Simple detection of polystyrene nanoparticles and effects in freshwater mussels: method development and in situ application to urban pollution
Abstract
The ubiquity of plastics in environments worldwide is raising concerns about their toxicity to organisms. The purpose of this study was to investigate simple means to determine the exposure and effects of nanoplastics (NPs) in the freshwater mussels Elliptio complanata (E. complanata). NP tissue levels were determined using a plasmonic nanogold sensor probe and effects were determined using the refractive index (RI) and thiol-reaction rates (TRR) in protein-dense tissue extracts. This method was adapted to quantitatively measure the concentration of NPs in tissues using a salting-out extraction in the presence of acetonitrile (ACN). Concentrated solutions of albumin were first spiked with NPs to evaluate changes in RI and TRR to determine crowding effects. The data revealed that NPs readily decreased the RI and TRR in albumin in vitro. These three simple assays were then applied on freshwater mussels caged for 3 months at various sites in a largely populated area. Mussels downstream of the city center and found at the street runoff discharge sites were highly contaminated by NPs and the RI and TRR were also reduced. In conclusion, simple and readily accessible assays to assess the NP contamination based on a visual nanogold sensor technology, and the effects of plastics are proposed for freshwater mussels.
Keywords
INTRODUCTION
Plastic materials are commonly found in many objects in our daily lives, such as clothing, personal care products, food packages, and drink containers. They provide numerous benefits in the fabrication of domestic and industrial products owing to properties such as their relative inertness, lightweightness, resistance to shock, and low cost of production[1]. Given the global and exponential consumption of plastics and the limited regulation of plastic waste disposal, it is estimated that five trillion fragments exceeding weights of 250,000 tons of plastics already found their way into oceans[2]. In the environment, plastics are gradually degraded into smaller particles termed microplastics (MPs, for sizes between 5 mm to 1 µm) and nanoplastics (NPs, for sizes between 1-1,000 nm[3]). The small size and high surface area of NPs make their analysis tedious in various environmental compartments[4,5]. While MPs are commonly analyzed using microscopic Fourier transform infrared, Raman spectroscopy, and thermo-analytical methods, NPs represent an ongoing challenge. At the moment, methods to detect NPs, which behave differently than MPs and dissolved contaminants, are lacking for organisms. Traditionally, methods used to study plastics involve specialized imaging techniques, separation by chromatographic/field flow techniques, and infrared spectroscopy. An important proportion of plastic materials are retained by municipal effluents and ongoing studies examine ways to remove and degrade plastic on-site[6]. These processes involve separation approaches such as absorption, filtration, and advanced oxidation to separate and degrade these materials at the wastewater treatment plant. The wide range of particle sizes adds to the complexity of global plastic contamination and raises questions as to the ability of the ecosystem to tolerate this pollution on such a large scale. In opposition to MPs, NPs can diffuse across cellular membranes and may perturb cellular physiology. A recent review highlighted that NPs are bioavailable and lead to oxidative stress, inflammation, and potentially cancers[7]. Hence the ability of NPs to diffuse across biological membranes and their ubiquity in the environment may pose risks to the sustainability of aquatic ecosystems and need to be further addressed.
