# Exposure of black soldier fly larvae to microplastics of various sizes and shapes: ingestion and egestion dynamics and kinetics

Christelle Planche, Siebe Lievens, Tom van der Donck, Jason Sicard, Mik van der Borgh

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Christelle Planche, Siebe Lievens, Tom van der Donck, Jason Sicard, Mik van der Borgh. Exposure of black soldier fly larvae to microplastics of various sizes and shapes: ingestion and egestion dynamics and kinetics. Waste Management, 2025, 203, pp.114852. 10.1016/j.wasman.2025.114852. hal-05015679

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Research Paper

# Exposure of black soldier fly larvae to microplastics of various sizes and shapes: Ingestion and egestion dynamics and kinetics

Christelle Planche<sup>a</sup>, Siebe Lievens<sup>b</sup>, Tom Van der Donck<sup>c</sup>, Jason Sicard<sup>a</sup>, Mik Van Der Borght<sup>b,*</sup>

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## ARTICLE INFO

**Keywords:**

Polyethylene

Food waste

Bioconversion

Hermetia illucens

Bioaccumulation

## ABSTRACT

Black soldier fly (BSF, *Hermetia illucens*) larvae can valorize food waste into high-valuable products including animal feed. However, these wastes may contain microplastics originating from food packaging, potentially compromising larval growth and their safety as feed. This study investigates the impact of microplastic sizes and shapes on their ingestion and egestion by BSF larvae during waste bioconversion. BSF larvae were reared for 10 days on artificial food waste spiked with three size ranges of spherical or irregularly shaped fluorescent polyethylene microplastics. Daily, several larvae were dissected (n = 9 per treatment) to isolate their gut and to finally determine the number of particles in their alimentary canal using a fluorescence microscope. The microplastics had no impact on larval growth and no bioaccumulation of microplastics was observed in the larval gut (bioaccumulation factors < 0.3). However, there was a significant difference in the ingestion rate based on the different particle sizes. While almost no particles exceeding 100 µm were ingested by the larvae, a steady increase in microplastics was observed in the larval gut for the smaller particles. However, a three-day starvation period reduced the number of microplastics in the larval gut by over 90 %. No significant difference was observed between spherical and irregularly shaped microplastics in terms of larval bioaccumulation. We proposed a kinetic model for representing the temporal dynamics of microplastics accumulation and elimination with respect to particle distribution parameters, enabling the results obtained in this study to be extrapolated to other microplastic sizes.

## 1. Introduction

Given the growing world population and the consequent need to increase food production while staying within our planet's limits, the industrial production of edible insects seems a relevant solution, considering the low resource use associated with their production and their ability to fit into circular food systems (Van Huis et al., 2021). In addition, edible insect farms are a way of valorizing biowaste by using it as a growing substrate for insects and converting it into high value-added products such as animal feed, biofertilizers, biopolymers, bio-enzymes or even biodiesel (Kee et al., 2023). In particular, black soldier fly (BSF, *Hermetia illucens*) larvae are considered one of the most promising insect species for the production of feed for livestock and

aquaculture. BSF larvae can grow on a wide variety of organic substrates (Fitriana et al., 2022; Lievens et al., 2021) and this capacity is currently considered as an environmentally-friendly way of recycling biowaste, offering an interesting complementary approach to already existing biowaste management solutions (Amrul et al., 2022; Rehman et al., 2023). Food waste streams, such as from supermarket or household waste, are a common feedstock but may be contaminated by a fraction of mis-sorted plastic packaging. This issue can become significant, as 300,000 pieces of microplastics have been detected per kg of food waste collected from grocery stores (Golwala et al., 2021). Similar high numbers were found by Tan et al. (2022) who reported 5,780 to 6,330 pieces of microplastics per kg of household food waste. Attempts are being made to reduce the amount of plastic contamination in food waste

<footnote><sup>a</sup> INRAE, UR370 Animal Products Quality Unit, F-63122 Saint-Genès-Champanelle, France</footnote>

<footnote><sup>b</sup> KU Leuven, Research Group for Insect Production and Processing, Campus Geel, Kleinhoefstraat 4, 2440 Geel, Belgium</footnote>

<footnote><sup>c</sup> KU Leuven, Department of Materials Engineering, Campus Arenberg, Kasteelpark Arenberg 44, 3001 Leuven, Belgium</footnote>

<footnote>* Corresponding author at: KU Leuven, Campus Geel, Faculty of Engineering Technology, Department of Microbial and Molecular Systems, Kleinhoefstraat 4, 2440 Geel, Belgium.</footnote>

<footnote>E-mail addresses: christelle.planche@inrae.fr (C. Planche), siebe.lievens@uliege.be (S. Lievens), tom.vanderdonck@kuleuven.be (T. Van der Donck), jason.sicard@inrae.fr (J. Sicard), mik.vanderborght@kuleuven.be (M. Van Der Borght).</footnote>
streams, and processing facilities use multiple approaches, such as me-
chanical depackagers (EPA, 2021). Even though manufacturers claim
purity rates of the organic fraction obtained after deconditioning of over
99 % (percentage of desired material), these approaches are not fully
effective. Porterfield et al. (2023) found plastic levels of up to 0.19 %
(w/w) in depackaged waste streams, while Lievens et al. (2022b)
observed levels up to approximately 1 % (w/w) in organic waste
streams. In addition, application of depackaging technologies may
negatively contribute to the defragmentation of larger plastic pieces into
microplastics, or even nanoplastics of various sizes and shapes (Bandini
et al., 2022).

