The abundance and localization of environmental microplastics in gastrointestinal tract and muscle of Atlantic killifish (Fundulus heteroclitus)

doi:10.21203/rs.3.rs-4916090/v1

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The abundance and localization of environmental microplastics in gastrointestinal tract and muscle of Atlantic killifish (Fundulus heteroclitus)

https://doi.org/10.21203/rs.3.rs-4916090/v1

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Microplastics (MPs) have been found in a diverse range of organisms across trophic levels. While a majority of the information on organismal exposure to plastics in the environment comes from gastrointestinal (GI) data, the prevalence of MP particles in other tissues is not well understood. Additionally, many studies have not been able to detect the smallest, most prevalent, MPs (1 µm – 5mm) that are the most likely to distribute to tissues in the body. To address these knowledge gaps, MPs in the GI tract and muscle of Atlantic killifish (Fundulus heteroclitus) collected from two sites on Buzzards Bay, Cape Cod, MA were quantified down to 2 µm in size. Fourier-transform infrared spectroscopy and Raman spectroscopy were used to identify all particles. Of the 2,008 particles analyzed in various fish tissue samples, only 3.4 % (69 particles) were identified as plastic; polymers included nylon, polyethylene, polypropylene, and polyurethane. MP abundance in the GI tract was greater than in the muscle. MPs detected in the GI tract samples also tended to be more diverse in both size and polymer type than those found in the muscle. We found that MPs <50 µm, which are often not analyzed in the literature, were the most common in both the GI tract and muscle samples. There was not a significant correlation between the MP content in the muscle compared to the GI tract, indicating that GI tract MP abundance cannot be used to predict non-GI tract tissue MP content; however, MP abundance in muscle correlated with fish total length, suggesting potential bioaccumulation of these small MPs.

microplastics

fish

translocation

Fourier-transform infrared spectroscopy

Raman spectroscopy

bioaccumulation

Understanding the fate of plastics in the ocean is challenging, in part, because plastics are an incredibly diverse suite of contaminants (Rochman et al., 2019a). The size, surface chemistry, polymer type, state of degradation, and additives present can all influence the distribution and fate of microplastics (1 µm < particle < 5 mm; MPs) in the environment (Rochman et al., 2019b). The exact influence of these factors on particle abundance and distribution in the environment is still poorly understood due to analytical limitations and a lack of harmonized methods (Koelmans et al., 2019).

Plastic ingestion is widespread, having been documented in more than 1,565 aquatic and terrestrial species (Santos et al., 2021). Even though ingestion is well-documented, the bioaccumulation potential of microplastics is not well understood. Bioaccumulation is classically defined as the buildup of a material in an organism over its lifetime (Nordberg, 2009; Suedel et al., 1994); however, the movement of MPs is likely to be more constrained due to their large particulate nature. The ultimate fate of these ingested MPs is both complex and unknown.

One of the key challenges is that small MPs (smaller than 50 µm), the most prevalent size of environmental plastics (Lebreton et al., 2019), are difficult to find and identify in environmental samples (Lebreton et al., 2019). Smaller particles can often be misidentified as MPs due to a lack of distinguishing characteristics as the particles decrease in size (Lenz et al., 2015; Remy et al., 2015). The abundance of MPs is expected to increase with decreasing size, but there is little information regarding the movement and fate of particles smaller than 50 µm in aquatic environments (Enders et al., 2015; Erni-Cassola et al., 2017). In addition to being more abundant in the environment, smaller MPs are also more likely to translocate from the gastrointestinal (GI) tract into internal tissues (Lusher et al., 2017). Therefore, having an accurate size distribution of ingested MPs is critical to understanding exposure and, ultimately, the risk that MPs pose to marine animals.

There is still much to discover regarding the characteristics of particles that govern MP translocation into non-GI tract tissues since the majority of published data examines plastic exclusively in the GI tract (Gouin, 2020; Pitt et al., 2024). Without this information, the implications of ingestion for bioaccumulation, trophic transfer, and biomagnification of MPs cannot be predicted (Provencher et al., 2019). Current research suggests that bioaccumulation of MPs is unlikely, but this prediction is limited to particles larger than 100 µm, emphasizing the need for research on the accumulation of smaller MPs in tissues (Covernton et al., 2022).

Smaller MPs are often not quantified in tissues due to analytical challenges (Hermsen et al., 2018; Provencher et al., 2019) and high levels of background contamination (Hermsen et al., 2018; Löder et al., 2017). The objective of this study was to quantify the full-size range of MPs using strict quality control procedures to reduce background contamination. Through this approach, we aimed to distinguish differences in characteristics of MPs detected in the GI tract and muscle of Atlantic killifish (Fundulus heteroclitus) and address the bioaccumulation potential of the under-reported small MPs.

2.1 Materials

Pyrex glassware was used whenever possible during this study. Whatman (grade 4; 25 μm pore size) and nitrocellulose (pore size of 1 μm) filter papers (Sigma Aldrich) were used for Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy analysis. Minnow traps were used for collecting Atlantic killifish.

