Micro and Nanoplastics size distribution in mussel tissues: first evidence, identification, and quantification in the nanogram range

doi:10.21203/rs.3.rs-2981733/v1

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Micro and Nanoplastics size distribution in mussel tissues: first evidence, identification, and quantification in the nanogram range

https://doi.org/10.21203/rs.3.rs-2981733/v1

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Microplastics (MPs) and nanoplastics (NPs) (MNPs) are hazardous to ecosystems, wildlife, and through seafood, also for human health. Due to biological, chemical, and physical characteristics, NPs can slip through cell membranes, being even more toxicologically important than MPs. Thermal Desorption - Proton Transfer Reaction - Mass Spectrometry (TD-PTR-MS) was used to analyse mussels from the Apulian region, Italy. All the analysed organisms have NPs (values ranging from 10 ng MNPs/ DW mg to 187 ng MNPs / DW mg). The highest mass of plastics was detected in the size groups > 2,2 µm (218 ng MNPs / DW mg) and 20–200 nm (187 ng MNPs / DW mg). An upscaling we performed estimated that people in Europe could ingest more than 2 mg of NPs per year through seafood consumption. The detected presence of nanoplastics in farmed mussels here presented contributes to establishing a baseline for monitoring these pollutants.

Earth and environmental sciences/Environmental sciences/Environmental chemistry

Earth and environmental sciences/Ocean sciences/Marine chemistry

microplastics

nanoplastics

size distribution

TD-PTR-MS

mussels

Plastic manufacturing started at the beginning of the 20th century and, after World War II became a cheap and useful alternative to more expensive products (Thompson et al.,2009). The earnings of plastics were evident, lightweight, strong, inexpensive, and durable material, moreover able to be transformed and modified to fit consumer demands (Napper and Tompson 2020).

In 2021 production of plastics touched 390 million tonnes worldwide. In Europe, 15% of the production is for packaging materials, and even if more the one-third is sent to recycling facilities, over 23% is still sent to landfills (Plastic The Fact 2022). Improper waste management or accidental discharge from activities like construction, farming, aquaculture, and mariculture are the principal sources of plastic that end up in the marine environment (Peng et al., 2020). Plastic can reach the sea directly as micro-or nano - range items intentionally produced for humans (primary microplastics (MPs); primary nanoplastics (NPs) or otherwise, as big pieces that, in the environment, undergo a decomposition process that can break down their bonds forming smaller sizes particles known as secondary MPs and eventually secondary NPs (Peng et al., 2020; Vitali et al., 2022; Thompson et al., 2015; González-Pleiter et al., 2019; Gaylarde et al., 2021). Up to date, MPs are being detected in organisms’ tissues and different ecosystems worldwide (Akdogan and Guven 2019; Piazzolla et al., 2020; Kumar et al., 2021; Koutnik et al., 2021; Kane et al., 2020; Gasperi et al., 2018; Girard et al,. 2021; Zhang et al., 2020; Moore et al., 2022; Panebianco et al., 2019) while NPs, even if their detection is still challenging, were found in every environmental compartment (air, water, soil) (Xu et al., 2020; Ter Halle et al., 2017; Wahl et al., 2021) even in the most remote places of the world (Materić et al., 2021; Materić et al., 2022), but still not in organisms.

Investigating nanoplastics is challenging due to instrument limitations, their low concentrations in the environment, and their conjugation with interfering substances. Some authors suggested the combination of different analytical techniques to reach the final goal of quantification, identification, and characterization of NPs (Zhang et al., 2022). One of the most promising approaches is the TD-PTR-MS technique, this recently proposed method couples high sensitivity and high mass resolution providing data on plastics presence in the samples with a limit of detection (LOD) of < 10 ng (Ivleva et al., 2021; Materić et al., 2020). Usually, the preferred way to express data about the presence of MNPs detected in environmental samples is number of particles/g dry weight or wet weight or number of particles/individual (Dellisanti et al., 2022; Vitali et al., 2022) due to limit of quantification (LOQ) and limit of detection (LOD) constrains posed by instruments. Primpke et al., 2020 proposed harmonization of size and shape information with mass data because particle size has a considerable influence on mass information: a 0.7–1 µg reduced to one single particle can correspond to a size between 50 and 200 µm (Primke et al., 2020).

