Abstract

The degradation of mismanaged plastic waste in the environment results in the formation of microplastics (MPs) and nanoplastics (NPs), which pose significant risks to ecosystems and human health. These particles are pervasive, detected even in remote regions, and can enter the food chain, accumulating in organisms and causing harm depending on factors such as particle load, exposure dose, and the presence of co-contaminants. Detecting and analyzing NMPs present unique challenges, particularly as particle size decreases, making them increasingly difficult to identify. Moreover, the absence of standardized protocols for their detection and analysis further hinders comprehensive assessments of their environmental and biological impacts. This review provides a detailed overview of the latest advancements in technologies for sampling, separation, measurement, and quantification of NMPs. It highlights promising approaches, supported by practical examples from recent studies, while critically addressing persistent challenges in sampling, characterization, and analysis. This work examines cutting-edge developments in nanotechnology-based detection, integrated spectro-microscopic techniques, and AI-driven classification algorithms, offering solutions to bridge gaps in NMP research. By exploring state-of-the-art methodologies and presenting future perspectives, this review provides valuable insights for improving detection capabilities at the micro- and nanoscale, enabling more effective analysis across diverse environmental contexts.

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Introduction

Plastic is a versatile material with indispensable applications in daily human life; however, it is increasingly becoming a significant environmental problem due to mismanagement and non-degradability [1,2,3]. Critical issues arise when plastics break down into NMPs. Particularly, MPs are defined as particles in the range between 1 µm and 5 × 103 µm, while NPs refer to particles with nanometric dimensions (< 1 µm) [4]. Once these particles are dispersed in the ecosystems, they can be ingested by animals and accumulated in tissues, and ultimately enter the food chain [5]. Primary NMPs are intentionally manufactured for various applications, including nano/microbeads, pellets, industrial cleaners and personal care products [6]. In contrast, secondary NMPs are formed by the fragmentation of plastic items during their usage and especially after their disposal due to UV radiation, mechanical degradation and biodegradation [7].

Environmental degradation of MPs can be initiated by biotic processes (involving enzymes, other biomaterials, bacteria, fungi, etc.), abiotic processes (such as thermal oxidation, photo-oxidation, mechanical degradation and atmospheric oxidation), or a combination of both [8]. Photodegradation, particularly through UV light, can cause fragmentation and breakage of polymers chains, resulting in reactive byproducts [9]. NMPs pollution is ubiquitous, extending beyond urban areas to remote regions [10,11,12].

Therefore, quantifying and characterizing NMPs using cost-effective and straightforward methods is essential. The dispersion of NMPs in the environment can be driven by climatic factors such as wind and rainfall, leading to both wet and dry deposition in remote areas [13, 14]. The presence of NMPs in surface waters and groundwater is primarily determined by hydrogeological conditions and proximity to major pollution sources [15]. Despite ongoing research, many aspects of the global circulation of plastics and NMPs in the environment remain unclear [13]. NMPs can enter the body and interact with cells, potentially leading to toxic effects influenced by factors such as plastic size, dose, and exposure duration. At the nanoscale, polymers with a density of less than 1 g cm-3 can float on water, while diffusion and aggregation significantly influence their behavior. Organisms can colonize larger pieces of plastic, while NMPs can be coated with biomolecules from the environment commonly defined as ‘bio-corona’. Smaller NMPs can also undergo hetero-aggregation, becoming coated and adsorbed on other large particles or bacteria [16]. Additionally, they can adsorb and retain other compounds, acting as carriers that increase complexity and reactivity with living organisms [8, 17]. Detecting NMPs is particularly important for investigating their interactions with biological organisms, as smaller particle sizes increase the likelihood of penetrating biological membranes [18].

Despite numerous reports on the presence of NMPs, a standardized protocol for sampling, pretreatment, quantification and classification remains absent [19, 20]. Sample pretreatment is a critical step to minimize the presence of non-plastic particles and focus on NMPs detection. Current challenges include: (a) separating NMPs from organic contaminants and inorganic sediment; (b) avoiding false-positive and false-negative during quantification and classification; (c) establishing universally accepted protocols and guidelines. While existing techniques may be effective at the micrometer scale, their efficiency diminishes for smaller contaminations or at the nanoscale, often becoming more expensive and less reliable [21, 22]. Furthermore, the heterogeneity of environmental samples complicates characterization and detection of NMPs, frequently leading to systematic errors [22, 23].