2.2 Quality Control

We followed the quality control criteria established by Hermsen et al. (2018). All water samples and solutions were filtered through a 0.2 μm filter prior to use. All sample processing apparatus (glass Erlenmeyer flasks and ceramic Buchner funnels) were combusted in a muffle furnace (Fisher Scientific Isotemp Programmable Forced-Draft Furnace) at 500 ℃ for 5 h. Glassware that could not be combusted in the furnace was rinsed three times with both acetone and filtered water prior to use. All work surfaces were wiped down with ethanol prior to working with samples. Sample manipulation took place in a laminar flow hood (AirClean 600 PCR Workstation). Samples were not exposed to ambient air. When the samples were not being actively manipulated, they were covered with aluminum foil. While working with samples, a 100% cotton jumpsuit was worn to prevent contamination from polyester clothing. During sample processing, occupancy of the room was kept to a single person. Procedural blanks were used for every 5 samples.

2.3 Fish collection and tissue sampling

Atlantic killifish were collected using minnow traps from two locations in Buzzards Bay, Massachusetts (Fig. 1). The sampling dates, coordinates, number of fish collected, and the environmental conditions are shown in Table S1. Fish were immediately euthanized using MS222 (1 g/L) buffered with sodium bicarbonate. Fish were kept in an aluminum-foil lined bucket during transport. The total length and weight, sex, and tissue wet weights were recorded upon collection. Prior to dissection, fish were rinsed three times with 0.2 μm filtered water to remove any loose particles on the fish’s skin. The GI tract and a section of the dorsal muscle (without skin) were collected. The total GI tract, including contents, were used in our analysis. Tissues were stored in plastic-free aluminum foil at -80 °C. All collected fish were considered mature as they exceeded 3.2 cm or 3.8 cm for males and females, respectively, and they were in good overall health according to the calculated condition factors (Abraham, 1985). Based on length-age relationships (Abraham, 1985), the collected fish from Bourne, MA appear to be a couple of years younger than the collected fish from Falmouth, MA.

At both sampling sites, we collected water samples in 1 liter glass jars. The jars were rinsed three times in water from the collection site prior to sample collection. To prevent air contamination, the jars were immersed underwater prior to opening. Duplicate water samples were collected from each site. Samples were stored at room temperature (20 - 25 ℃).

2.4 Sample Digestion

A flow chart of the sample processing and analysis is shown in Figure 2. Tissue samples were digested with 10% KOH (3x sample wet weight) at 60 ℃ for 48 h (Fig. 2). KOH digestion at this temperature has previously been shown to not degrade MPs (Gulizia et al., 2022). Digests were neutralized with a combination of sodium bicarbonate (0.05 g/mL) and 10% HCl (0.54 mL HCl/mL KOH) prior to filtration. Particles in the samples were size fractionated by filtering initially through a 25 μm pore-size filter followed by a 1 μm filter (Fig. 2). Filters were stored in plastic-free aluminum tins until analysis. The 25 μm pore-size filter was used for FTIR spectroscopy analysis and the 1 μm filter was used for Raman spectroscopy (Fig. 2). Size fractionation was used to analyze samples more efficiently. Raman spectroscopy is more accurate at identifying particles smaller than 20 μm in length, and FTIR is a more efficient method for particles >20 μm in length (De Frond et al., 2023).

2.5 Particle Recovery experiments

Particle recovery experiments were conducted using polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polyethylene (PE) particles (250 μm) dyed with 300 μL of Nile Red (10 μg/mL). Blue mussel (Mytilus edulis) tissue was used to determine the particle recovery from the MP isolation process from biological tissues. Fifty particles from each polymer type were manually added to whole mussel tissue (Mytilus edulis) in Erlenmeyer flasks. The mussels were then digested for 48 h as in section 2.4. The resulting digests were then filtered through 25 μm pore-size filters. Each flask was washed three times with filtered water. The number of particles on the filter were counted under a dissecting microscope. There was 80% recovery of particles regardless of the polymer type (n=2 experiments per polymer) (Fig. S1).

2.6. FTIR spectroscopy Sample Analysis

The 25 μm filters were scanned for particles under a dissecting microscope at 8.6x magnification. The whole filter was examined, and any particles found were imaged (ThorCam Imaging Software V.3.5.1.1). Particles were then transferred to a piece of double-sided tape in a glass petri dish and numbered. These particles were analyzed with a diamond attenuated total reflection (ATR) attachment on the Cary 630 FTIR (Agilent Technologies Inc). Particles were placed individually on the detection area. MicroLabPC software was used to collect the spectra.

2.7. Raman spectroscopy Sample Analysis

The 1 μm filter was used for Raman analysis. The particles were analyzed with a Renishaw inVia Raman microscope using Wire 3.4 software to collect images and spectra of the particles. Spectra were collected with a 532 nm excitation laser. The filter was scanned at 20x magnification. Eight transects were made in a straight line across the filter covering 8% of the filter (Fig. 2). Particles were imaged prior to spectra collection. If particles were smaller or larger than the 20x field of view, 50x or 5x magnification was used to image the particle.