Several studies have revealed that microplastics (plastic particles < 5 mm) can have various adverse effects on the environment and living organisms, including humans (Chen et al., 2023; Fournier et al., 2023; Lu et al., 2022; Osman et al., 2023; Wang et al., 2021). Microplastics can release toxic additives and monomers to the organisms or biota and act as carriers of various harmful adsorbed contaminants including heavy metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and per- and polyfluoroalkyl substances (PFAS) (Godoy et al., 2019; Golwala et al., 2021; Zhang et al., 2022). The concentration of pollutants accumulated on microplastics can be 6 – 70 times higher than that of the surrounding environment (Golwala et al., 2021). Given the widespread contamination of microplastics in our food waste streams and the wider environment, it is important when using food waste streams as a substrate for the industrial production of BSF larvae to ensure that the presence of microplastics does not impact larval growth and survival, and above all does not compromise the safety of the larvae as feed ingredient for fish, chickens or pigs. Recent studies have shown that BSF larvae are not affected in terms of growth performance, survival, and bioconversion by the presence of polyvinylchloride (PVC) microplastics up to a mass fraction of 5 % in their substrates (Lievens et al., 2022a). Further research questions arise as the ingestion and the bioaccumulation of these microplastics in BSF larvae seem to be dependent on initial particle load, larval mouth size, and consequently on larval age (Lievens et al., 2023a).

Given the diversity of microplastics that can be found in the envi-
ronment, it is now necessary to expand on these initial results, firstly by
studying a wider spectrum of microplastic sizes and shapes, and sec-
ondly by developing mathematical models that can simulate various
practical cases while limiting animal experiments. Indeed, while bio-
waste potentially used as substrates for industrial BSF larvae rearing
may contain very heterogeneous microplastics, no study has yet
addressed the combined impact of the size and shape of these micro-
plastics on the production of larvae subsequently used as feed and the
associated risk if they enter the food chain. To close this research gap
and to enhance the risk assessment associated with the presence of
different microplastics in food waste used for insect-based bioconver-
sion, this research was performed. The aim of this study was to deter-
mine the impact of different microplastic sizes and shapes on BSF larval
growth, while monitoring the microplastic ingestion, egestion and bio-
accumulation. The study focused on polyethylene, which accounts for
34 % of the total plastics market, and is one of the most widely used
types of polymer for food packaging (Golwala et al., 2021; Matthews
et al., 2021). In the first part of this study, three different sizes of
spherical fluorescent blue-labelled microplastics were spiked in an
artificial food waste and their ingestion and egestion dynamics of the
BSF larvae were investigated. In the second part, self-produced fluo-
rescently labelled irregularly shaped microplastics were used to simulate
realistic microplastics that can be found in biowaste. The impact of the
shape on the ingestion and egestion dynamics by BSF larvae was
assessed, in order to identify whether certain microplastics might be
more problematic than others in the BSF larvae biowaste recycling
sector. Finally, to extrapolate these results to other conditions, such as
other microplastic sizes, while avoiding the multiplication of animal
experiments, which today raise ethical and animal welfare issues, a ki-
netic model was developed for the first time to describe the

concentration of microplastics in BSF larvae throughout the rearing and
starvation periods as a function of particle size and shape.

## 2. Materials and Methods

2.1. Microplastic production and characterization

2.1.1. Spherical microplastics

Fluorescent blue polyethylene microspheres with a density of 1.13 g cm⁻³ were purchased from Cospheric (Santa Barbara, USA) in three different size ranges: 38 – 45 µm, 53 – 63 µm and 90 – 106 µm. Supplier data indicate 90 % of the particles to be within the size range boundaries. Assuming normal particle size distribution, this corresponds to a standard deviation of 2.1 µm, 3.0 µm and 4.9 µm for the three size ranges, respectively.

2.1.2. Irregular microplastics

2.1.2.1. *Production of polyethylene microplastics.* Irregularly shaped microplastics were self-produced according to Lievens et al. (2023b) from a linear low-density polyethylene (LLDPE) foil ($\rho$ = 0.87 g cm⁻³; 15 µm thickness) obtained from Flexfilm (Schoonhoven, The Netherlands). Briefly, the microplastics were artificially produced by a cryogenic grinding procedure using a Spex Freezer-Mill (6875, Instrument Solutions, Nieuwegein, the Netherlands). After production, the irregularly shaped microplastics were separated into different size fractions using a Retsch vibrating wet sieving tower (Verder NV, Aartselaar, Belgium), to finally obtain three size fractions of interest. These fractions correspond to the sieving mesh size ranges of 45 – 75 µm, 75 – 106 µm, and 106 – 150 µm, which were further used during the study.