Recent reports address generalized aspects of sampling and separation of NMPs [26,27,28]; however, they often lack critical discussions on advancements, challenges and innovative solutions, alongside practical research examples. Many literature reviews mainly focus on analytical tools, such as microscopy and spectroscopy, for NMPs detection. However, they often fail to provide insights into how these techniques can be improved. Discussions on significant research outcomes addressing these limitations are also limited [27,28,29]. This work aims to fill these gaps by providing a comprehensive and critical overview of the current state-of-art technologies for sampling, pretreatment, and analytical detection of NMPs. It highlights recent advancements, particularly nanotechnology-based approaches and combined spectro-microscopic techniques, where nanomaterials are effectively employed in separations methods. Finally, this review explores the role of artificial intelligence (AI)-based classification algorithms, which remain underexplored in the existing literature, despite their potential to enhance accuracy and efficiency in NMPs detection.

Sample preparation

Sediments, sewage sludge, air, water and biological samples collected from the environment often contain organic and inorganic substances that must be removed to enhance accuracy in detecting, quantifying and classifying NMPs. This challenge is compounded by the absence of global regulations defining environmental limits for these emerging pollutants, making standardized measurements and assessments particularly difficult [30].

Sampling methods advancements

Common methods for sampling surface water include the use of nets, filters, or sieves. These approaches are effective in highly contaminated environments where large quantities of MPs or mesoplastics are present. However, they may not provide a complete representation of the overall water quality [31]. Alternatively, raw water samples can be collected in glass bottles or jars and later filtered in the laboratory, particularly when the focus is on detecting smaller MPs or NPs. A sequential filtration process is often employed, progressively removing larger fragments to facilitate the final isolation of smaller particles.

Sampling groundwater samples is more complex than surface water due to the limited availability of accessible sampling points, such as springs, water wells or monitoring wells. Spring water can be directly collected at the spring’s mouth without perturbing the aquifer’s natural dynamics. In contrast, collecting groundwater from monitoring wells typically requires pumps or volumetric samplers, which can increase the risk of cross-contamination [32]. Another critical challenge in groundwater sampling is determining the minimum volume required to achieve statistically reliable results [33]. For aquifers with very low contamination levels, low-flow pumping systems connected to in situ filtration are particularly useful, as they allow for collection of hundreds of liters of groundwater. Conversely, in more contaminated sites, a few liters of groundwater can be collected using volumetric samplers [34]. While general methods for sampling soil can be adapted, there is a pressing need for standardized techniques specifically designed for the quantification of nanoplastics and microplastics in groundwater and other environmental matrices [35].

Airborne NMPs can settle due to gravity and be collected using containers, which can be connected to a funnel at top as a passive sample collector device [36]. Collecting indoor dust by brush deals with NMPs as well as other dust components mixture and hence other impurities must be separated from NMPs. While active sampling devices can be effective, they are not suitable for difficult-to-access areas, such as remote mountains. These types of devices are complex and expensive [37].

Separation and pretreatment advancements

After collection, samples must undergo pretreatment to prepare them for analysis. This process depends on the type of analysis to be performed and generally involves the separation, concentration and purification of the specimens. Pretreatment methods vary depending on the sample type but typically aim to isolate NMPs from other components in complex environmental matrices, such as soil particles, dissolved ions and molecules, and organic residues. Common strategies for pretreatment include size selection (via sieving), digestion (using oxidation, enzymatic digestion, or acid-alkaline digestion), density separation (with salt solutions), and filtration.

Separation is primarily achieved through density separation and filtration. Density separation often relies on flotation, but this approach is less effective for NPs because, at the nanoscale, buoyant forces are minimal, and particle density can be altered by surface fouling. Froth flotation, another separation technique, is generally unsuitable for plastics due to significant particle loss caused by bubble interactions [38]. The effectiveness of density separation also depends on the density of the floating solution. For example, raw water can only separate low-density plastics such as polystyrene (PS), polyethylene (PE), and polypropylene (PP) [39].