2.8. Data Validation and Analysis

The collected spectra from FTIR and Raman spectroscopy were baseline corrected and smoothed prior to identification. OpenSpecy (Cowger et al., 2021) and a custom library database were used for identifying the particles based on their spectra. Pearson’s correlation coefficient (Pearson’s r) of greater than or equal to 0.8 was used as a statistical cutoff for a good fit between the reference and the particle spectrum (Giani et al., 2023; Zhu et al., 2023). All the plastic particle spectra (Pearson’s r > 0.8) were then manually checked to ensure that the spectral peaks were well matched with the reference peaks from both the libraries (Renner et al., 2019). Particles (Pearson’s r > 0.8) were not used in the subsequent analysis if they had peaks that either did not match the overall pattern in the reference spectrum or had a low enough signal-to-noise ratio that it was challenging to interpret the true signal. Examples of spectra that were included or excluded in the analysis are shown in Figure 3,

Spectral peaks were identified according to commonly reported Raman shifts in the literature. Plastic degradation or weathering peaks were identified in all of the samples. Non-plastic particles were compared to reference spectra for a variety of different materials, including fur, cellulose, cotton, sand, chitin, and plant material. The non-plastic particles were identified by a Pearson’s r of 0.8 or greater without manually comparing the spectra matches.

2.9. Statistics

Fulton’s condition index (K) (Equation 1) was calculated to determine the overall health of the fish (Ricker, 1975).

Equation 1: K=(W/TL³)* 100; W is the fish weight and TL is the total length.

MP abundance and occurrence data were non-normally distributed, so nonparametric analyses were used. To predict the occurrence of MPs in the muscle, a Random Forest model was generated using the R CARET package’s leave-one-subject-out train() function (Kuhn, 2008). Fish length, sex and plastic content in the GI tract were used as input variables. Spearman’s rank correlation was used to determine the correlation between fish total length and GI tract and muscle MP abundance. Graphpad Prism (10.0.2) was used to calculate the Spearman’s rank correlations and to generate the visualizations. Significant correlations were accepted if the p-value of the Spearman’s rank correlation was less than 0.05.

The Random Forest analysis used MP occurrence in muscle samples, and the Spearman’s rank correlation used the muscle MP abundance.

All concentrations are reported as the number of particles per gram of tissue wet weight. Data from the Raman analysis are based on the whole filter plastic count estimates extrapolated from the 8% of the filter that was analyzed. No corrections were made for the FTIR data since the whole filter was analyzed.

In the eighteen fish analyzed, 69 MPs were identified in the GI tract or the muscle. The fish collected from Falmouth generally contained more MPs than the fish collected from Bourne. Given the developing nature of the field, we describe the different metrics used to verify our sample analysis below. In the next section, the differences in MP morphologies are compared between the GI tract and muscle samples. Following that, we examine the impact of fish total length and sex on MP abundance.

3. 1. Methodology Verification

All encountered particles were analyzed using either Raman or FTIR. Of the 2,008 particles analyzed in various fish tissue samples, only 3.4% (69 particles) were identified as plastic (Table S2). All particles identified as plastic had a Pearson’s r correlation of at least 0.82, and an average correlation of 0.91 (Table S3). In Falmouth fish, there were 50 plastic particles in the GI tract samples and nine in the muscle tissue. The Bourne fish had nine plastic particles in the GI tract and only one in the muscle tissue.

In our procedural blanks, only one particle was identified as plastic (polypropylene). No polypropylene was detected in the fish samples analyzed concurrently with the contaminated procedural blank. Due to the detection of only a single plastic particle out of 11 procedural blanks, no blank correction was performed. Of the 180 particles analyzed with a correlation of at least 0.8, 70 particles were included in the microplastic analysis. Particles not included could be plastic particles that had altered spectra due to particle oxidation or the presence of additives. The analysis we used is highly conservative, due to uncertainty surrounding the 110 particles not included in the analysis.

Numerous plant-based, chitin and or bone particles were detected with FTIR analysis (Fig. S2A & S2B). Fish collected from Bourne had more plant-based particles in their GI tracts than Falmouth fish (Fig. S2A). Some cellulose and cotton particles were also detected in the samples (Fig. S2). Raman spectroscopy analysis detected primarily minerals, metal compounds, and fragments of soot or black pigment (Fig. S2C & S2D).

3.2. Plastic particle identification

3.2.1. Plastic Concentrations

MP occurrence varied widely between the water and fish tissue (GI tract and muscle) samples. The water samples had no detected MPs (Fig. 4A). GI tract samples contained MPs more frequently than the muscle samples (Fig. 4A). Most Falmouth fish contained MPs, with 87.5% of the GI tract samples and 62.5% of muscle samples containing MPs (Fig. 4A). The Bourne sampling site had a lower MP occurrence, with 40.0% of the GI tract samples and 10.0% of muscle samples containing MPs (Fig. 4A).