2.1.2.2. Fluorescent staining of polyethylene microplastics. In order to study the fate of irregular self-produced microplastics in BSF larvae, fluorescently staining of these microplastics was carried out with Nile red according to a slight modified procedure of Gao et al. (2022). A Nile red stock solution (Carl Roth, Karlsruhe, Germany) was prepared having a concentration of 1 mg mL⁻¹ in methanol (analytical grade, VWR International, Radnor, USA) and subsequently diluted to 2 µg mL⁻¹ with ultrapure water (arium pro, Sartorius, Belgium). The microplastics were then mixed with the Nile red dye, in a ratio of 5:1 (mg:ml), for 3 h in a glass beaker placed in a 70 °C water bath. Following the dye uptake period, the beaker was then placed for 30 min in an ice bath to cool down. To remove any dye residue attached to the surface, the microplastics were rinsed seven times with ethanol (Technisolv, VWR International, Radnor, USA) and then two times with ultrapure water until a transparent solution was obtained. Finally, the microplastics were dried in an oven at 35 °C and stored in the dark at room temperature preceding further characterization and usage.

2.1.2.3. Characterization. The particle-size distribution of produced microplastics was measured using a laser diffraction particle size analyzer (Mastersizer 3000, Malvern Instruments Ltd, Worcestershire, U.K.). Microplastics were suspended in ethanol and stirred at 3100 rpm (Technisolv, VWR International, Radnor, USA), until an obscuration of approximately 5 % was obtained. The particle size at 10 % (Dv10), 50 % (Dv50, median diameter), and 90 % (Dv90) of the volume distribution were obtained. Each measurement was carried out in triplicate. Similarly to the spherical particles, a Gaussian function was fitted to the obtained size distributions to apply the kinetic model generalized for normally distributed particles. The obtained standard deviation was larger than that of the purchased microspheres (Fig. S2, Table S2).

For the SEM analysis, the microplastics were placed on an aluminium
stub and coated with a 5 nm-thick platinum-palladium layer using a high
vacuum (turbomolecular pumped) coater (Q150T, Quorum). The SEM
images were then produced at 5 kV acceleration voltage with a CBS
(concentric backscatter, BSE) detector in a Nova Nanosem 450 (ThermoFisher Scientific, Waltham, USA), while all other parameters like magnification, working distance (WD) and horizontal field width (HFW) are displayed on the respective image.

## 2.2. Production and spiking of the rearing substrates

Rearing substrate for BSF larvae containing fruits, vegetables, bread, and dairy products was produced according to Lievens et al. (2022b) in order to mimic a food waste stream (25 % dry matter). All ingredients were purchased at a local supermarket in Belgium. The rearing substrate was then spiked with spherical or irregular microplastics. The same number of particles was introduced into the substrate each time, regardless of particle size (Table 1). The substrate was then gently homogenized using a k-beater for 15 min at low disk speed in a food household mixer (Major Titanium KMM020, Kenwood, UK).

## 2.3. Rearing black soldier fly larvae

BSF larvae were obtained from the research colony of the Centre of Expertise Sustainable Biomass and Chemistry (Thomas More University of Applied Sciences, Geel, Belgium) and reared according to Broeckx et al. (2021). Briefly, eggs were collected, and approximately 1.0 g was hatched on 200 g chicken feed (AVEVE, Belgium) having a moisture content of 55 %. After five days, another 600 g of chicken feed was added and, eight days after hatching (DAH), 150 larvae were counted and transferred into a round glass container (Ø = 7.5 cm, H = 10 cm; 3.3 larvae cm⁻²) having a lid with an opening of approximately 4 cm in diameter for ventilation. The larvae received a single batch of control or spiked substrate in the amount of 100 mg larvae⁻¹ day⁻¹. Larvae were then kept for 10 days of rearing followed by 3 days of starvation at 27 °C and relative humidity of 60 %. These rearing experiments were carried out in triplicate, both for the controls and for the different sizes of spherical or irregularly shaped microplastics.

## 2.4. Sampling procedure of the larval gut

During the rearing and starvation phase, daily, three BSF larvae from each container (control or spiked with microplastics, each container being in triplicate) were sampled and washed using tap water. Afterwards, the larvae were weighed, anaesthetized on ice, and their guts were immediately isolated in a phosphate-buffered saline solution (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4 according to Bonelli et al. (2019)) using a stereo microscope (Leica Microsystems, EZ4W, Germany). The obtained guts were then placed on a microscopical object slide, covered with PBS buffer and a coverslip to prevent dehydration preceding the visualization and quantification of the microplastics in the lumen of the gut.

## 2.5. Microplastic visualization and quantification in larval guts

Microplastics were visualized in the lumen of larval guts using a fluorescence microscope (Zeiss, Axioplan, Germany) equipped with a MoticamPro 252A camera (Motic, Hong Kong). The fluorescent micro-

scope featured a halogen lamp with a 96 HE BFP or a 09-filter set (VWR, USA) for the spherical and irregular microplastics respectively. The specific filter set used determines both excitation and emission wavelengths, with values of 450 – 490 nm (09-filter) and 340 – 390 nm (96 HE BFP), and 515 nm (09-filter) and 440 – 450 nm (96 HE BFP), respectively. Microplastic particles were quantified by manually counting and the bioaccumulation factors (BAF) were calculated using Equation (1) (Lievens et al., 2023a):

$$BAF = \frac{w_{BSFL}}{w_s} = \frac{100 N_{BSFL} m_{MP}}{m_{BSFL} w_s} \quad (1)$$

where: $w_{BSFL}$ = final microplastic mass fraction in BSF larvae (g/100 g), $w_s$ = initial microplastic mass fraction in substrate ingested (g/100 g), $N_{BSFL}$ = mean number of microplastics found in larval gut (9 repetitions), $m_{MP}$ = estimated mass of one microplastic particle (g), $m_{BSFL}$ = mean BSF larval wet mass (g, 9 repetitions).