Solutions containing salts like NaCl, CaCl2, KHCO2 can reach densities between 1.2 and 1.3 g cm-3, enabling the separation of mid-density plastics such as some types of polyvinyl chloride (PVC), PS, and acrylic polymers [40, 41]. Higher density salts such as NaI [42], ZnCl2 [43], Na6(H2W12O40) [44] can produce solutions with densities ranging from 1.6 to 1.8 g cm-3, sufficient to suspend a broader range of plastics, including all types of PVC and polyethylene terephthalate (PET).

Preliminary sieving is typically used to remove larger materials from the sample, but is insufficient for complex sediments, as it can lead to artifacts related to particle polydispersity. The subsequent step usually involves the digestion of organic matter, often performed using the wet peroxidase method or Fenton’s reagent, which effectively removes organic residues to improve the accuracy of NMP analysis. Additional care is requested in case of use of Fenton’s reagent because the intense oxidative reaction can alter or destroy NMPs [45]. Although the wet peroxidase method does not affect the NMPs, some studies have shown that this method may alter or digest nylon (polyamide, PA) and low-density polyethylene (LDPE) [46]. Another approach in the organic matter digestion is based on enzymatic digestion. This method is cheaper but time-consuming; and enzymes may interact with other impurities present in sample limiting their efficacy [41].

Despite the challenges and drawbacks of existing approaches for sample pretreatment and separation, recent studies highlight promising developments, particularly for NPs, which are significantly affected by the aforementioned methods. This section discusses significant results achieved in separation processes, including filtration and centrifugation, as well as nanotechnology-based methods that incorporate novel nanomaterials. To understand the development in NMPs detection in a timely manner, Fig. 1 summarizes the technical evolution from 2000 to the present, illustrating the methods used to study NMPs and their effects on the environment and living organisms. It also highlights various analytical and detection tools for NMPs in different environments and organisms, illustrating the growing understanding and detection capabilities regarding the distribution and impacts of NMPs over time.

Fig. 1

Timeline of improvements in NMP detection

Filtration and centrifugation advancements

Centrifugation is one of the most feasible and cost-effective techniques for density separation of NMPs that is performed prior to filtration or other separation techniques to reduce sample mass and minerals or can be employed independently for the purpose of separation. Ideally filtration and centrifugation are most widely used techniques, demonstrating high removal efficiencies of 74% and 98%, respectively [47]. In this section, we will discuss recent advancements in these two techniques.

Advancements in filtration process through engineering filtration membranes enables a more feasible way for NMPs removal. However, filtration technologies are still unable to show great potential to reach a commercial level from lab scale studies. These challenges can be overcome through using improved pre-treatments, modifications in membrane or filter media, integration with other technologies such as coagulation, photocatalysis, advanced oxidation process, etc. Owing to these issues, Ali et al. [51] highlighted wide range of filtration technologies explaining various types of filters, advantages, limitations and their overall capabilities towards NMPs separation. Yang et al. [48] reported a separation and detection method by using membrane filtration (Fig. 2a) for standard PS NMPs at different sizes and concentrations (50 nm to 103 nm and 0.1 g L−1 to 10−7 g L−1). In this approach, silver nanowires were prepared by polyol method and self-assembled on a commercial quantitative filter paper (15–20 μm). During membrane filtration, it can be observed that 1 μm PS MPs are retained on the silver membrane. This method addresses the issue of separation and loss during sample transfer process by providing methodological support for low concentration NMPs detection. While the technique offers simple operation and high sensitivity, detecting trace amounts of NMPs remains a challenge. In another attempt, Juraji et al. [52] fabricated polyurethane-based (PU) electro spun composite membrane the separation of NMPs via filtration. However, since the pore size of composite membrane is 624 nm, it is difficult to separate NPs with small size. In parallel direction, Lepointe et al. [53] reported iron grafted cellulose fibers for removal of contaminants from wastewater. Figure 2b represents a schematic about separation of NMPs (size: 58 – 255 nm; concentration: 1.67–2.08 μg L−1) from tap water using micro/nano-porous membrane. In the first step, 0.45 μm glass fiber filter (borosilicate) is employed to separate large particles and bacteria, the filtration proceeds with Whatman Anopore (Al2O3) with pore size of 200 nm, 100 nm and 20 nm. Following the filtration process, NPs fractions are retained on the membrane due to electrostatic charge. Cleaning filters to remove NMPs by suitable chemical method without affecting membrane should be further optimized.