MP concentration varied by tissue and sampling site, with the Falmouth GI tract samples containing the largest concentration of plastics (Fig. 4B). The median concentration of MPs in the GI tract of fish from Falmouth was 83 particles/g w.w. while the muscle samples had a median concentration of only 11 particles/g w.w. tissue. Both the GI tract and muscle samples from Bourne fish had a median concentration of 0 particles/g w.w. with the GI tract having a 2.6x greater range compared to the muscle samples (Fig. 4B). Many fish did not contain any MPs (Table 1), leading to large standard deviations.

3.2.2. Plastic Sizes

Table 1

Average Concentration and Size of MPs in fish tissues from Falmouth and Bourne. The mean concentration ± 1 standard deviation is shown. For size characteristics, the medians are shown with range in parentheses.
Collection Site	Falmouth		Bourne
Tissue	GI Tract	Muscle	GI Tract	Muscle
MP Occurrence (# MP samples/total sample number)	7/8	5/8	4/10	1/10
MP Concentration (Particle Count/g w.w.)	85.5 ± 70.2	11.4 ± 12.5	12.2 ± 18.1	1.69 ± 5.36
Length (µm)	24.8 (2.60–1291)	15.5 (4.26–28.1)	24.6 (5.19–728)	8.73
Width (µm)	12.6 (2.10–140)	9.52 (1.79–13.8)	13.8 (2.35–248)	8.95
Aspect Ratio (Length/Width)	1.41 (0.61–51.0)	2.13 (0.97–10.8)	2.21 (1.28–6.78)	0.98

The MP size distributions were similar between the two collection sites (Table 1), so the data from the sampling sites were combined to more closely examine how MP characteristics differ between tissues. Particles 2 µm − 5 mm in length were analyzed in the collected samples. Length is defined as the longest dimension of the particle in a 2-dimensional plane. The particle lengths ranged from 2.6 µm − 1291 µm. The median MP length in the GI tract was 24.7 µm, and the median MP length for the muscle samples was 14.5 µm (Fig. 5A). MPs in the GI tract had a greater size range of 1289 µm compared to the muscle MP’s size range of 23.9 µm (Fig. 5A); however, for both tissue types most of the particles (81.2%) were smaller than 50 µm in length (Fig. 5A).

3.2.3. Plastic Shape

The particle aspect ratio was determined by dividing the length of the particle by the particle width. The particle aspect ratios varied more widely in the GI tract than in the muscle samples (Fig. 5B). The median aspect ratios for the GI tract and muscle were approximately 1.6 and 1.9, respectively (Fig. 5B).

An aspect ratio of three or greater was used to define a particle as a fiber (Li et al., 2020). Any particles with an aspect ratio below three were defined as a fragment. The identified MPs were predominantly fragments (Fig. 5C). The GI tract and muscle samples had similar percentages of fragments and fibers. The GI tract particles were 78.6% fragments and 21.4% fibers, and the muscle particles were 80.0% fragments and 20.0% fibers (Fig. 5C).

3.2.4. Polymer Types

Nylon was the major polymer present in the samples (Fig. 6). Polyethylene was found in the GI tracts collected from both Falmouth and Bourne sites in roughly equivalent percentages (Fig. 6A). We rarely found polypropylene and polyurethane in the samples (Fig. 6A). The GI tract particle composition consisted of nylon (84.8%), polyethylene (11.9%), polypropylene (1.7%), and polyurethane (1.7%) (Fig. 6B). The MPs found in the muscle samples were all nylon (Fig. 6B).

The full range of both the length and aspect ratio for each particle is shown in Figure S3A. The length of the nylon particles was significantly correlated (r = 0.5; p-value < 0.001) with aspect ratio (Fig. S3A & S3B; Linear regression R² = 0.74). The length of the polyethylene particles, on the other hand, was not correlated with the aspect ratio (r = 0.42; p-value 0.35; Fig. S3B). The polyethylene particles and nylon particles had similar proportions of fragments (Fig. S3C).

3.2.5. Plastic Weathering

Identified MPs frequently had two spectral peaks (800–900 and 1600–1700 cm^− 1) suggestive of particle oxidation (Fig. S4; Matsui et al., 2000; Phan et al., 2022). An example of a spectrum displaying these oxidation peaks is illustrated in Figure S4A & S4B along with a picture of the particle and its identified chemical structure. Many particles found in both the GI tract and the muscle samples contained both of those peaks, indicating oxidation of the particles (Fig. S4C). Fewer MPs had the additional 1600 cm^− 1 peak compared to the 800–900 cm^− 1 peak (Fig. S4C). It should also be noted that only 10 MPs were detected in the muscle samples, which could skew the reported peak proportions.

