currently, spherical
polystyrene nanobeads are the most commonly used model, with some variations in shape (such
as bead or blackberry-like forms) (Mitrano et al., 2019; Pessoni et al., 2019). However, there is
an urgent need to produce nanoplastics from other polymers that are more environmentally
relevant, namely, i.e., polydispersed with sizes ranging from 1000 nm to 1 nm and an open, heterogeneous shape, exhibiting colloidal stability but aggregating depending on the physicochemical conditions (Julien Gigault et al., 2018; Ter Halle, 2017). While nanoplastics from PE and PP exist, their diversity remains limited. Some researchers have proposed methods to generate nanoplastics by milling polymers or plastics collected from the environment (Balakrishnan et al., 2019; Blancho et al., 2021a; El Hadri et al., 2020; Magrì et al., 2018; Mitrano et al., 2019; Pessoni et al., 2019). These methods enable the production of models from any polymer, including those containing additives, and better replicate the surface properties of environmental plastic debris compared to raw polymers.

Another challenge with producing nanoplastics from environmental plastics is the potential introduction of biases, such as the release of nanoparticles like TiO₂, which is commonly used as an additive in formulated plastics. Depending on their size, these particles may not be removed through ultrafiltration and can remain in nanoplastics suspensions, potentially interfering with results and complicating data interpretation. These nanoparticles can alter the behavior of nanoplastics in environmental and biological systems, making it necessary to either eliminate these contaminants or account for their influence.

In ecotoxicology, understanding the spread of nanoplastics requires visualization within organisms and tissues, often using techniques like X-ray, IR spectroscopy, or fluorescence. Currently, metal- or isotope-labeled (Au, Pt, Gd, 14C) or fluorescent PS nanobeads are widely used, but they are all spherical (Al-Sid-Cheikh et al., 2020; Marigliano et al., 2021; Mitrano et al., 2019; Smith et al., 2024), introducing bias in absorption mechanisms. Therefore, it is crucial to produce labeled nanoplastics in large quantities, with asymmetric shapes and surface charges that more accurately reflect those formed under environmental conditions.

The second challenge rely in the ability to identify and characterize nanoplastics among an extreme heterogeneity of molecular compounds and elements and according to their multiple physicochemical properties (size, shape, surface, structure, composition, stability) (Gigault et al., 2021). Although many techniques exist for the qualitative analysis of nanoparticles (Ivleva, 2021; Mourdikoudis et al., 2018; Nguyen et al., 2019; Thomas et al., 2024), few are specifically suited for identifying, characterizing, and quantifying nanoplastics in the environment. Given the significant heterogeneity in the multitude of sources, types (carbon-based vs. metallic), behaviors (i.e. hetero-aggregation with natural organic matter), and physicochemical properties of
nanoplastics (size, shape, structure), only non-targeted analysis (NTA) approaches can provide a comprehensive and unbiased means of detecting a wide range of nanoplastics, including unknown or unexpected ones, which are not developed for addressing such issues.

Based on our knowledge on manufactured nanoparticles in the environment, the ideal non-targeted analytical approach begins with an initial step of sorting and characterizing various particle fractions by size, shape, and composition, ranging from a few nanometers to several hundred micrometers. This includes microorganisms containing nanoparticles and nanoparticles dispersed as colloids in environmental matrices such as water, ice, and sediments. Among these approaches, microfluidic techniques such as gravitational force coupled flow fractionation and asymmetrical flow field-flow fractionation have been pivotal to separate and fractionate those nanoparticles and microparticles based on their density and Brownian diffusion, respectively (Abdolahpur Monikh et al., 2019; Correia and Loeschner, 2018; Davranche et al., 2020; Gigault et al., 2017). The second step involves simultaneously quantifying and characterizing the physical properties (size, shape) and the molecular, elemental, or multi-metallic composition of these assemblies, either at the atomic scale as individual nanoparticles (using single-particle methods) or as microparticles associated with microorganisms (using single-cell methods). The ideal analytical strategy would relies on the coupling of the fractionation techniques (i.e. G3F and A4F) described above with: (i) size- and shape-based detectors, such as static- and dynamic-light-scattering (SLS, DLS) or single-particle-extinction-scattering (SPES) detectors, and (ii) elemental or molecular composition analysis using advanced mass spectrometry techniques, integrated with machine learning to classify and determine their mean elemental/molecular compositions and cluster composition (Baalousha et al., 2024). These advanced mass spectrometry techniques include single-particle- and single-cell- mass-spectrometry for elemental characterization of metal- or metal-oxide NPs used as additives in plastics or directly molecular identification of plastic using Pyrolysis-Gas-Chromatography Mass Spectrometry (Blancho et al., 2021b; Le Juge et al., 2023a; Okoffo and Thomas, 2024; Ter Halle et al., 2017). Indeed, in recent years, "single-particle" (SP) and "single-cell" (SC) methods, combined with high-resolution mass spectrometry techniques, machine learning and statistical approaches, have enabled the deconvolution of the presence of certain anthropogenic and natural nanoparticles in complex environmental matrices (Alshehri et al., 2023; Erhardt et al., 2019; Gonzalez de Vega et al., 2021; Holbrook et al., 2021; Wu et al., 2023). The coupling of these inorganic techniques
with methods specifically designed to detect nanoplastics (such as polystyrene, polypropylene, or polyethylene terephthalate) directly like Py-GC-MS or more recent thermo-desorption based mass-spectrometer would enable the identification and source-tracing of nanoplastics through their additives (Le Juge et al., 2023b; Materić et al., 2022).