Fig. 2

Recent advances in filtration and centrifugation: a membrane made up of silver nanowire for NMPs filtration [48]; b NMPs separation from tap water [49]; c continuous flow centrifugation for NMPs [50]

Hildebrandt et al. [50] reported two continuous-flow centrifuges (scheme in Fig. 2c) combined with two titanium rotors having 300 mL sedimentation capacity for NPs (about 160 nm) separation. Upon recirculating water sample twice or thrice, retention efficiency of > 90% was obtained. Pd doped NMPs were used in this study to estimate efficiency of this approach. Applying two continuous flow centrifugations in a sequence of different speeds can allow density/size selective separation of NMPs in terms of colloidal fractions. Separation efficiency of NMPs was tested in presence of interfering agents such as plankton; and it was found that lower speed retains planktons while NMPs were retained in secondary speed actuation. Grause et al. [54] attempted to develop a protocol for MPs separation from agricultural soil using centrifugation. In this study, Fenton oxidation was employed to oxidize biological matter, and NMPs were efficiently separated from heat-altered soil by centrifugation, achieving a recovery rate of 94% wt. However, the method was found to be unsuitable for high-density polymers due to their reduced buoyancy.

Nanotechnology-based advancements

Nanotechnology can play a central role in water treatment as well as in detecting pollutants in the air. NMPs at the micrometer scale are relatively easy to separate, but their removal at the nanoscale presents greater challenges. Nanotechnology offers potential new strategies to enhance separation processes.

Modak et al. [55] reported chromium-based metal organic framework (Cr-MOF) for removal of PS NPs (size < 1 μm), where 96% removal efficiency, 800 mg g−1 were achieved as shown in Fig. 3a. Electrostatic interaction between PS NMPs and Cr-MOF is the significant mechanism for adsorption of NMPs on the surface of Cr-MOF. In the present study, the adsorption behavior of PS NMPs on Cr-MOF, synthesized by hydrothermal method, investigated across varying pH levels. The adsorption mechanism was elucidated based on experimental observations. Further regeneration and reusability were tested. The results indicated that the adsorption increased in acidic conditions, particularly between pH 2–5, but decreased between pH 5–10. At lower pH levels, enhanced electrostatic interaction is attributed to the negatively charged PS NMPs and positively charged Cr-MOF surface. In contrast, at higher pH levels, zeta potential of Cr-MOF approaches its isoelectric point, leading to particle aggregation, which reduces the number of available active sites and consequently decreases adsorption efficiency. In another related study as shown in Fig. 3b, functionalized magnetic nanoparticles were used to separate PS NMPs via a magnetic flow cell method [56]. Furthermore, Liang et al. [57] detected PS NMPs by using a contactless conductivity detector (C4D) made up of a glass microfluidic chip with controlled geometric parameters with a detection limit and a quantization limit of 0.25 μg mL−1 and 0.8 μg mL−1, respectively. An experimental emission parametrization was also developed through well order experimentation and aerosolization for understanding NMPs size, density and concentration impact in water.