The mass of a microplastic particle (g) was estimated using Equation (2):

$$m_{MP} = \frac{4 \pi r^3 \rho_{MP}}{3} \quad (2)$$

where: r = median radius of the microplastic (m) and $\rho_{MP}$ = microplastic density (g m⁻³).

## 2.6. Kinetic model

A kinetic model has been developed based on existing models for other animals likely to be highly contaminated by environmental microplastics (Liu et al., 2021; Weinstein et al., 2022).

Based on these previous models, the microplastic ingestion and egestion kinetics were assessed separately. The egestion kinetics was first modelled based on the 3-day starvation phase (during which there is no further ingestion by the larvae) while the ingestion kinetics was then modelled based on the previous rearing phase (during which ingestion and egestion simultaneously occur). The model was implemented in Matlab R2023b.

### 2.6.1. Egestion kinetics model

A first order reaction rate model was used for the approximate egestion of microplastics in the BSF larvae. In the symbols, the subscript indicates in which product the microplastic particles are considered, i.e. “s” for the substrate or “BSF” for black soldier fly larvae. The number of microparticle entities per larval mass, subsequently called concentration ($C_{BSFL} = N_{BSFL}/m_{BSFL}$, unit g⁻¹) during egestion can hence be determined with Equation (3) (McQuarrie and Simon, 1997), with $k_e$ the egestion rate constant (d⁻¹):

$$\frac{dC_{BSFL}}{dt} = -k_e C_{BSFL} \quad (3)$$

It follows that from the time when exposure to the spiked substrate ends $t_{eoe}$ (end of exposure time), microplastic larval concentration reduces from the end of exposure value $C_{BSFL}^{eoe}$, respecting the following equation (McQuarrie and Simon, 1997):

### Table 1

Bioaccumulation factors (BAF) for spherical and irregularly shaped microplastics (MP) in 17 DAH old BSF larvae reared on a spiked artificial food waste stream. Data represent the mean value ± SD (standard deviation over 9 repetitions).

<table><thead><tr><th>MP shape</th><th>MP size (µm)</th><th>Number of MPs in substrate (g⁻¹)</th><th>MP mass fraction in substrate (w/w%)</th><th>BAF</th></tr></thead><tbody><tr><td>Spherical</td><td>38 – 45</td><td>(26.0 ± 0.1) × 10³</td><td>0.11</td><td>(2.8 ± 0.9) × 10⁻¹ a</td></tr><tr><td>Spherical</td><td>53 – 63</td><td>(26.0 ± 0.1) × 10³</td><td>0.30</td><td>(8.9 ± 6.2) × 10⁻² b</td></tr><tr><td>Spherical</td><td>90 – 106</td><td>(26.0 ± 0.1) × 10³</td><td>1.45</td><td>(3.4 ± 2.4) × 10⁻³ c,d</td></tr><tr><td>Irregular</td><td>45 – 75</td><td>(26.0 ± 0.1) × 10³</td><td>0.37</td><td>(5.9 ± 2.5) × 10⁻² b</td></tr><tr><td>Irregular</td><td>75 – 106</td><td>(26.0 ± 0.1) × 10³</td><td>0.86</td><td>(7.6 ± 2.6) × 10⁻³ c</td></tr><tr><td>Irregular</td><td>106 – 150</td><td>(26.0 ± 0.1) × 10³</td><td>2.62</td><td>(3.7 ± 3.8) × 10⁻⁴ d</td></tr></tbody></table>

Different superscript letters indicate significant differences among BAF values (p < 0.05).
$$C_{BSFL} = C_{BSFL}^{\text{teoe}} e^{-k_{e}t} \quad (4)$$

$k_e$ can be directly estimated using a linear regression of the natural logarithm transformed concentration of microplastics in larvae versus egestion time to obtain the slope (McQuarrie and Simon, 1997).

$$\ln(C_{BSFL}) = \ln(C_{BSFL}^{\text{teoe}}) - k_{e}t \quad (5)$$

The degree of linear correlation between observation and this prediction, goodness-of-fit indicator, is provided through the adjusted coefficient of determination for egestion $R_{Adj}^2$ in the results.

The half-life of egestion (d) is complementarily provided in Table 2, and it is constant and equal to (McQuarrie and Simon, 1997):

$$t_{1/2} = \frac{\ln(2)}{k_e} \quad (6)$$

### 2.6.2. Ingestion kinetics model

Knowing that for BSF larvae, microplastic ingestion depends on the size of the larval mouth opening, which must be larger than ingested particles and increases with larval age (Lievens et al., 2023a), this additional factor was taken into account in this study, unlike models already developed on other animals (Liu et al., 2021; Weinstein et al., 2022). Larval mouth opening size was estimated through Lievens et al. (2023a) data for the size of mouth in the transverse plane, generally the most restrictive plane. During the 8–17 DAH rearing period considered in this study, their measurements showed a mostly linear relationship with time ($R^2 = 0.88$). We hence assumed a linear growth of the mouth.

The maximum microplastic size accessible for ingestion by larvae $S_{max}^{access}$ (µm) then is:

$$S_{max}^{access} = at + b \quad (7)$$

with a the slope of the mouth transverse growth (5.37 µm d⁻¹) and b the mouth transverse size at the beginning of the rearing period, 8 DAH (44.14 µm).