Geochemical approaches remain significantly underutilized in nanoplastics tracing, even though they hold great potential for identifying sources, transport processes, and transformation mechanisms. Recently, we demonstrated the value of rare earth elements and lead in explaining the reactivity of nanoplastics, as well as strontium for tracing the presence and behavior of nanoplastics in soil (Blancho et al., 2024, 2022; Wahl et al., 2024a). Other elements, such as mercury and iron, could provide new insights on NPs behaviour. For instance, the mass-independent fractionation of mercury isotopes is influenced by specific physicochemical conditions like UV radiation (Rose et al., 2015) or microbial activity (Jackson et al., 2008; Janssen et al., 2016). Similarly, the structure, speciation, and isotopic distribution of metal compounds could help determine unique patterns of nanoplastic presence and diffusion in the environment. However, these approaches have yet to be fully explored for nanoplastics.

One approach that requires further development is Compound-Specific Isotope Analysis (CSIA) of $\delta^{13}$C, a powerful analytical technique used to study the isotopic composition of individual compounds in complex mixtures by measuring the ratios of carbon, nitrogen, and hydrogen isotopes in specific compounds, rather than in bulk materials. Recent studies have shown that $\delta^{13}$C can be specific to certain volatile organic compounds adsorbed onto plastics and can also differentiate between various plastic compositions. Birch et al. demonstrated differences in $\delta^{13}$C values between plant-derived and petroleum-based polymers, as well as distinctions based on the origin of the plastics (e.g., recycled vs. non-recycled) and their degradation pathways (e.g., photodegradation) (Birch et al., 2021). We have also shown that coupling a thermo-desorption purge and trap system to a GC and IRMS allows for the quantification and source identification of volatile organic compounds adsorbed onto microplastics (Le Juge et al., 2023b). As CSIA $\delta^{13}$C is widely used in environmental science, geochemistry, and forensics to trace the origins, sources, and transformation processes of both organic and inorganic contaminants, this technique presents a promising opportunity to enhance non-targeted analysis approaches for nanoplastics of their (bio)degradation products that are still not documented. As recently demonstrated with microplastics (Amaral-Zettler et al., 2020; Omura et al., 2024), bacteria secrete degradative
enzymes that cleave molecular chains of plastic materials and break them down into monomers and oligomers or even potentially fragmented them into nanoplastics. While this latter process is not yet demonstrated, we need to perform representative experimental studies focusing on the nanoplastics transformation induced by microorganisms to obtain new series of markers (monomers, oligomers) that could be implemented into the non-targeted analysis approach.

The final step of the non-targeted approach would be to aggregate all these data and develop machine learning methods to identify the nanometric nature of nanoplastics, detect their presence, and, most importantly, deconvolute their potential sources in various environmental compartments. Recent advances in the study of metallic nanoparticles, particularly through the development of single-particle ICP-Q-TOF-MS (Baalousha et al., 2024), offer new perspectives for nanoplastics. The development of new single-particle methodologies using carbon as the primary unit of measurement could help overcome the analytical challenges associated with plastics.