The accumulation of NPs in crowded intracellular compartments can disrupt the complex fractal organization of protein networks[8,9], resulting in the loss of protein function and ultimately impair enzyme activity. These interactions involving hydrophobic surfaces of NPs and local ionic properties of proteins can result in the coating of various proteins [e.g., lysozyme, albumin, and lactate dehydrogenase (LDH)] and can lead to changes in the protein organization. During the aging of NPs, the hydrophobic surface is oxidized by UV or ozone-forming carboxylates at the surface, which can also add to the interaction with the hydrophobic and local positive charges of proteins[10]. The refractive index (RI) of proteins represents a simple and cheap analytical measure to determine the changes in protein concentration, conformation, and composition[11,12]. Changes in the RI per protein mass reveal changes in the conformation and composition of proteins at concentration scales approaching those found in cells
This study aimed to suggest simple and quick markers for the assessment of exposure and effects of PsNPs in aquatic organisms. For this study, we chose a freshwater mussel Elliptio complanata (E. complanata), endemic to the Saint-Lawrence River, as a sentinel species to reflect the NP contamination in various urban contexts. To assess the exposure of mussels to NPs, a newly developed plasmonic nanogold (nAu) sensor probe allowing the visual detection and quantitation of NPs, requiring only a cell phone camera and free application for color analysis[16], was adapted in biological tissues. Effects of NPs were determined by changes in the RI and thiol-reaction rates (TRR) first with concentrated solutions of albumin in vitro. We also tested these markers, along with neutral lipids, aldehydes, and amyloids, in the previously exposed
MATERIAL AND METHODS
In vitro effects of polystyrene nanoparticles
The influence of PsNPs on the RI and TRR in concentrated albumin was examined in vitro to highlight potential biophysical properties of interest in the development of NP biomarkers. A solution of bovine serum albumin (100 mg/mL; MilliporeSigma Canada Ltd., Oakville, Canada), which corresponds to the concentration range of proteins in eucaryotic cell cytoplasm[17] (100-400 mg/mL), was prepared in phosphate-buffered saline (PBS, 140 mM NaCl, 1 mM KH2PO4, 1 mM NaHCO3, pH 7.4). The aliquots of 200 µL were exposed to increasing concentrations of PsNPs (0, 0.05, 0.1, and 0.15 µg/mL; Polyscience Inc, Warrington, USA) for 5 min at RT. The PsNPs were uncoated spheres with a diameter of 20 nm, and dilutions were prepared in MilliQ deionized water to limit aggregation. The RI was determined in the albumin-rich solutions using PBS as blank (RI = 1.3326) using an electronic RI device (HDR-P5, Fisher brand, Canada). This preliminary experiment revealed that PsNPs alone did not influence the RI. Then, the TRR was determined by adding 20 µL of 1 mM 5,5’-dithiobis-2-nitrobenzoate (DTNB; MilliporeSigma Canada Ltd., Oakville, Canada) to 200 µL of albumin concentrate[18]. The absorbance was recorded at
Gold nanoparticle - mercaptoundecanoic acid assay
The detection of NPs was achieved using a recently developed nAu plasmonic sensor methodology initially designed for the visualization of NPs in drinking water and from the leaching of heated tea bags or other materials[16]. This methodology was adapted to biological tissues by introducing a preliminary PsNPs salting-out extraction step and recuperation with acetonitrile (ACN) as previously described[19]. The nAu suspension (mean diameter of particles: 14 nm) was commercially obtained from nanoComposix (San Diego, USA) as 50 µg/mL suspension containing 1 mM citrate. The nAu suspension (10 mL) was treated with 0.01% mercaptoundecanoic acid (MUA; prepared in ethanol 100%) for 12 h at RT in the dark to allow binding of the lipophilic acid MUA to nAu to form nAu-MUA complexes [Figure 1]. This solution was filtered through a 0.22 µm cellulose membrane and centrifuged (15,000 × g, at 20 oC) for 5 min. The supernatant was discarded, and the pellet was resuspended in half the volume (5 mL) of distilled water (10% ethanol or water) to remove the unbound MUA and citrate. The assay consisted of adding 10 µL of the test sample to 200 µL of the nAu-MUA suspension, followed by the addition of 10 µL of 100 mM HCl (MilliporeSigma Canada Ltd., Oakville, Canada). To investigate the responsiveness of the assay to various types of plastics, we examined the following types of plastics: PsNP of different sizes (20, 50, and 100 nm diameter), polyethylene terephthalate (PET) leachates from a 4-year-old water bottle, and tire wear nanoparticles (TWNP) leachates. The different sizes of PsNPs were examined to determine the influence of size on the plasmonic signals at 523 and 610 nm (see below). The TWNP leachates were prepared from drilling dust in new tires with a diamond-coated drilling probe (0.5 cm diameter) and from an ethanol-based commercial formulation of recycled used tire crumbs (particle size: 0.5-1 mm, 40 mg/mL) as previously described[20]. This suspension was exposed to the following cycle three times: frozen at -85 oC, thawed at RT, and sonicated for 1 h on the highest setting. Then, the suspension was filtered through a
Figure 1. Methodology overview for the nAu sensor for the detection of NPs. Gold nanoparticles (nAu) of 10 nm diameter are first labeled for 24 hr to mercaptoundecanoic acid (MUA) to form nAu-MUA (A). The nAu-MUA complexes are separated from unreacted MUA by centrifugation and resuspension in 10% ethanol. The nAu-MUA is colored violet in the presence of 5 mM HCl in the absence of NPs (B). When it binds to the hydrophobic NPs, the violet color (610 nm) changes to a red wine color (550-580 nm) in a concentration-dependent manner[16]. MUA: Mercaptoundecanoic acid; NPs: nanoplastics; nAu: nanogold.