We then look for the concentration of particles in the substrate ($C_s$, g⁻¹) whose size is below this maximum and which can therefore potentially be ingested by the larvae. This accessible fraction of particles ($F_{access}$), can be evaluated by means of the cumulative distribution function, which is for normally distributed particles the following equation, with $μ$ the mean particle size (µm) and $σ$ the standard deviation (µm) (Downey, 2011):

$$F_{\text{access}} = \frac{1}{2} \left( 1 + \text{erf} \left( \frac{S_{\max}^{\text{access}} - \mu}{\sigma\sqrt{2}} \right) \right) \quad (8)$$

Egestion during the rearing phase is supposed to function in the same way as during the starvation phase, hence this model assumes that larval starvation does not affect the biological functions related to egestion.

Accounting for those previous considerations, we provide in the results section a function $f$ respecting Eq. (9), matching with experimental data.

$$\frac{dC_{BSFL}}{dt} = F_{\text{access}}C_s - f(t) - k_e C_{BSFL} \quad (9)$$

<caption>**Table 2**</caption>

Kinetic parameter values obtained after modelling the egestion and ingestion of the different sizes of spherical and irregular microplastics. Parameter incertitude is propagated from one standard deviation of experimental data. The adjusted coefficient of determination ($R_{Adj}^2$) and the relative mean absolute error (RMAE) are provided as goodness-of-fit indications respectively for the starvation and for the rearing phase.

<table><thead><tr><th>MP shape</th><th>MP size (µm)</th><th>k<sub>e</sub> (± SD)</th><th>t<sub>1/2</sub> (± SD)</th><th>R<sup>2</sup><sub>Adj</sub>e</th><th>log<sub>10</sub>(k) (± SD)</th><th>k<sub>i</sub> (± SD)</th><th>RMAE (%)</th></tr></thead><tbody><tr><td>Spherical</td><td>38 – 45</td><td>2.42<sup>+13.33</sup><sub>-0.79</sub></td><td>0.29<sup>+0.14</sup><sub>-0.24</sub></td><td>0.96</td><td>-1.52<sup>+0.27</sup><sub>-0.58</sub></td><td>0.36<sup>+0.10</sup><sub>-0.03</sub></td><td>14</td></tr><tr><td>Spherical</td><td>53 – 63</td><td>4.60<sup>+9.73</sup><sub>-0.32</sub></td><td>0.34<sup>+0.01</sup><sub>-0.10</sub></td><td>1.00</td><td>-2.04<sup>+0.27</sup><sub>-0.71</sub></td><td>0.43<sup>+0.04</sup><sub>-0.01</sub></td><td>32</td></tr><tr><td>Spherical</td><td>90 – 106</td><td>3.75<sup>+7.25</sup><sub>-0.51</sub></td><td>0.34<sup>+0.03</sup><sub>-0.12</sub></td><td>1.00</td><td>-2.29<sup>+1.73</sup><sub>-0.54</sub></td><td>0.39<sup>+0.06</sup><sub>-0.39</sub></td><td>73</td></tr><tr><td>Irregular</td><td>45 – 75</td><td>2.02<sup>+0.02</sup><sub>-0.04</sub></td><td>0.34<sup>+0.01</sup><sub>-0.01</sub></td><td>0.99</td><td>-2.29<sup>+0.04</sup><sub>-0.03</sub></td><td>0.39<sup>+0.03</sup><sub>-0.05</sub></td><td>21</td></tr><tr><td>Irregular</td><td>75 – 106</td><td>1.05<sup>+0.75</sup><sub>-0.22</sub></td><td>0.66<sup>+0.17</sup><sub>-0.27</sub></td><td>0.94</td><td>-4.23<sup>+0.13</sup><sub>-0.20</sub></td><td>0.70<sup>+0.01</sup><sub>-0.01</sub></td><td>19</td></tr><tr><td>Irregular</td><td>106 – 150</td><td>2.61<sup>+4.10</sup><sub>-0.67</sub></td><td>0.27<sup>+0.09</sup><sub>-0.16</sub></td><td>1.00</td><td>-8.57<sup>+1.48</sup><sub>-2.33</sub></td><td>1.66<sup>+0.12</sup><sub>-0.31</sub></td><td>8</td></tr></tbody></table>

Numeric values of kinetic model parameters were fitted on measured concentrations through mean squared error minimization. Results are provided with the impact of the propagation of a single positive and negative standard deviation from the mean experimental value, with rounding towards the most unfavourable value. This helps highlighting the sensitivity of the model's parameters to variations in input values. Relative mean absolute error (RMAE), calculated as the sum of absolute errors divided by the sample size and the mean experimental value over the period, is provided as a goodness-of-fit indicator in results.

### 2.7. Statistical analysis

Statistical analyses were carried out with Jamovi 2.3.21 and with GraphPad Prism version 10.0.3 for Windows, with a 5 % significance level. The assumption of normality was tested with a Shapiro–Wilk test, and the assumption of homoscedasticity with Levene's test (Cattaneo et al., 2023; Zarantoniello et al., 2024). To compare BSF larval weights, for which the hypotheses of normality and homoscedasticity were satisfied, a one-way ANOVA followed by Tukey's multiple comparison test were performed (Cho et al., 2020; Lievens et al., 2023a). For the comparison of the number of microplastics in the guts of larvae and bioaccumulation factors, for which the hypothesis of normality was not satisfied, the nonparametric Kruskal–Wallis test followed by the Dwass–Steel–Critchlow–Fligner pairwise comparison test was used (Martínez Marín et al., 2023; Monteiro Dos Santos et al., 2023).