## Sampling Dilemmas and Innovations needed for tracking Nanoplastics in Environmental Matrices

Despite advancements in analytical methods for characterizing and quantifying nanoplastics at trace levels, the development of reliable sampling methodologies remains a significant gap. Prior research on engineered nanoparticles often prioritizes the characterization of individual nanoparticles dispersed in the environment. However, sampling techniques that effectively collect and concentrate nanoplastics without altering their physico-chemical properties are urgently needed. Moreover, among the organic components, microorganisms such as phytoplankton and bacteria play a significant role as natural collectors of nanoplastics. These organisms, through their biological processes, absorb or attach nanoplastics, forming complex hetero-associations. These interactions not only alter the size, shape, and behavior of nanoplastics but also pose major challenges to environmental monitoring and analysis. As nanoplastics transform from isolated particles into complex aggregates, they exhibit different physical and chemical behaviors. Consequently, sampling strategies must account for both free nanoplastics and those associated with organic matter, as traditional methods often fail to capture particles embedded within biological and organic matrices.
Table 1 summarizes the ideal sample quantities needed to detect nanoplastics using various analytical techniques employed to date. In most environmental samples, nanoplastics are present at very low concentrations, typically ranging from 0.1 to 10 µg L⁻¹, with the proportion of nanoplastics to organic matter varying between 0.01 wt% and 1 wt%. This disparity underscores the critical need for pre-concentration techniques to achieve detection limits in the 5-50 ng range, as required by methods like Pyrolysis-Gas Chromatography–Mass Spectrometry (Py-GC-MS). Depending on the sample type, the concentration factor necessary for accurately detecting and quantifying nanoplastics in environmental matrices can range from 5 to 1000. For instance, isolating 5 µg of nanoplastics from seawater may require 20 liters of sample, whereas soil or sediment samples may require 500 mg to several grams of material for detection using Py-GC-MS.

In soils and sediments, the challenge is twofold, involving both the soil itself and the (sub)surface runoff water. In both cases, sufficient volumes must be collected to extract detectable and quantifiable mass of nanoplastics. For soil waters, the main challenge is collecting enough solution across varying hydrological periods. This highlights the need for long-term collection devices, particularly those capable of gathering large volumes of soil solutions and runoff waters. Current soil solution collection systems, such as suction-cup methods and lysimeters, are well-established but have significant limitations (Grossmann and Udluft, 1991). One major issue is the interference with water flow through macropores, which leads to incomplete sampling. These systems can also alter samples through "filter effects," where colloids, and thus nanoplastics are retained. For surface runoff, erosion measurement devices equipped with sample collectors (e.g., GENEQ UGT) could potentially be used, but the collected volumes are small (around 1000 mL). Additionally, no devices currently exist for subsurface flow. Quantifying nanoplastics in soil waters is crucial to assess their potential for mobilization and transport to other environmental compartments. In soils and sediments, refining extraction methods is crucial. These methods must be specifically designed for nanoplastics, which often exist as aggregates or colloids. Unfortunately, nanoplastics are frequently grouped with microplastics under the term "MNPs," leading to the use of extraction methods designed for microplastics (Patil et al., 2022), which are not suitable for nanoplastics. For example, traditional density-based separation methods used for microplastics cannot be applied for nanoplastics. Additionally, nanoplastics in soils tend to form heteroaggregates, which must be broken down to
release measurable quantities (in mass or concentration). The use of deflocculating agent to break down soil aggregates and release nanoplastics is essential. Although this process could alter the surface properties of nanoplastics, it is a necessary step to recover quantifiable concentrations from soil matrices. Organic matter degradation techniques must also be adjusted to avoid damaging nanoplastics (Blancho et al., 2021a), which are more fragile than microplastics. It is also important to establish appropriate cutoff thresholds for nanoplastics recovery and to identify organic solvents capable of isolating a broad range of polymers with high efficiency, ensuring reliable quantitative analysis.

<table><caption>Table 1: Sample quantity required to detect nanoplastics using Pyrolysis coupled to GCMS according to the matrix of the sample and the method of sample treatment used.</caption><thead><tr><td>Matrix</td><td>Sample Quantity Required per Analysis (Pre-Extraction)</td><td>Collection &amp; Extraction Methods</td></tr></thead><tbody><tr><td>Nanoplastics freely dispersed in water</td><td>20-50 liters (freshwater or seawater)</td><td rowspan="2">Filtration using solid-phase filters or ultrafiltration (front/tangential) for liquid phase isolation</td></tr><tr><td>Nanoplastics hetero-aggregated with natural organic matter</td><td>1-20 liters (depending on carbon concentration: nmol C L<sup>-1</sup> to mmol C L<sup>-1</sup>)</td></tr><tr><td>Nanoplastics adsorbed or incorporated with microorganisms</td><td>20 µg – 1 mg of microorganisms</td><td>Filtration using phytoplankton nets to isolate nanoplastics integrated with microbial biomass</td></tr><tr><td>Nanoplastics in soils or sediment matrices</td><td>500 mg – several grams of soil/sediment (based on density)</td><td>Soil/sediment size sorting and density separation techniques to isolate nanoplastics from denser materials</td></tr></tbody></table>