Extraction and detection of NPs from mussel homogenates
As stated before, this method was further adapted for biological mussel tissues by conducting a preliminary salting-out extraction of NPs using saturated NaCl (5 M)/ACN[19]. The freshwater mussels were collected at a reference site, a pristine lake 100 km north in the Laurentians area, far from anthropogenic activity. Following an acclimation period of 3 weeks, they were caged (from July to October 2017) at the following sites: (1) a rainfall overflow site (OVF); (2) a site 15 km downstream from the center of Montréal, QC, Canada (Down-city); and (3) a site in the Saint-Lawrence River 8 km downstream of the municipal effluent dispersion plume of the city of Montréal (Down-Effluent). We also selected Down-Effluent, a site where wastewater treatment occurs and where a lower plastic exposure is expected. For each site, three cages containing 20 mussels were placed at 1 m depth. At the end of the 3-month exposure period, mussels were brought back to the laboratory (Montréal, Québec, Canada) and purged in clean water overnight at 15 oC, and their digestive glands were excised and homogenized in Hepes-NaOH buffer
Comparison of the nAu-MUA methodology in the presence of EtOH or ACN
Added PsNP | Absorbance1 (λ = 570 nm) | Absorbance2 (λ = 570 nm) | RI2 |
0.0 mg/mL (blank) | 0.00 | 0.00 | 1.3365 |
0.6 mg/mL | 0.10 | 0.09 | 1.3362 |
1.3 mg/mL | 0.15 | 0.13 | 1.3363 |
2 mg/mL | 0.23 | 0.21 | 1.3363 |
Calibration of the nAu-MUA assay for PsNPs
Concentration | R signal | Absorbance (λ = 570 nm) | RI | Extrapolated concentration | Concentration standard addition |
Direct calibration (external) | |||||
0 (blank) | 162 | 0.00 | 1.3362 | ||
20 µg/mL | 168 | 0.04 | 1.3365 | ||
40 µg/mL | 174 | 0.07 | 1.3363 | ||
60 µg/mL | 193 | 0.10 | 1.3363 | ||
Standard addition (internal) | |||||
0 (sample) | 170 | 30 | 27.4 µg/mL | ||
+20 µg/mL | 176 | 53 | |||
+40 µg/mL | 181 | 63 | |||
+60 µg/mL | 189 | 97 |
Refractive index, neutral lipids, and amyloid estimations
The changes in RI and TRR were also determined in the digestive gland fractions (10,000 × g, 20 min, 2 oC). The levels of proteins were determined in the homogenate and in S10 digestive gland fractions (10,000 × g, 30 min, 4 oC) using the Coomassie blue dye binding principle, and bovine serum albumin was used for calibration[22]. The RI was directly measured in homogenate fractions (between 8 and 16 mg/mL protein concentration) as described above. To measure the TRR, the homogenate samples were diluted to 1 mg/mL in PBS and 1 mM of DTNB was added and 412 nm absorbance changes followed for 40 min as described above[18].
The levels of neutral lipids were determined in the homogenates using Nile red dye[23]. Briefly, a 20 µL of the homogenates was mixed with 200 µL of Nile red dye (10 µM, in PBS) and fluorescence was measured (excitation 485 nm, emission 520 nm) with a fluorometer (model: TB-380, Turner Biosystems, San Jose, USA). Calibration was achieved using 0.01% Tween-20 and the data were expressed as mg lipids/mg proteins.
The levels of amyloid proteins were estimated by the centrifugation methodology using Congo red (MilliporeSigma Canada Ltd. Oakville, Canada) as previously described[24]. The principle of the assay resides in the observation that amyloid proteins (insoluble in saline media such as PBS but soluble in distilled water) decrease the absorbance of the dye at 490 nm in a concentration-dependent manner. Briefly, the homogenate 1,500 × g fraction was centrifuged at 10,000 × g for 10 min and the pellet resuspended in PBS. This process was repeated 4 more times, and the pellet was washed once in distilled water and centrifuged again. The pellet was resuspended once more in distilled water to release amyloids and centrifuged again. The presence of amyloids was determined by the decrease in absorbance at 490 nm (1/490 nm) after adding 10 µM Congo Red in the supernatant and the data were expressed as the inverse of the absorbance/mg proteins in the digestive gland.