## 3. Results

### 3.1. Confirmation of the size and structure of the self-produced microplastics

The mean particle sizes of irregularly shaped microplastics are 67.9 µm, 89.8 µm, and 129 µm, respectively for the 45–75 µm, 75–106 µm, and 106–150 µm size ranges (Table S2). The size distribution of these irregular particles is slightly asymmetric with more large particles compared to a Gaussian distribution (Fig. S2).

Staining the particles had a negligible effect on their erratic shapes (Fig. 1 and Fig. S3). However, it was observed that the edges of the particles became less sharp and grainy for all size fractions. This is probably because of the staining process used, which might first induce swelling of the polymer, resulting in a slightly smoother surface.

### 3.2. Overall larval growth on microplastic contaminated substrates

When looking at the effect of both spherical and irregularly shaped microplastics, no significant difference was observed between the weight of larvae reared on control or spiked substrate ($p > 0.05$, one-way ANOVA) (Fig. 2). The presence of polyethylene microplastics in the substrate has therefore no impact on the growth of BSF larvae. On average, the larval weight increased from 11.0 ± 3.7 mg to 221.4 ± 16.5 mg during rearing on substrate, while starvation resulted in an average larval weight reduction of 30.5 %.
<figure><img src="image_4.png" /><figcaption>Fig. 1. SEM images (magnification: 1000 × ) of self-produced polyethylene microplastics before (left) and after (right) the application of the staining procedure. Size range fraction of the microplastics shown is 45 – 75 µm.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 2. Daily average weight (mg) of BSF larvae reared on an artificial food waste: (a) control (unspiked), spiked with spherical microplastics of three size ranges (38 – 45 µm; 53 – 63 µm; 90 – 106 µm), and (b) spiked with irregularly shaped microplastics of three size ranges (45 – 75 µm; 75 – 106 µm; 106 – 150 µm). A starvation phase was carried out between 17 and 20 days after hatching (DAH). Data points and error bars represent the mean value (9 repetitions) and the standard deviation, respectively.</figcaption></figure>

### 3.3. Impact of spherical microplastic size on ingestion and egestion by BSF larvae

The blue-labelled particles observed under the fluorescence microscope (Fig. 3) remained spherical throughout the whole experiment. Regarding their number in the guts of BSF larvae, a significant difference can be observed at the end of the rearing period between the three different spherical particle sizes (Fig. 4). On average, 1725 ± 397, 555 ± 384 and 19 ± 13 particles were counted for the 38–45 µm, 53–63 µm and 90–106 µm size ranges, respectively (Table S1). For 38–45 µm and 53–63 µm spherical particles, an increase in the number of microplastics in the larval gut was observed from 9 DAH and 13 DAH, respectively. In contrast, for 90–106 µm spherical particles, the number of particles in the larval gut remains low (less than 20 particles) throughout the rearing experiment.

The starvation, which was carried out from 17 DAH to 20 DAH, allowed to reduce the number of spherical microplastics present in the larval gut by 92 % (for 38 – 45 µm spherical particles) to 99 % (for 90 – 106 µm spherical particles) (Fig. 4). However, this period also resulted in an average larval weight reduction of 30.5 % (Fig. 2).

Calculation of bioaccumulation factors (BAF) at 17 DAH demonstrates that even without incorporation of a starvation stage, there is no bioaccumulation of spherical microplastics during the rearing experiment, whatever the size of the microplastics (Table 1). The BAF at 17 DAH appeared to be (2.8 ± 0.9) × 10⁻¹ for 38 – 45 µm spherical particles, (8.9 ± 6.2) × 10⁻² for 53 – 63 µm spherical particles and (3.4 ± 2.4) × 10⁻³ for 90 – 106 µm spherical particles.

### 3.4. Impact of microplastic shape on BSF larval ingestion and egestion

As with spherical particles, a significant difference could be observed under a fluorescence microscope (Fig. 5) between the three irregularly self-produced microplastic sizes regarding the uptake by BSF larvae. At the end of the rearing period, an average of 317 ± 128, 42 ± 16 and 1.9 ± 1.9 particles were detected in larval guts (Table S1). The latter correspond to BAF values of (5.9 ± 2.5) × 10⁻², (7.6 ± 2.6) × 10⁻³ and (3.7 ± 3.8) × 10⁻⁴ (Table 1) for the 45 – 75 µm, 75 – 106 µm and 106 – 150 µm size ranges, respectively. At the end of the rearing period, no significant difference in the number of particles was found in larval guts between the similar size ranges of spherical and irregularly shaped
<figure><img src="image_6.png" /><figcaption>Fig. 3. Light microscopy images, fluorescence microscopy images and overlay of both images of three size ranges (38 – 45 µm, 53 – 63 µm, 90 – 106 µm) of spherical blue marked polyethylene microplastics found in the guts of BSF larvae (magnification: 10 × ).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Number of polyethylene particles found in the guts of BSF larvae reared on an artificial food waste: control (unspiked), spiked with spherical polyethylene microplastics of three size ranges (38 – 45 µm, 53 – 63 µm, 90 – 106 µm), and spiked with irregularly shaped polyethylene microplastics of three size ranges (45 – 75 µm, 75 – 106 µm, 106 – 150 µm). A starvation stage was carried out between 17 and 20 days after hatching (DAH). Data points and error bars represent the mean value (9 repetitions) and the standard deviation, respectively.</figcaption></figure>

particles (53 – 63 µm spherical vs 45 – 75 µm irregularly shaped particles, and 90 – 106 µm spherical vs 75 – 106 µm irregularly shaped particles) (Fig. 4, Table S1). In addition, as with spherical particles, the starvation period reduces the number of microplastics present in the larval gut by more than 90 % (Table S1).