Nevertheless, the interference of the surrounding matrix is still a concern while using this technique (Materić et al., 2020). In particular, with biota samples removing the organic matrix sufficiently without damaging the polymers is an essential step. The optimization of a digestion protocol effective on the matrix preserving the plastic integrity is a crucial point to proceed in the quantification and identification with sophisticated and highly sensitive approaches such as TD-PTR-MS.

Microplastics and nanoplastics (MNPs) can negatively impact all biological organization levels from ecosystem to subcellular (Galloway et al., 2017). MNPs can also offer a suitable surface for the settlement of microorganisms or invasive species, enabling their migration to distinct locations (Zettler et al., 2013). Moreover, has been proved that decreasing the size increases the toxicity, thus NPs having a high surface-volume ratio can severely interact with toxic organic compounds (Galloway et al., 2017). MPs and NPs can be ingested by aquatic organisms and bioaccumulate along the trophic chain (Sana et al., 2020). Their nanoscopic dimension allows their passage through biological membranes entering cells and tissues (Hollóczki et al., 2019). These conditions made nanoplastics an even more severe threat to all organisms than MPs, raising serious concerns about human exposition (Yee et al., 2021).

Bivalves are largely consumed by humans, and as sessile benthic filter feeders, organisms are highly affected by MPs pollution. These organisms are widely distributed all over the globe and can provide a useful and interesting assessment of NPs’ presence in the surrounding environment acting as bioindicators (Gonçalves and Bebienno 2021). Within the Bivalvia class, mussels are well-known studied animals, used as model organisms for ecotoxicological studies, as well as for sentinel organisms for marine pollution for several years (Gonçalves and Bebianno 2021; Goldberg 1986) and were proposed as a target group to study the accumulation and toxicity of NPs as well (Sendra et al., 2020). Nevertheless, up to date, no information about the presence of NPs in organisms has been reported. Most of the performed studies, are focused on the polystyrene NPs’ toxic effects (Sendra et al., 2021; Gonçalves et al., 2022; Merzel et al., 2019) along with feeding-controlled experiments.

To fulfil this lack of data, nanoplastics presence in organisms was investigated, and to provide a comprehensive size distribution of MNPs in mussels, the TD-PTR-MS technique was combined with a cascade filtration, and five mussels were analysed. In the present work, the micro and nanoplastics size distribution in farmed mussel tissues is provided, quantifying plastics in the nanogram range and chemically identifying them. The measured values were then upscaled to estimate the nanoplastics uptake by Europeans in a year by mussels consumption.

2.1 Nanoplastics in all mussels tested.

Mussels used in this study were bought in a local fish market in the Apulian region (Italy). The size ranges 20– 200 nm were analyzed in all five mussels. These five specimens have similar sizes (details reported in Supplementary Table 2). The mussel M4 is the smallest one followed by M5 and M2. A high quantity of nanoplastics was registered in each organism (details in Table S4). The highest amount of plastic was found in M3, with 187 ± 27 ng /mg DW, followed by M4 (132 ± 36 ng NPs/ mg DW) and M1 (89 ± 16 ng NPs/ mg DW). Plastics were chemically identified, and a high diversity of nanoplastics was registered in each mussel. PE, PVC, and PS were detected in all the analyzed individuals. PP was present in M2, M3, M4, while PET in M1, M4 and M5 (0.7 ng MNPs / mg DW) (Fig. 1). The most frequent and abundant polymer is PE, followed by PVC and PP. The less-represented polymers are PET (7%) and TIRE particles (8%) (Fig. 2).

2.2 Micro- and Nanoplastics size distribution

The organism with the highest amount of MNPs was selected for a more complete study of the MNP size distribution, and the relevant results are summarized in Fig. 3. Some of the most common polymers found in the marine environment (Polystyrene PS; Polypropylene PP; Tire; Polyethylene PE; Polyvinyl-chloride PVC) were detected in each size class. Most of the plastics were detected in the highest size group > 2.2 µm (218 ng/mg DW), which was the first step of the cascade filtration, and in the lowest size group 20–200 nm (187 ng/mg DW). The size group with less amount of plastic was 700–1400 nm (25 ng/mg DW).