Fig. 3

Unique strategies for NMPs separation using nanomaterials: a PS NMPs removal using MOF (metal–organic framework) from water [55]; b functionalized magnetic nanoparticles-based separation of PS NMPs with respect to optimization and magnetic flow cell method [56]

Recently, Liu et al. [58] demonstrated that mixing nanolayered double hydroxides (LDHs) with PDMS and epoxy resins and coating on PU sponges can maintain superhydrophobicity, electrostatic attraction property, capillary action and chemical bonding. Such properties demonstrated that this material can serve as efficient adsorbent for the removal of NPs in oil, offering both mechanical durability and excellent reusability. In a literature study, Wang et al. [59] critically discusses potential strategies for implementing bio-electrochemical systems (BES), i.e., bio-electrochemical systems to mitigate the inhibitory effects of NMPs during wastewater treatment procedures. This approach presents possibilities in electrochemical degradation of NMPs as well as attenuation via increased microbial metabolism in NMPs degrading bacteria. However, extensive research is still required, as this field remains in its infancy regarding the potential of BES for treating NMP-contaminated water.

Advances in current measurement methods

Microscopic methods advancements

Optical microscopy

Optical microscope uses a lens system to magnify small objects. The objective lens, with a short focal length of a few millimeters, creates an image of the object in an intermediate image plane, which is then captured by a detector [60]. Optical microscopy, despite its simplicity, offers several advantageous for detecting NMPs. Its ease of operation, accessibility, cost-effectiveness, non-destructive nature, and ability to provide real-time observations make it a highly effective tool for this purpose. It can efficiently scan large areas and detect large MP fragments. However, its main limitation is the resolution, around 200 nm, making it difficult to detect NPs smaller than that limit [61]. Optical microscopy cannot determine the chemical composition of particles, making it challenging to distinguish or conclusively identify different types of plastics [62].

Without fluorescence or labeling, differentiating NMPs from similarly sized particles like organic debris or minerals can be challenging, leading to misidentification. Staining with agents like Nile Red (NR), a lipophilic dye, helps distinguish NMPs by binding to hydrophobic substances and activating fluorescence under specific wavelengths. This preferential staining of plastic particles reduces the risk of mistaking them for non-plastic materials and aids in identifying smaller particles below the diffraction limit [63]. Nile red, for instance, was applied to detect NMPs in complex matrices and tissues from living organisms [64]. Unfortunately, Nile red can suffer from false-positive artifacts, as it can bind to other hydrophobic materials such as lipids, therefore requiring additional confirmation steps.

Further research will focus on improving the efficiency and selectivity of NMPS detection. Advanced staining techniques, including solvatochromic dyes that change color based on their environment, are being developed for NMPs. ATTO dyes (a class of highly fluorescent dyes from ATTO-TEC GmbH, Germany), such as ATTO 647N, have been used in fluorescence microscopy to label and track NMPs [65]. These dyes offer strong fluorescence signals, which help in the detection of small particles. A novel low-cost twisted intramolecular charge transfer-based staining agent has been developed for fluorescent labeling MPs [66]. This method provides a simple, cost-effective way to fluorescently label MPs. Co-staining with dyes like DAPI (4’,6-diamidino-2-phenylindole is a fluorescent dye used for nucleic acids staining) and propidium iodide can further improve specificity by differentiating particle types based on fluorescence properties [67].

Despite labeling and fluorescence, chemical identification of different polymers remains challenging since staining agents are not polymer-specific. Optical microscopy often requires manual counting, which is time-consuming and prone to error, especially without automated analysis tools. Its limited depth of field also complicates focusing on 3D structures in thick samples. However, optical microscopy is easily integrated with other techniques to enhance accuracy of detection.

Electron microscopy

Electron microscopy uses an electron beam for high-resolution imaging. Scanning electron microscopy (SEM) reveals surface texture and morphology of MPs, while transmission electron microscopy (TEM) provides ultra-high-resolution images of internal structures at the nanometer scale. Both techniques are crucial for investigating the physical properties of MPs and frequently serve as complementary methods to spectroscopy for chemical identification [68].

SEM offers high magnifications and resolutions, allowing observation of fine surface details like texture, defects, and contamination in MPs. When combined with energy-dispersive X-ray spectroscopy (EDS/EDX), SEM can provide elemental composition data, identifying additives or contaminants. However, EDS mapping cannot distinguish between different polymer/additives types in complex materials [[68](https://link.springer.com/article/10.1186/s12302-024-01044-y#ref-CR68 “Silva AB, Bastos AS, Justino CIL, da Costa JP, Duarte AC, Rocha-Sa