3.5. Kinetic model and parameter values for various microplastics

The egestion kinetics was first modelled based on the 3-day starvation phase. The egestion rate constant $k_e$ was determined for each particle size and shape and the fitting quality evaluated through the adjusted coefficient of determination for egestion $R_{Adj}^2$ (Table 2). Based on these results, the ingestion kinetics was then modelled. Whatever the shape and size of the microplastics, we observed a rapid accelerating increase of the microplastic uptake rate with the progression of time, conflicting with classic first or second order reaction models. Despite accounting for the increasing larval mouth opening size, the ingestion rate is rising at an exponential rate over time, hence the ingestion kinetic have been modelled through the following equation, with $C_s$ the microplastic concentration in the substrate ($g_{substrate}^{-1}$), and $k_i$ ($d^{-1}$) and $\kappa$ ($g_{substrate}^{-1} d^{-1} g_{BSFL}^{-1}$) parameters of the ingestion, respectively the ingestion rate constant and the pre-exponential parameter of the ingestion rate.

$$ \frac{dC_{BSFL}}{dt} = F^{\text{access}}C_s - e^{k_i t} - k_e C_{BSFL} \quad (10) $$

This model leads to a continuously increasing microplastic concentration in larvae during the rearing phase. While it is reasonable to believe that given enough time the concentration should stabilize
<figure><img src="image_8.png" /><figcaption>Fig. 5. Light microscopy images, fluorescence microscopy images and overlay of both images of three size ranges (45 – 75 µm, 75 – 106 µm, 106 – 150 µm) of stained irregular self-produced polyethylene microplastics found in the guts of BSF larvae (magnification: 10 × ).</figcaption></figure>

towards a steady-state value, in a sigmoid fashion, this model cannot be used to predict this concentration at steady-state, as it is limited to a reasonable representation for the 8 to 17 DAH rearing period.

Equation (11) details the solution to this differential equation after substitution of Faccess (estimated according to Lievens et al. (2023a) for a normal distribution of particles), representing the time-dependent concentration of microplastic in larvae during the ingestion phase, for zero initial microplastics.

$$C_{\text{BSFL}} = \frac{\kappa C_s}{2(k_i + k_e)} (h_1 e^{k_i t} - h_2 e^{-k_e t} + (h_3 - h_4) e^{(h_5 - h_6)}) \quad (11)$$

where

particle distribution, C_s (particles g<sub>substrate</sub><sup>-1</sup>) the concentration of particles in the substrate, k_e (d<sup>-1</sup>) the egestion rate constant, k_i (d<sup>-1</sup>) and κ (g<sub>substrate</sub> d<sup>-1</sup> g<sub>BSFL</sub><sup>-1</sup>) parameters of the ingestion.

Kinetic parameters determined through mean squared error minimization for each studied microplastic type are provided in Table 2.

The performance of the model is lower (higher RMAE) for the spherical particle shape, especially for larger sizes, and statistical analysis have revealed no correlation between the parameters and mean particle size for spherical particles. However, there is a linear covariance between mean particle size and the ingestion parameters k_i and log<sub>10</sub>(κ) for irregular particles (R<sup>2</sup> = 0.99 and 0.98 for k_i and log<sub>10</sub>(κ) particles, respectively). Table 2 provides further insights into the influ-

$$h_1 = 1 + \text{erf}\left(\frac{\sqrt{2}(at + b - \mu)}{2\sigma}\right), \quad h_2 = 1 + \text{erf}\left(\frac{\sqrt{2}(b - \mu)}{2\sigma}\right), \\ h_3 = \text{erf}\left(\frac{\sqrt{2}\left(\frac{a(\mu - b) + \sigma^2(k_i + k_e) - a^2t}{2}\right)}{a\sigma}\right), \quad h_4 = \text{erf}\left(\frac{\sqrt{2}\left(\frac{a(\mu - b) + \sigma^2(k_i + k_e)}{2}\right)}{a\sigma}\right), \\ h_5 = \frac{(k_i + k_e)(2a\mu + (k_i + k_e)\sigma^2)}{2a^2}, \quad h_6 = \frac{(k_i + k_e)b + ak_e t}{a}$$

with a (5.37 µm d<sup>-1</sup>) and b (44.14 µm) respectively the slope of larval mouth growth in the 8 – 17 DAH period and the larval mouth size at 8 DAH, µ (µm) and σ (µm) respectively the mean and standard deviation of

ence of shape. It indicates that, for particles of similar size, the temporal factor k_i tends to be higher for irregular particles than for spherical ones. This indicates that the shape of the microplastics may slightly modify the
early ingestion kinetics (with a less curved profile of the larval ingestion curve for spherical particles), as it influences both parameters related to ingestion, $k_i$ and $\log_{10}(\kappa)$. However, those parameters have concurrent impact on larval global ingestion speed, which results in the absence of significant differences in the bioaccumulation at 17 DAH (Table 1).