The most frequent polymers are PP, PS, PVC and PE. PP and PS are present in all the size classes while PVC and PE were absent in few size ranges. However, when present, PE is the most abundant polymer detected, always followed by PP, except for the 400–700 nm size class in which PVC and PS are equally present constituting the second most abundant polymers (22%). The lowest plastics diversity was measured in the 700– 1400 nm size class, with the dominance of PP (75%) and the presence of PS (25%), PP was the dominant polymer (38%) also in the 1400– 2200 nm size group. It is interesting to notice that PE is widely present in all the size classes except for the above ones, indicating that in the analyzed organisms were not present PE particles of those dimension and in absence of PE, PP is the most abundant polymer detected. In both, the smallest 20–200 nm and the biggest size class > 2200 nm there are small differences between PE and PP contribution, representing the most abundant plastics found (Fig. 4).

2.3 Comparison between MPs and NPs

To compare MPs and NPs quantity in M3 the microplastics size threshold was set at the size class 700– 1400 nm, considering NPs below 700 nm then the data were summed up. The mass of NPs is similar or higher (PE and TIRE) to the mass of MPs for all the polymers. PE and TIRE are more abundant in the nano range of our data (Supplementary Fig. 2S).

Figure 6 shows the polymer contribution of both MPs and NPs, which is quite similar. It is not surprising that PE and PP are the most represented polymers, with a higher contribution of PE in the nanometer range (37%) followed by PP (27%), while in MPs the presence of PE and PP is analogous (about 30%). PE (including LDPE and HDPE) and PP are the two most produced plastics in 2021 (Plastic the fact 2022), and the most abundant and frequently found polymers in the Mediterranean Sea and consequently in organisms (Sharma et al 2017; Wang et al., 2021). The third produced polymer in 2021 was PVC which is also abundant in both MPs and NPs but its contribution in higher in the micro range (Plastic the fact 2022). While the presence of PS percentage is almost the same in the micro and nanometric range, there is a big difference in the quantity of TIRE found in NPs and MPs, in the nano range this polymer type contributes by 5% to the total of polymer found while in micro range its percentage is 0.94%.

Mussels are commonly used to assess toxicity and bioaccumulation of environmental pollutants (Franzellitti et al., 2019; Goldberg et al., 1986; Li et al., 2018). Laboratory studies have been performed feeding mussels with MPs and NPs (especially PS) to investigate the possible physiological responses (Cappello et al., 2021; Green et al., 2019; Sendra 2020; Rist et al., 2019; Gonçalves et al., 2022; Auguste et al., 2020). Some authors report the mussel’s ability to excrete foreign particles through pseudofeces (Wagner et al., 2012), but there is evidence that small particles can cross the gastro-intestinal barrier reaching the tissues (2–4 µm) while particles larger than 10–20 µm are not able to cross the barrier, remaining in the intestinal tract and being expelled subsequently (Ribeiro et al., 2019). NPs can cause different responses in mussels, but there is no information about their excretion. Wang et al., 2021 evaluated the effects of 70 nm PS particles and reported their accumulation in the digestive tract of mussels. Sendra et al., 2020 suggested different uptake routes in mussel hemocytes depending on size, with a higher internalization of smaller (50 nm) particles while the distribution in tissues was independent of size. NPs at the laboratory concentration can cause severe stress to the bivalve immune system compromising also larval development and fertility (González-Fernández et al 2018; Cole et al., 2020).