3.3. Impact of Fish Length on Microplastic Abundance

MP abundance correlated with fish total length in the muscle and GI tract; however, key differences between the fish from the different sampling sites complicated this analysis. Only female fish were caught at the Falmouth site (8/8 fish), and mostly male fish were caught at the Bourne site (8/10 fish). The fish collected at the Falmouth site were also larger than those collected at the Bourne site (Fig. 7). These site differences made it challenging to disentangle the impact of sex and fish total length from the different collection sites (Fig. 7). Fish collected from both sites had equivalent Fulton’s condition factors.

Additionally, the water samples from both sites had MPs below the limit of detection, indicating that MP prevalence is low at both collection sites. Therefore, for the following analyses the information from both collection sites has been combined.

Fish total length correlated with MP abundance in both the GI tract (r = 0.51; p-value 0.03) and the muscle (r = 0.48; p-value 0.04; Fig. 8A & 8B). Both comparisons showed a moderately positive relationship. GI tract MP abundance did not correlate with muscle MP abundance (r = 0.25; p-value 0.31; Fig. 8C). Female fish had a greater MP abundance than male fish in both the GI tract and muscle (Fig. 8D). In fact, only female fish had MPs present in the muscle (Fig. 8D). The random forest analysis determined that fish total length was the most important variable for predicting muscle MP occurrence, followed by fish sex and then GI tract MP abundance (Table S4).

The bioaccumulation potential of environmental MPs is not well understood, in part because MP detection is a developing field with little harmonization amongst methods. This study sought to draw on existing recommendations from the field while expanding on both classification techniques and the detectable size range of MPs. Using this rigorous approach, we compare the characteristics of MPs found in the GI tract and muscle. Of particular focus in the following sections are the small size of detected MPs and the correlation of MP abundance and fish total length. Other factors are also discussed to ensure a thorough characterization of the data for future research to draw upon.

4.1. Importance of Methods in MP Assessment

The concentrations and occurrence frequencies of MPs from different field studies are difficult to compare due to the lack of standardized methods across the field. It is unclear how much differences in MP identification approaches (Abbasi et al., 2018; Akhbarizadeh et al., 2018; Curtean-Bănăduc et al., 2023; My et al., 2023; Piskuła & Astel, 2023; Qaiser et al., 2023), collection methods (Guilhermino et al., 2021; Matupang et al., 2023; Menéndez et al., 2023; Piskuła & Astel, 2023; Sultan et al., 2023), and quality control measures (Esmaeilbeigi et al., 2023; Kumari et al., 2023; Nawar et al., 2023; Piskuła & Astel, 2023; Qaiser et al., 2023; Raza et al., 2023; Sabilillah et al., 2023; Widyastuti et al., 2023; Wu et al., 2023) have influenced these results. These differences highlight the often reported (Provencher et al., 2017, 2020) need for harmonized methods of MP analysis and improved methods for detecting smaller MPs.

In our study, no significant background contamination was seen, with only one MP detected from the procedural blanks. Recovery assessments with 250 µm particles yielded an 80% recovery rate for three different polymers tested. All of the tested polymers were fragments or spheres, so the recovery rate for fibers in this study is unknown. Particles smaller than 250 µm were not tested due to difficulty in small particle manipulation. Protocols to assess the recovery of different sizes and shapes of MPs are essential to increase the reproducibility of these results.

MP sub-sampling was done in this study to reduce the analysis time of each sample; however, the use of sub-sampling can introduce misestimates of MP concentration (Brandt et al., 2021). Sub-sampling recommendations are still being developed, especially for MPs smaller than 50 µm. Previous recommendations suggest that our results should be representative of MPs present but might not capture MP polymers that occurred rarely in our sampling environment (De Frond et al., 2023; El Khatib et al., 2023).

4.2. Comparison of MPs in Muscle and GI Tract

We predicted that the size of the MPs is the most important parameter for potential bioaccumulation, but we also considered other factors that are known to impact particle uptake, such as particle shape, polymer composition, and degradation status.

4.2.1. Concentrations of MPs

We detected no plastic particles in our water samples using an established method for detecting small MPs in water (Covernton et al., 2019; Dent et al., 2023; Green et al., 2018; Qaiser et al., 2023; Sabilillah et al., 2023). Despite our water samples having non-detectable levels of MPs, MPs were detected in the sampled fish, indicating MP presence in our sample sites.

Table 2

MP Concentrations in Fish: A comparison of the average concentrations of MPs found in the GI tract and muscle. Concentrations are shown as the average number of MPs per gram of wet weight ± 1 standard deviation. If a range is present, multiple species were sampled in the study. A dash indicates the tissue was not analyzed. N.D. = not detected
Study	MP Size Limit of Detection (µm)	GI Tract Concentration (MPs/g W.W.)	Muscle Concentration (MPs/g W.W.)
Current Study	2	45 ± 60	6 ± 10
Di Giacinto et al., 2023	1–10	-	0.14–0.27
Su et al., 2019	20	0.1 ± 0.1–8.8 ± 7.4	N.D.
Park et al., 2023	20	3.42 ± 3.2	-
Soltani et al., 2023	100	0.04 ± 0.05–0.47 ± 0.28	-
Wu et al., 2023	100	0.02 ± 0.05	0.01

Both collection site and tissue type affected MP occurrence frequencies. MPs occurred more frequently in the GI tract than in the muscle samples. This trend is generally agreed upon in the literature (Jeyasanta et al., 2023; My et al., 2023; Pandey et al., 2023) with one study finding the opposite trend (Dent et al., 2023).