Filtration remains the most used method for nanoplastics collection, but it often faces limitations, such as rapid clogging caused by natural particles, which reduces its ability to process large sample volumes. Additionally, the physico-chemical properties of nanoplastics may change during filtration, leading to potential analytical biases when characterizing the particles post-filtration. These challenges are particularly pronounced when using low cut-off filters, as they can alter the shape and size of nanoplastics, especially when associated with organic or inorganic matter. To mitigate these issues, advanced techniques such as tangential and frontal ultrafiltration have been developed (Wilkinson and Lead, 2007). These methods enable the concentration of colloids and nanoparticles on ultrafiltration membranes with very low cut-off thresholds (down to a few nanometers), even when processing large sample volumes. Tangential ultrafiltration offers the advantage of horizontal flow across the membrane, which minimizes clogging and
fouling. This technique is especially effective in recovering both freely dispersed nanoplasics in water columns and those that are hetero associated with organic and mineral matter. The size range captured can extend from several hundred nanometers to tens of micrometers, making it a versatile tool for nanoplasics analysis across a wide range of environmental matrices.

The challenge of sampling and characterizing nanoplasics in complex environmental matrices requires an integrated approach that combines advanced sampling strategies with robust analytical techniques. The hetero association of nanoplasics with organic matter, microorganisms, and other environmental particles necessitates specialized methods capable of isolating these particles without altering their properties. Techniques such as ultrafiltration offer promising solutions but must be tailored to the specific matrix and environmental context. As research progresses, the development of more precise and efficient sampling methodologies will be key to improving our understanding of nanoplasics' environmental behavior, fate, and impact.

## A Broader View of Nanoplastic Pollution

For over twenty years, the growing interest in manufactured nanoparticles and nanotechnologies has led the scientific community to question their potential presence in nature. It quickly became apparent that nanoparticles from these technologies are far less prevalent than those generated naturally or those produced accidentally by human activities. Ironically, the focus on nanoplasics has drawn attention to the need for the scientific community to investigate the presence, reactivity, and behavior of nanoparticles in the environment, especially those of anthropogenic origin. Behind the term "nanoplasics" lies a wide array of possible reactivities that must be understood to better assess the associated environmental issues. A more appropriate term might be "reactive anthropogenic nanoparticles", which includes not only nanoplasics but also other accidentally produced nanoparticles, such as mineral oxides (titanium dioxide, iron oxides) or refractory carbon particles (like black carbon or certain composite materials). These particles can interact with each other as well as with chemical, physical, and biological elements in their environment, including salts, organic matter, and microorganisms (phytoplankton, bacteria, viruses). Recent studies have shown that nanoplasics found in the environment also contain metal oxide nanoparticles (Alshehri et al., 2023). At a time when humanity is producing more materials than the Earth generates biomass (Elhacham et al., 2020), it is likely that these
reactive anthropogenic nanoparticles will compete with naturally reactive nanoparticles, even as
the latter continue to increase.

As illustrated in Fig. 2, among the significant sources of reactive nanoparticles that surpassing
nanoplastics, wildfires and wildland-urban interface fire play a major role, generating gigatonnes
of reactive particles that disperse into soils, air, and water over varying distances (from a few
meters to several tens of kilometers). These soot particles are actually aggregates of refractory
carbon nanoparticles, ranging from black carbon to carbon nanotubes (Lara-Romero et al., 2017),
including nanoplastics (Gigault et al., 2024), often associated with metallic nanoparticles
(Alshehri et al., 2023). These aggregates are highly reactive with trace metals as well as organic
pollutants like polycyclic aromatic hydrocarbons (PAHs) or dioxins (Guillon et al., 2013; Lopez
et al., 2023). Studies have also shown a strong correlation between PAH concentrations in the
Arctic and the presence of soot from wildfires (Silver et al., 2024). The consensus within the
scientific community is that nanoplastics may not be inherently toxic in trace amounts in the
environment, but they act as vectors for contaminants. This "colloidal vectorization" allows
contaminants trapped in one environmental compartment to migrate to others, a mechanism first
highlighted by McCarthy et al. in the 90's (McCarthy and Zachara, 1989), when they
demonstrated the role of colloids in the transport of trace elements in soils. In the context of
increasing human impact on ecosystems, focusing solely on nanoplastics as the primary source
of nanoparticle pollution is a narrow approach. To obtain a comprehensive understanding of
nanoparticle contamination, it is essential to integrate nanoplastics with other reactive
anthropogenic nanoparticles to better evaluate their relative presence and abundance in the
environment.
<figure><img src="image_3.png" /><figcaption>Figure 2: Global production of carbon in 2023 (in Million tons, Mt) from ashes induced by wildfire and plastics from pollution.
Sources: NASA (“CMS | Home,” n.d.) and Plastics Europe (“Plastics Europe • Enabling a sustainable future,” n.d.)</figcaption></figure>