Although several studies have been carried out to assess the toxicity of NPs particles, often together with MPs, in laboratory experiments, no information is available on their presence in organisms in environmental conditions. This lack of data makes it impossible to build a baseline of NPs concentration in the marine environment. Indeed, the NPs in the marine environment are smaller compared to the quantities tested in lab experiments (Ter Halle et al., 2017; Materić et al., 2022), also our data confirm the actual amount of MNPs found in mussels is lower than the particles’ concentration used in laboratory experiments. We measured distinct levels of NPs contamination in the five tested mussels, ranging from tens to hundreds of ng NPs/mg DW. Our data may reflect the variability of the real contamination; however, more measurements are needed before robust conclusions can be made. Mussels are not able to discriminate between natural particles or artificial particulate contaminants, ingesting both regardless of their chemical composition (Bhrem et al., 2022). However, it is possible that mussels in natural conditions can manage the MNPs uptake by modulating their filtration when exposed to increasing external stressors in the marine environment (Capolupo et al., 2021). Recently Halback et al., 2022 assessed the mass content of MPs in historical series of mussel samples from the North and the Baltic seas. Summing values of different pooled organisms reported masses between 2–12 µg/ g DW indicating the most represented polymers as PET and PVC. We measured much higher content of plastic in our samples and a different polymer composition. This difference can be explained by local pollution which can vary several orders of magnitude at regional scale (Halback et al., 2022), for example the measured microplastics polymer composition and abundance along the North Sea is different within the North Sea itself, and from the Mediterranean Sea, known as one of the most plastic polluted sites on the world (Suaria et al., 2016; Alessi et al., 2018; Lorenz et al., 2019). Mussels feeding strategy is filtration, and the plastic content within their tissues is directly influenced by the surrounding environment, indeed the abundance of MPs in seawater and the abundance of MPs in bivalves have a reported positive correlation (Wang et al., 2021; Halback et al., 2022¸Ward et al., 2019).

Bivalve mollusks are highly exposed to plastics during farming. In aquaculture and mariculture, most floating structures are made of plastics (often Expanded polystyrene (EPS) or plastic buoys (PVC) and are stabilized by ropes and lines (usually made of PE and PP). In mussel farming, plastics are ubiquitous, especially PP and PE: hybrid ropes built with natural materials and mixed with synthetic filaments (PE and PP) are used to attract more larvae during the recruitment period, as well as anti-predator nets to protect shellfish are mostly made of PP (followed by PE) (FAO 2017 Plastic in fishery and aquaculture).

Mussels are one of the most widely consumed foods worldwide and are of serious concern as a potential source of NPs for humans. Although there are still some aspects that need to be investigated in detail, our results show a concerning situation that needs to be addressed. To ensure a safe seafood product for consumers, mussels must be grown in clean water and undergo a depuration process according to HACCP recommendations (FAO and WHO 2020 Code of Practice). To date, micro- and nanoplastics have not been included in the list of potential human hazards associated with mussel consumption. The depuration time is several days, but our results underline that this process needs to be regulated differently or improved to "clean" these organisms from MNPs before they reach the consumer. There are several works dealing with MPs in mussels from the fish market (Cho et al., 2019; Nalbone et al., 2021; Fraissinet et al., 2021; Bom et al., 2022; Masiá et al., 2022). Masiá (2022) reported that, on average, a consumer on the Asturian coast could ingest up to 109 MPs particles per serving of mussels, while Cox et al., (2019) calculated that an American adult male consumed approx. 142 MPs particles daily, mainly from seafood. In UK consumers eat approx. 70 MPs particles in a 100 g portion of processed mussels (Li et al., 2018). These animals are farmed to fit human demands being the most farmed product in aquaculture facilities after finfishes, in 2020 the global production touched 1108,3 Kilotons LWE (live weight). In Europe, mussels are the second main farmed product consumed, principally supplied by Spain (which provides 80% of EU production). Recently (2018–2019), the consumption of mussels slightly exceeded 1,20 kg LWE per capita in Europe (EUMOFA 2021). Upscaling our data we estimated the presence of approx. 2 tons of NPs (DW) (2114 Kg DW) in the total world mussel production and, that a European consumes slightly more than 2 mg of NPs (DW) (2.2 x 10^− 6 Kg) per year. In absence of a consistent dataset regarding NPs presence in farmed bivalves it is impossible to understand possible contamination pathways through the trophic chain, and knowing the actual extend of the problem.

Comparing micro and nanoplastics in our sample there are no differences in polymer composition. Even if these results are referred to just one mussel, they are still quite interesting. We found PP, PE, and PVC as the most abundant and frequent polymers in both micro- and nanorange, and a possible correlation between a source of contaminants and their presence in the organisms tested. Generally, bivalves contain comparable amount of small and large microplastics, but the smallest plastic size found in bivalve organisms is 3.6 µm (Vital et al., 2021; Ding et al., 2022). Despite the global attention posed to these contaminants their presence in the environment is progressively increased, suggesting a gap of knowledge and consequently in pragmatical actions, making more difficult the proposal of mitigation measures especially for nanoplastics. (Sarkar et al., 2022).