The present study’s estimated GI tract MP concentration (45 particles/g w.w) is higher than those in other studies (Table 2). The muscle samples had a lower MP concentration than the GI tract samples, but the muscle sample concentration was still greater than most of those previously reported (Table 2). A difference in size limits of detection could partly explain this difference, as we were able to detect smaller particles than most previous studies.

The absorption of MPs into the GI tract epithelium cannot be determined from our results since both GI tract and GI tract contents were digested together. It has previously been shown that GI tract contents contain higher MP concentrations than just the GI tract epithelium (Curtean-Bănăduc et al., 2023; Rosas et al., 2023), suggesting that few particles are absorbed by the epithelium from ingested food; however, uptake is known to be in part size-dependent, and the retention and rate of tissue translocation of the smaller MPs found in our study are unknown.

4.2.2. Plastic Sizes

The size of MPs found in both the environment and in biota ranges widely throughout the literature. MPs range from 1 µm − 5 mm in commonly agreed definitions (Rochman et al., 2019a); however, few studies measure the full-size range of these particles. It is common for studies to have minimum size limits of detection that are much higher than the 1 µm theoretical minimum size, or to not report the size limits of detection (Reviewed in Pitt et al., 2024). For example, we found that in studies reporting a minimum size detection limit, the median value was 206 µm, with most studies not reporting a minimum size detection limit (Pitt et al., 2024). These differences in size detection limits make it challenging to compare the abundance of MP sizes detected in fish tissues, such as the muscle, which is predicted to preferentially accumulate smaller MPs. The current study was able to measure particles 2 µm − 5 mm in size.

Studies often find that smaller particles are the most prevalent in the atmosphere (Liao et al., 2021), water (Shim et al., 2022), and organisms (Valente et al., 2023). Our study found the GI tract and muscle MP median lengths to be 24.7 µm and 14.5 µm respectively, sizes that are smaller than the most commonly reported minimum size detection limits for biological samples (Amini-Birami et al., 2023; Collard et al., 2017; Matluba et al., 2023; McIlwraith et al., 2021; Widyastuti et al., 2023). Other studies in non-GI tract tissues have found MPs larger than those found in the current study (Abbasi et al., 2018; Curtean-Bănăduc et al., 2023; Esmaeilbeigi et al., 2023; Guilhermino et al., 2021; Matias et al., 2023; McIlwraith et al., 2021; My et al., 2023; Nawar et al., 2023; Sabilillah et al., 2023; Sultan et al., 2023; Wu et al., 2023), but the uptake pathway used by these larger particles is unclear, as in some cases, they are above previously established uptake limits (Lusher et al., 2017).

The MPs found in the muscle were both smaller in size and size range than those found in the GI tract, implying a size restriction for particles to enter the muscle. It has been suggested that particle translocation from the GI tract through a mechanism called persorption has an upper size limit of 150 µm (Lusher et al., 2017). Persorption is the paracellular movement of particles in the desquamation region of epithelial cells (Volkheimer, 1974). This mechanism of transport has been previously critiqued as being unlikely and has not been demonstrated since its inception (Hussain et al., 2001; O’hagan, 1996). The actual upper limit of translocation is uncertain, but previous research suggests that only particles smaller than 21 µm are capable of translocating into tissues (Deng et al., 2017; Lusher et al., 2017; Huang et al., 2022; Jin et al., 2021).

In the present study, eight (out of ten) of the MPs detected in muscle were smaller than 21 µm. The maximum size for particles found in the muscle was 28 µm. How these particles reached the muscle is unclear, but some phagocytic cells are 25–30 µm in size and might be capable of engulfing and transporting these particles (Lendeckel et al., 2022). GI tract absorption of particles is the most likely route of uptake, as particles larger than 10 µm are unlikely to be internalized from the lungs or gills (Prata, 2023). We suggest that the MPs detected in the muscle were moved there following phagocytic cell engulfment from the GI tract.

The present study is the first to look for the full-size range of MPs in non-GI tract tissues without significant contamination issues. Previous environmental studies with size detection limits lower than 21 µm either did not look for larger MPs (Di Giacinto et al., 2023; Ferrante et al., 2022) or had a significant amount of background contamination (Akoueson et al., 2020; Rasta et al., 2021; Su et al., 2019); however, it is clear from previous studies that particle translocation is governed by factors other than size, as particles theoretically small enough to translocate have not moved through the intestinal wall (Kim et al., 2020; Pyl et al., 2022).