The levels of aldehydes were determined in the 10,000 × g supernatant of the homogenate using the 4-amino fluorescein fluorescence methodology[25]. Briefly, 20 µL of S10 fraction was mixed with 200 µL of 10 µM
Data analysis
The experiments were repeated 3 times (n = 3), and the data were presented as mean (± standard error). The data were subjected to the Kruskal-Wallis non-parametric analysis of variance followed by the multiple comparison Conover-Iman test to find differences between groups. Only differences with the reference site were discussed. Correlation analysis was performed using the Pearson-moment procedure. All statistical tests were performed using the StatSoft software package (version 13). The level of significance α of 0.05 was used.
RESULTS AND DISCUSSION
In this study, we adapted and used a simple visualization assay using a nAu sensor probe[16] to assess the exposure of mussels in situ to different urban effluents. In addition, we investigated the effects of NPs
Methodological improvement of the nAu-MUA assay
The levels of NPs were determined in biological tissues using the nAu-MUA assay initially developed for the estimation of plastics in simple matrices such as water or plastic tea bag leachates[16]. However, more complex matrices such as digestive gland homogenates inevitably require a primary fractionation step. Therefore, a simple salting-out extraction was designed in the presence of ACN to recuperate NPs in mussel tissues. The nAu-MUA assay was tested in the presence of ACN (5% final concentration). The absorbance at 570 nm and the R color measurements were taken directly in microplates with a smartphone camera or a microplate reader [Table 1]. The absorbance did not change compared to the original method (10% ethanol), suggesting that ACN could also be a suitable solvent for this assay. Additionally, the RI did not change with the addition of PsNPs to nAu-MUA reagent, suggesting negligible aggregation of nAu-MUA. The calibration data revealed that the R color (and the absorbance at 570 nm) increased with the addition of increasing concentrations of PsNPs [Table 2]. Calibration by the standard addition method of a spiked tissue sample revealed the same results as the external calibration method, with regression coefficients ranging from 0.96 to 0.99 [Table 2]. For the standard addition calibration, the test sample was a NaCl/ACN extract of the digestive gland of mussels E. complanata. The estimated concentration of PsNPs in the sample was estimated at 30 and 27 µg/mL with the direct and standard addition methodologies, respectively. This suggests that the extraction of PsNPs in ACN from tissues did not introduce important matrix effects. The aggregation of nAu-MUA could be favored by the presence of salts at concentrations over 10 mM, such as HCl or NaCl[16,26]. However, the assay was conducted in ACN fraction devoid of salts. The saturated NaCl/ACN extraction is based on the salting out of large molecules. In this context, less dense plastic particles (usually 0.75-0.95 g/mL) will tend to float at the surface. The ACN step will extract and concentrate these lighter particles. Larger molecules such as peptides/proteins and other large molecules (humic and fulvic acids present in natural organic matter in surface waters) would then precipitate during the centrifugation step[27]. Although the ACN can extract less polar organic compounds (e.g., neutral lipids) along with PsNps (20 to 100 nm in diameter), interference from large amounts of neutral lipids could interfere in the distances between the nAu-MUA-NPs complex. However, significant differences between the direct calibration method and the standard addition method were not found, suggesting the absence of interferences in the conditions used in this study. Moreover, changes in lipids in digestive gland homogenates were not significantly correlated with NPs as determined by the plasmonic nAu-MUA assay. Interference from dissolved organic matter (> 15 mg/L) was observed with the nAu-MUA assay when used directly on surface water samples[27]. In amounts exceeding 15 mg/L, only 15%-20% of the NPs were detected when the assay was used directly on the water samples. An extraction step should be considered when the assay is to be applied directly to the sample with high levels of dissolved organic carbon, as used in the present study.
Responsiveness of the nAu-MUA assay to various plastics
The nAu-MUA assay was tested against various types of plastics such as PsNPs (20, 50, and 100 nm), water from 4-year-old PET water bottles, and tire/recycled tire leachates [Table 3]. As expected, the test responded to PsNPs. Tire wear leachates also elicited a contrasted positive signal compared to the blanks, suggesting that this assay also responds to petroleum-based rubber materials from tire wear and recycled tires. The nAu-MUA assay was tested on water from 4-year-old PET drinking water bottles and the assay also detected the presence of NPs. The nAu-MUA assay responded well to different types and sizes of plastics such as PS, rubber (tire), and PET. The detection of NPs in water bottles corroborates previous findings[16], i.e., NPs < 100 nm are released from plastic bottles. This assay also proved to work with polypropylene and polyethylene (tea bags) samples and seemed unaffected by the positive or negative charges at the surface of PsNPs, which are likely to be found in aged plastics.