To conclude, there is an urgent need to establish viable approaches that can provide an assessment of the actual MNPs contamination of the marine environment, especially since MNPs have already been found in human blood, proving that we are in close contact with these pollutants by breathing, drinking and eating (Leslie et al., 2022; Sangkham et al., 2022). Bivalves have a high tolerance to environmental stressors and have already been shown to ingest and accumulate NPs in laboratory experiments. Mussels are sessile powerful filter-feeders, widespread and abundantly farmed in diverse aquatic environments, investigate MNPs’ presence in these animals can help to better understand eventually transferring to humans as for other contaminants or MPs (Jovic et al., 2012; Carbery et al., 2018; Smaal et a., 2002; Beyer et al., 2017). Our findings show important levels of contamination reflecting the actual concern for NPs’ interaction with fauna and perhaps humans and pose a new benchmark for comprehension of the problem extension. This is critical information needed to assess the ecological risks of NPs and the potential impacts on marine wildlife.

2.1 Chemicals and materials

KOH pellets, H₂O₂ (30%) solution, and NH₄OH (25%) solution were purchased from Sigma-Aldrich (Milano, Italy). All the solutions were prepared with ultrapure water. The digestion solution was freshly prepared every day. Glass fiber filters, Quartz fiber filters and, Anodisc filters were supplied by Sigma-Aldrich. PS standard spheres of 1 µm in diameter (PS-ST-1.0, Microparticles GmbH, Berlin, Germany).

2.2 Sample preparation

Several organisms were bought in a local market and stored at -20°C. Five specimens of similar sizes were (average shell length 5.56 cm ± 0.52) selected and digested. Information about the mussel’s length and dry weight (DW) are provided in Supplementary Table 1. One organism within five was randomly chosen and all filters corresponding to the 6 different particle’s size fractions were analyzed covering the entire size range from > 2.2 µm to 20–200 nm. Then for all the digested mussels, the lower size fraction (20–200 nm) was analyzed providing information about the presence of nanoplastics in seafood from the fish market.

Tissues were defrosted and the whole animal was dried in an oven at 60°C for 48 h, then grounded to a powder and weighed, the powder was placed in glass bottles.

Samples were treated slightly changing Fraissinet et al., 2021. A digestion solution prepared with KOH 2.5% and H₂O₂ 5% was added to the bottles that were then placed in a water bath at 60°C for 3 h. After 3h bottles were left cooling and the solution was centrifugated for 5 minutes at 3000 rpm. The supernatant was neutralized with 10% citric acid added dropwise, while the precipitate was treated using an acidic solution. After 30 minutes at room temperature, also the acid solution was neutralized using 2.5 mL of 25% NH₄OH. Both supernatant and precipitate were filtered on the same membranes using a water jet (vacuum) pump (KNF- LABOPORT® N 86 KT.18).

Six steps of filtration were performed using decreasing pore size filters starting from 2.2 µm (Quartz filter) -1.4 µm (Glass fiber filter) – 0.7 µm (Glass fiber filter) – 0.4 µm (Glass fiber filter) – 0.2 µm (Anodisc filter); 0.02 µm (Anodisc filter). Each bottle was rinsed three times with prefiltered ultra-pure water and the rinsing water was filtered on the same membrane. After each filtration, the filter was washed with prefiltered MilliQ water, and placed in a glass petri dish.

Each filter was analyzed in triplicates. For pore sizes from 2,2 µm to 700 nm replicas were obtained punching the filters using a metal tool having 1 mm perfect circular pieces, while for the 400 nm pore size filters a 2 mm puncher was used. While Anodisc filters (with 200 nm and 20 nm pore size) were cracked using a metal needle, each piece was photographed and stored in a glass vial. From the collected pictures, each Anodisc filter piece area was measured using ImageJ software (Schneider et al., 2012).

Knowing the dry weight of the sample and the filter’s area is possible to estimate the amount of sample on each filter piece for each size fraction (Supplementary Table 2). The same approach was used to calculate the mass content of plastic in all five mussels at 20 nm, values reported in Supplementary Table 3. Then, we used the mass content of plastics to upscaling the data to the global production and the global consumption of mussels, trying to estimate a possible contamination at worldwide scale. The former data are expressed in live weight which means the weight of the animal alive, while our data are referred to dry weight of the animal without the shell. Knowing by literature that in approx. 1kg of mussels the 36,5% of weight is represented by shell (Moore et al., 2021) and the mussel water content is up to 80% (Prato et al., 2019), the live weight was converted in dry weight, then this value was used to estimate the NPs content in produced and consumed mussels.