4.2.3. Particle Shape

The higher proportion of fragments, versus fibers, in our samples compared to previous studies could be due to the smaller size of MPs detected in our study versus these earlier studies (Akoueson et al., 2020; Guilhermino et al., 2021; Mai et al., 2023; Matias et al., 2023; Matupang et al., 2023; McIlwraith et al., 2021; Menéndez et al., 2023; Piskuła & Astel, 2023; Sánchez-Guerrero-Hernández et al., 2023). Previously, fibers have tended to predominate in larger size fractions of MPs, while fragments make up the majority of particles < 100 µm (Liao et al., 2021; Piskuła & Astel, 2023). On the other hand, some studies that quantified particles down to 20 µm in size have detected primarily fibers, emphasizing that size is not necessarily the major predicting factor for determining particle shape (Kumari et al., 2023; Pandey et al., 2023; Park et al., 2023; Sabilillah et al., 2023; Wu et al., 2023; Zhu et al., 2023).

Previously, it has been speculated that fibers are more likely to translocate or be internalized by cells compared to other MP shapes (Ramsperger et al., 2023); however, our study contradicts that finding as fibers were not found at a higher frequency in muscle.

4.2.4. Polymer Types

Nylon was the predominant polymer (80–88%) in the GI tract samples. While nylon has occasionally been reported as the major polymer in samples spanning a large range of trophic levels (Amini-Birami et al., 2023; Covernton et al., 2022; Nawar et al., 2023; Sánchez-Guerrero-Hernández et al., 2023), polypropylene and polyethylene are usually the most common polymers reported in marine organisms (Dent et al., 2023; Di Giacinto et al., 2023; Giani et al., 2023; Kor et al., 2023; Mahu et al., 2023; Matluba et al., 2023; Park et al., 2023; Zhu et al., 2023). Polypropylene and polyethylene are less dense than water, and so species found near the surface of the water could be more likely to encounter these polymer types. Nylon, on the other hand, is slightly denser than seawater, and so would be more likely to sink to the benthic and benthopelagic environment where killifish dwell.

In contrast to the GI tracts, nylon was the only polymer found in the muscle samples. One other study has found nylon at an increased proportion in muscle compared to the GI tract (Sultan et al., 2023) with other studies seeing no difference (Menéndez et al., 2023). Our results suggest that nylon more readily penetrates into muscle than polyethylene.

4.3. Implications for MP Bioaccumulation

We did not calculate a bioaccumulation factor from our results due to a lack of information regarding the water and sediment concentrations of MPs at the collection sites. Our results suggest that few MPs are ultimately internalized to the muscle. MP deposition in the muscle was potentially impacted by the sex and total length of the fish.

4.3.1. Relationship between Fish Length and MP Abundance

The long-term sequestration of larger MPs (> 100 µm) has previously been shown to be unlikely (Covernton et al., 2022; Giani et al., 2023; Matias et al., 2023; McIlwraith et al., 2021; Valente et al., 2023; reviewed in Pitt et al., 2024). The bioaccumulation potential, especially for smaller MPs (< 100 µm), has remained unclear, largely due to a lack of information about the distribution and abundance of MPs in non-GI tract tissues; however, our study has started to address this knowledge gap.

A positive relationship was observed between fish length and the MP abundance in the GI tract. This finding agrees with the majority of studies (Esmaeilbeigi et al., 2023; Guilhermino et al., 2021; Jeyasanta et al., 2023; Mai et al., 2023; Makhdoumi et al., 2021; Matupang et al., 2023; McIlwraith et al., 2021) despite some contradictory evidence (Dent et al., 2023; Kor et al., 2023; Park et al., 2023; Sultan et al., 2023). It has been suggested that one reason studies report a positive correlation could be that as fish grow, they can ingest a wider size range of MPs and, consequently, are exposed to more MPs in the environment (Bhattacharjee et al., 2023). Larger fish also have increased dietary needs, and so might ingest more prey, thus increasing their incidental MP ingestion.

A positive relationship was also seen between fish length and MP abundance and occurrence in the muscle. This contrasts with most of the existing literature, which did not see this trend for non-GI tract tissues (Guilhermino et al., 2021; Makhdoumi et al., 2021; McIlwraith et al., 2021). The lowest reported MP size for some of these studies was approximately 63 µm, which is 31.5 times larger than our lowest reported MP size (Guilhermino et al., 2021; McIlwraith et al., 2021). It seems likely that MP size influences trends seen regarding fish length. Our results suggest that small MPs, which are often not quantified, are the primary MPs to be found in tissues like the muscle. Killifish increase in length as they age (Abraham, 1985), so the correlation between fish total length and muscle MP abundance also could be indicative of bioaccumulation.