Response of the nAu-MUA assay to various types of plastics
Materials1 | Response factor2 | |
Blank | 1.00 | |
Distilled water | 1.00 | |
PsNPs | 20 nm3 | 1.21 |
50 nm3 | 1.17 | |
100 nm3 | 1.20 | |
Tire wear particle leachates4 | 1.20 | |
Tire crumb4 | 1.14 | |
Water from 4-years-old PET bottle5 | 1.17 |
Detection of NPs in mussel digestive glands
The relative levels of NPs were determined in the extract of the digestive gland of the freshwater mussel
Case study with caged lake mussels downstream a large city, rainfall overflow, and municipal treated effluent in the Saint-Lawrence River
Caging sites | nAu-MUC method (µg/g) | Lipid (mg/g tissue) | Aldehydes (RFU/mg proteins) | Amyloids 1/ (Abs × mg proteins) |
Reference lake | 0.02 ± 0.01 | 16 ± 3 h | 110 ± 30 | 4.9 ± 0.02 h |
Down-city | 0.11 ± 0.01* | 31 ± 5* | 200 ± 30* | 4.7 ± 0.47 h |
Down-Effluent | 0.05 ± 0.02* | 21 ± 5 h | 450 ± 53* | 4.3 ± 0.20 h |
OVF | 0.06 ± 0.01* | 21 ± 3* | 330 ± 27* | 5.5 ± 0.04* |
Biological and biochemical markers in the mussel digestive glands
In addition to NPs, other biomarkers such as the RI, TRR, aldehydes, neutral lipids, and amyloids in the digestive gland fractions were assessed. Globally, exposure of mussels to urban pollution increased levels of lipids, aldehydes, RI (digestive gland homogenates), and denatured proteins (amyloids). Their levels were highest at the rainfall overflow and downstream the city center sites for lipids and amyloid levels [Table 4]. In our study, we observed a significant increase in neutral lipids at the overflow site and downstream of the city, but not following exposure to the wastewater effluent site [Table 4]. Furthermore, an increased signal for aldehyde formation was observed at all sites [Table 4]. We observed that the RI was significantly lower in mussels downstream of the city as shown below. Interestingly, the highest levels of NPs were also found at this same site. In our study, we also noticed a significant increase in the amyloid contents in mussels exposed to the overflow site [Table 4]. Amyloid formation is involved in several degenerative diseases as a result of a long-term accumulation of oxidatively denatured protein plaques in various tissues and organs[15] (e.g., brain, muscle, and liver) and was referred to as a typical cellular disorder in the physiopathology of aging[34]. To this date, we ignore why amyloids were increased in these mussels, but we observed a significant correlation between amyloids and the RI (r = 0.66; P < 0.01). This observation strengthens the possible association between these two markers in the digestive gland homogenates.
Figure 2. Refractive indexes and TRR following in vitro exposure to plastic nanoparticles (A and B) or in E. complanata digestive gland extracts following in situ exposures (C and D). The RI (A) and the TRR (B) were determined in concentrated albumin solutions. Mussels were encaged for 3 months at various sites and dissected, and digestive glands homogenates were assessed for RI and TRR. OVF: Overflow site; PsNP: polystyrene nanoplastic; RI: refractive index; TRR: thiol-reaction rates.
In vitro investigations of effects of PsNPs in albumin-rich solution and biological matrices
To further investigate the observed changes in the RI and TRRs following exposure to NPs, we conducted an exploratory in vitro test to determine the effects of PsNPs on RI and TRRs in an albumin-rich solution and the digestive gland homogenates of E. complanata [Figure 2A-D]. Thus, a concentrated albumin solution was prepared at 100 mg/mL near the reported density of intracellular environments[18] and spiked with increasing concentrations of PsNPs. The presence of 50 nm PsNPs decreased the RI of the albumin solution in a concentration-dependent manner (between 0 and 0.15 µg/L, Figure 2A). It is important to state that the addition of PsNPs (without albumin) did not influence the RI of the solution. Furthermore, the TRRs in albumin-rich solutions were determined and revealed a decrease in the reaction rates when over 100 ng of PsNPs were added [Figure 2B]. In turn, this rearrangement could lower the free albumin in the dissolved phase and explain the decreased RI. Overall, these results obtained in albumin-rich solutions contribute to a better understanding of the effects of NPs in protein-rich samples and strengthen the link between the effects of NPs and the biological and biochemical effects observed in the exposed mussel’s digestive gland homogenates.