2.3 TD-PTR-MS Analysis

The analysis was performed using TD-PTR-MS 1000, IONICON Analytik, Austria, where the setting was: ramping from 35 to 360°C at 40 degrees/min, E/N ~ 105 Td.

Data were processed using PTRwid.13 custom-made software (Holzinger 2015). The TD-PTR-MS signal was integrated over 7 min starting when the TD temperature reached 200°C, to include in the analysis the thermal degradation products of each polymer (which are different at different temperatures).

Each plastic polymer shows a specific ion signal at certain temperatures. TD-PTR-MS can monitor these signals at high resolution quantifying each organic ion produced by the thermal desorption process (an example of mass spectrum and thermogram of detected polymers types related to a mussel sample are provided in Supplementary material Fig. 2S and 2S). This approach has been exploited to study environmental pollutants in different environmental matrices (Oyama et al., 2015; Salvador et al., 2020; Allen et al., 2022; Velimirovic et al., 2021; Materić et al., 2022; Hale et al., 2022; Kirchsteiger et al., 2022; Materić et al., 2021). TD-PTR-MS measurements and fingerpinting analysis were performed as described in previous works (Materić et al., 2020; Materić et al., 2021; Materić et al., 2022; Materić et al., 2022b)

2.4 QC/QA

Working in a laboratory is almost impossible to avoid plastic materials, so we tried to minimize the contamination as much as possible. During the distinct phases of this work, particular attention to preserving samples was paid: 1) Sample preparation: The procedures were performed under a laminar flow cabinet, which was carefully cleaned before any activity using paper and prefiltered water. Operative staff wore cotton coats (or clothes) and nitrile gloves. Glass materials were baked before use. All the containers were covered with aluminum foil to reduce exposure to air. Along with the sample preparation, a procedural blank was performed; 2) TD-PTR-MS analyses were carried out in triplicates, both for samples and blanks. Three blanks per experiment were included: a) procedural blanks to assess the contamination during the laboratory operations; b) material blanks to assess the possible contamination due to the filter material; c) system blanks to assess the contamination of vials and instruments; 3) Data analysis and fingerprinting: all the mass spectra were corrected for blank signal, and masses below the 3σ detection limit were not considered for the analysis. The fingerprinting was then performed by four algorithms using a high number of ions (40) with m/z > 120, following Materić et al., 2020.

Procedural blanks were analyzed along with mussel samples. In blank samples, all the size ranges tested showed plastic presence (fingerprinting output provided in Supplementary material). Nonetheless, often the plastic present in the blanks showed a fingerprinting weak match (e.g. in the size range 1400– 2200 nm) or their mass were quite lower than the quantity registered in mussels (e.g. in the size range 20– 200 nm). Just for the size range 400 nm – 700 nm due to technical problems during the analysis blanks were not available.

Moreover, to assess the possibility of false negative responses, on the 700–1400 nm size fraction, a spiking experiment was performed using PS nanoparticles. A water solution (concentration 20 ng/µl) was prepared to have a final load of 100 ng on samples. Three sample and blank replicas were spiked with 100 ng of PS. In mussels’ samples, PS was detected in both spiked and not spiked filters, due to the PS contamination of organisms tissues as we showed in the results section, while the blank filters were completely devoid of PS. However, in this test experiment a positive increment of PS quantity in spiked filters (recovery 30%) was detected (Supplementary Fig. 4S).

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Micro and Nanoplastics size distribution in mussel tissues: first evidence, identification, and quantification in the nanogram range

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Abstract

Figures

1. Introduction

2. Results

2.1 Nanoplastics in all mussels tested.

2.2 Micro- and Nanoplastics size distribution

2.3 Comparison between MPs and NPs

3. Discussion

Material and Methods

2.1 Chemicals and materials

2.2 Sample preparation

2.3 TD-PTR-MS Analysis

2.4 QC/QA

References

Additional Declarations

Supplementary Files

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