4.3.2. Relationship between MP Abundance in the GI Tract and the Muscle

There was no observed relationship between the abundance of MPs in the GI tract and in the muscle. This finding agrees with the majority of previous studies (Arafat et al., 2023; Gedik et al., 2023; Raza et al., 2023) with a few conflicting reports (McIlwraith et al., 2021; Nawar et al., 2023). Generally, this suggests that the MP residence times differ between these tissues, and therefore one tissue cannot be used to predict the content of another. GI tract residence time is short, with previous studies showing rapid elimination of larger MPs (> 250 µm) in killifish (Ohkubo et al., 2020). Given the short residence time of MP in the GI tract and the lack of correlation between GI tract and muscle MP abundance, bioaccumulation assessments should not include GI tract data when considering the long-term sequestration potential of MPs.

4.3.3. Other Factors

There were several differences between the sites that potentially contributed to the different patterns in MP abundance that we saw including: proximity to a residential area, connection to the ocean, differences in recent weather patterns, and differences in the characteristics of the collected fish; however, the MP water concentrations were not detectable from both collection sites, indicating similar low contamination levels.

Only female fish had MPs in muscle samples, and they also had greater GI tract MP abundance. This finding could be confounded by several biases, such as the collected females being predominantly from the Falmouth site and that female fish are larger than male fish; however, a couple of other studies have observed the same trend (Esmaeilbeigi et al., 2023; Matupang et al., 2023). Overall, relatively few studies have compared male and female fish in terms of the MP content of GI tract and tissues. In the future, more research is needed to investigate the origins of this trend.

4.4. Conclusions

There continues to be a dearth of information regarding the distribution of smaller MPs to non-GI tract tissues. This study only detected smaller MPs in the muscle tissues, suggesting a potential upper limit on the sizes of MPs in muscle tissue. This MP abundance was positively correlated with fish total length, indicating that the smaller MPs detected in this study might bioaccumulate. Future studies should focus MP quantification efforts on small MPs in non-GI tract tissues to further investigate this trend.

MPs = Microplastics

GI = Gastrointestinal

FTIR = Fourier-transform infrared spectroscopy

PVC = Polyvinyl chloride

PET = Polyethylene terephthalate

PE = Polyethylene

ATR = Attenuated total reflection

5.1 Availability of Data and Materials

The data supporting the findings of this study are available within the paper and its supplementary material and data files. Raw data files (Raman and FTIR spectra) are available from the corresponding author upon reasonable request.

5.2 Competing Interests

The authors declare no competing financial interest.

5.3 Funding

This research was supported in part by Woods Hole Sea Grant (Award No. NA18OAR4170104, project R/P–89) to M.E.H., N.A., and J.A.P. and by an NSF Graduate Research Fellowship to J.A.P. Additional support was provided by Woods Hole Sea Grant (Award No. NA18OAR4170104, project R/O-59 to S.M.G.) and by the Woods Hole Center for Oceans and Human Health (NIH/NIEHS grant P01ES028938 and NSF grant OCE-1840381 to M.E.H. and N.A.).

5.4 Authors' Contributions

JAP: Conceptualization, Formal analysis, Funding acquisition, Methodology, Visualization, Investigation, Writing - original draft, Writing - review & editing. SMG: Methodology, Software, Funding acquisition, Resources, Writing – Review & Editing; SY: Methodology, Software, Writing – Review & Editing; APMM: Methodology, Resources, Writing – Review & Editing; MEH: Conceptualization, Funding acquisition, Methodology, Supervision, Writing - review & editing; NA: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing - review & editing

5.5 Ethics approval

All procedures performed in the study were in accordance with the ethical principles described in the Guide for the Care and Use of Laboratory Animals (U.S. National Research Council, Eighth Edition, 2011). Euthanasia was performed according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2013 Edition). The research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Woods Hole Oceanographic Institution (Assurance D16-00381 from the Office of Laboratory Animal Welfare (OLAW) at the U.S. National Institutes of Health).

5.6 Consent to publish

Not applicable

5.7 Consent to participate

Not applicable

5.8 Acknowledgments

Not applicable

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The abundance and localization of environmental microplastics in gastrointestinal tract and muscle of Atlantic killifish (Fundulus heteroclitus)

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Quality Control

2.3 Fish collection and tissue sampling

2.4 Sample Digestion

2.5 Particle Recovery experiments

2.6. FTIR spectroscopy Sample Analysis

2.7. Raman spectroscopy Sample Analysis

2.8. Data Validation and Analysis

2.9. Statistics

3. Results

3. 1. Methodology Verification

3.2. Plastic particle identification

3.2.1. Plastic Concentrations

3.2.2. Plastic Sizes

3.2.3. Plastic Shape

3.2.4. Polymer Types

3.2.5. Plastic Weathering

3.3. Impact of Fish Length on Microplastic Abundance

4. Discussion

4.1. Importance of Methods in MP Assessment

4.2. Comparison of MPs in Muscle and GI Tract

4.2.1. Concentrations of MPs

4.2.2. Plastic Sizes

4.2.3. Particle Shape

4.2.4. Polymer Types

4.3. Implications for MP Bioaccumulation

4.3.1. Relationship between Fish Length and MP Abundance

4.3.2. Relationship between MP Abundance in the GI Tract and the Muscle

4.3.3. Other Factors

4.4. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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