Indeed, albumin could interact with PsNPs, forming local and dense albumin-coated PsNP clusters that restrict the diffusion of substrates[9,35]. Analysis of the reaction rates revealed that the reaction rates over time (a measure of the spectral dimension of the reaction in crowded environments[9]) decreased more strongly in mussels downstream of the urban area compared to the reference lake (results not shown), consistent with the formation of elongated aggregate arrangements of percolation organization. Similarly, TRR could be decreased by the rearrangement of albumin in the presence of PsNPs and perhaps by a non-specific binding of the reagent on the hydrophobic surface of PsNPs. Additionally, the RI and the TRR were also measured in the digestive gland extracts [Figure 2C and D]. The analysis revealed that the RI of the homogenate (normalized to protein content) was significantly decreased at the site with the highest levels of NPs (downstream city center site). Although other contaminants might be at play in these complex environments, it is plausible that NPs contribute to the decreased RI as a result of alteration in protein organization and reaction rates in the digestive gland extracts. Such changes in protein organization were also measured by anisotropic changes in the digestive gland homogenates, consistent with nematic crystal formation in mussels exposed to PsNPs and municipal effluents[36]. The complexation of albumin to PsNPs was previously shown to result from the hydrophobic environment of both albumin and PsNPs[37] and could be positively influenced by the presence of anionic charges on the surface of PsNPs[38]. This complexation is likely to happen given the local cationic patch that albumin displays on its surface and the hydrophobic environment of the NPs, especially when anionic charges are present on aged/weathered NP surfaces. Anionic charges (R-COO-) were reported to form during the weathering/degradation of plastic materials by UV and ozone[10]. The addition of these anionic charges on the surface of NPs could expectedly weaken the interaction with anionic natural organic matter (e.g., humic acids and alginates) and support interactions with cationic proteinaceous compounds usually found in wastewater. Ultimately, these interactions with NPs could lead to spatial changes in proteins and protein networks and consequently alter enzyme activity. Such influence of PsNPs on the enzymatic activity of LDH was previously reported in vitro and mussels exposed to PsNPs[13]. The changes in LDH activity by these NPs were consistent with the altered fractal organization of complex protein networks. Indeed, the calculated fractal dimension of LDH reaction rate was reduced by PsNPs as with F-actin, the natural ligand for LDH, suggesting a loss of affinity for its substrate pyruvate and NADH (increased affinity constant KM). Indeed, the spectral dimension of the reaction rates reached 1.2, also indicative of a percolation structure (continuous linear dust fragments). Thus, NPs interacting with albumin or protein networks in cells would form percolation-like structures, leading to reduced RI and TRR.
CONCLUSION
In this study, we adapted a simple extraction and detection method for NPs, and we investigated the in situ exposure of the freshwater mussel E. complanata in polluted urban environments. We also assessed the biochemical effects using several simple markers in exposed mussels and conducted an exploratory
DECLARATIONS
Acknowledgments
The authors are thankful to the Saint-Lawrence Action Plan and Plastics research initiative of Environment and Climate Change Canada for supporting this research. The technical assistance for the mussel caging experiments of Hiba Qchiqach is duly recognized in this project.
Authors’ contributions
Conceptualization, methodology, validation, formal analysis, investigation, writing - original draft: Gagné F
Experimentation, analysis, writing -review and editing: Gagné F, Gauthier M, André C
Conceptualization, supervision, resources: Gagné F
Availability of data and materials
Not applicable.
Financial support and sponsorship
None.
Conflicts of interest
All authors declared that there are no conflicts of interest.
Ethical approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Copyright
© The Author(s) 2023.
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Gagné, F.; Gauthier, M.; André C. Simple detection of polystyrene nanoparticles and effects in freshwater mussels: method development and in situ application to urban pollution. Water Emerg. Contam. Nanoplastics. 2024, 3, 4. http://dx.doi.org/10.20517/wecn.2023.60
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