## Conclusion

Nanoplastics represent a growing environmental concern, with their widespread presence in diverse ecosystems and potential to disrupt biological and geochemical processes. Despite the increasing number of research on microplastics and nanoplastics, significant gaps remain in our understanding of the behavior, transport, and toxicity of nanoplastics in real-world environments. Current approaches, which often treat nanoplastics and microplastics as a homogeneous group, fail to account for the unique properties and complex interactions of nanoplastics across different ecological contexts.

To address these challenges, the Table 2 summarizes the solution we propose. Given the existing gaps and limited knowledge, it is essential to develop advanced detection techniques and adopt integrated experimental approaches that reflect the complexity of natural ecosystems. By combining field studies with mesocosm and microcosm experiments, as well as utilizing novel technologies such as microfluidics, we can achieve more accurate assessments of nanoplastics' fate and impact. Moreover, long-term and transgenerational studies are crucial for understanding the chronic effects of low-concentration NP exposure on organisms and ecosystems.

Looking ahead, interdisciplinary collaboration and the development of innovative methodologies will be crucial for fully understanding the environmental behavior and ecotoxicological impacts
of nanoplastics. By building a comprehensive framework that integrates biological, chemical, and physical factors, we can better inform strategies for monitoring, mitigating, and managing nanoplastics in the environment, ultimately safeguarding ecosystems from the long-term effects of this emerging pollutant.

<table><caption>**Table 2: Summary of key scientific gaps and technical challenges related to the presence, fate, and impact of nanoplastics in ecosystems, along with proposed responses.**</caption><thead><tr><th>Key Scientific Gaps</th><th>Proposed responses</th></tr></thead><tbody><tr><td>Lack of data on nanoplastics distribution in environmental compartments</td><td>Conduct field studies tracking sources and transport pathways of nanoplastics</td></tr><tr><td>Limited understanding of nanoplastics' behavior, fate, and toxicity</td><td>Implement field studies that integrate different ecosystems and environmental conditions</td></tr><tr><td>Current models rely on standard nanoplastics that fail to represent real-world nanoplastics</td><td>Development environmentally relevant nanoplastics models for more accurate experimentation</td></tr><tr><td>Scarcity of data on nanoplastics reactivity and fate under dynamic, real-world conditions</td><td>Design experiments simulating dynamic environmental conditions from the nano-scale to the meso-scale</td></tr><tr><th>Key technical challenges</th><th>Proposed responses</th></tr><tr><td>Inability to identify and quantify nanoplastics in environmental samples</td><td>Development extraction-size fractionation methods, polymer identification techniques, and machine learning-based approaches with tracer integration</td></tr><tr><td>Limited ability to detect unknown or unexpected nanoplastics in complex matrices</td><td>Adopt non-targeted analytical approaches to detect a wide range of nanoplastics, including unknown particles</td></tr><tr><td>Current devices for studying sorption provide only macroscale data, missing nanoscale and nanoseconds mechanisms</td><td>Introduce microfluidic devices combined with on-line and in-situ characterization methods</td></tr><tr><td>Inability of current models to integrate sorption, kinetics and particle-particle interactions</td><td>Combine models for speciation (PHREEQ-C), aggregation (SINFONIA), and sorption kinetics</td></tr></tbody></table>
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September 25th, 2024,

To Editor of Nanolmpact

Dear Editor,

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Sincerely,
Julien Gigault, Ph.D.
CNRS Research Scientist
ULaval Associate Professor
TAKUVIK Laboratory
**Highlights**

1. Exposes critical gaps in nanoplastics research, calling for better interdisciplinary approaches.

2. Recommends microfluidics and non-targeted analysis to advance NP investigation presence and fate.

3. Emphasize the urgent need for long-term studies on nanoplastics' impact on ecosystems

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