# 4. Discussion

## 4.1. Overall larval growth on microplastic contaminated substrates

Our results show that the different sizes and shapes of polyethylene microplastics do not have any effect on the growth of the BSF larvae. This confirms the results obtained by Lievens et al. (2023a) who spiked food waste with blue fluorescently labelled polyethylene microplastics of 53 – 63 µm size range. Further, no impact was observed due to the presence of microplastics in the rearing substrate of BSF larvae for a mass fraction of up to 3 %. Similar results were also obtained for other microplastics such as polyvinyl chloride (Lievens et al., 2022a), poly-styrene (Cho et al., 2020) or polylactic acid (Heussler et al., 2023), whose presence in rearing substrates neither had an impact on the growth of BSF larvae. Therefore, rearing BSF larvae on biowaste contaminated with microplastics does not affect the productivity of manufacturers in this sector. Given that monitoring larval growth would not enable contamination to be identified, this would suggest the need for regular monitoring of the microplastic content of biowaste before it is used as a substrate for rearing BSF larvae. It is important to note that these results are specific to the case of industrial recycling of biowaste by BSF larvae, but recent work has shown that the effect of exposure to microplastics on insect weight is species and dose dependent (Sucharitakul et al., 2024). In addition, the lack of visible effects can potentially be explained by the rather short exposure time of 10 days, which limits the chances of effects being visibly expressed. Moreover, potential compensatory physiological mechanisms, such as modulation of the gut microbiota or energy reallocation, described in other animal species exposed to microplastics (Duan et al., 2024; Urbina et al., 2023), could mask the effects on larval growth. Therefore, it might be interesting to assess whether a 10-day exposure duration could be sufficient to have an impact on the composition of the larvae's intestinal microbiota, as suggested by a preliminary study carried out on larger irregular PVC microspheres (approximately 150 – 190 µm in diameter) (Piersanti et al., 2024). Although it's likely that only the fraction of their PVC powder containing the smallest particles could have been ingested by BSF larvae (Lievens et al., 2023a), this study demonstrated that the presence of PVC microplastics selectively affects the abundance of specific bacterial and fungal taxa in the guts of BSF larvae. Knowing that intestinal bacteria significantly influence BSF growth and development (Li et al., 2023), it would then be interesting to study whether changes induced by the presence of microplastics on the composition of the intestinal microbiota could have an impact on the growth of subsequent generations of BSF larvae.

## 4.2. Ingestion and bioaccumulation of microplastics in BSF larvae

Regarding blue-labelled spherical particles, the results obtained are in line with previous studies. Firstly, the particles remain spherical throughout the whole experiment, which confirms the previous observation of Lievens et al. (2022a; 2023a), where no degradation of the particles appeared to happen by BSF larvae during rearing. Secondly, for the same 53 – 63 µm spherical particles as in the present study, the final number of microplastics in BSF guts at 17 DAH found by Lievens et al. (2023a) was 264 ± 462 for a microplastic mass fraction of 0.1 % and 2067 ± 1578 for a microplastic mass fraction of 0.5 %. In the present study, the results are intermediate to these with a mass fraction of 0.3 %, which confirms that the final particle amount correlates with the initial microplastic content in the substrate.

When comparing similar size ranges of spherical and irregularly

shaped particles (53 – 63 µm spherical vs 45 – 75 µm irregularly shaped particles, and 90 – 106 µm spherical vs 75 – 106 µm irregularly shaped particles), no significant difference in the number of particles is found in larval guts. In a previous study, Choi et al. (2018) exposed fish (Cypri-nodon variegatus) to spherical or irregularly shaped microplastics for 4 days, and no significant difference in the fate of these microplastics was observed. Although microplastics accumulated in the intestinal lumen, they were not detected in other fish organs, regardless of their shape. On the other hand, these authors observed in exposed fish significant differences in the transcriptional levels of key genes linked to detoxification, antioxidant enzymes and the immune system, depending on the shape of the microplastics. These differences could be explained by the roughness of irregularly shaped microplastics, which are more likely to cause injury and increase inflammation and cellular stress in fish (Choi et al., 2018; Hamm and Lenz, 2021). It should however be noted that caution should be exercised when comparing these results with those of the current study, since different animal species were exposed. Further, it was documented that the use of spherical microplastics, which are easily available commercially, seemed to be a good model for monitoring the fate of irregularly shaped microplastics in organisms. However, its use would not be relevant for toxicological studies, especially as microplastics originating from for example packaging foil can also contain various additive chemicals. Although this impact seems to be limited, it is still underexplored thus far (Lievens et al., 2024).

Whether for spherical or irregularly shaped particles, we can observe that the smallest particles are those that can be ingested earlier during rearing and ingestion decreases with increasing particle size. Lievens et al. (2023a) noted that as BSF larvae age, their mouth opening size increases, influencing their ability to consume microplastics. This may explain why smaller microplastics can be ingested earlier than larger ones, for which it is necessary to wait until the larvae grows and its mouth opening outpaces the particle diameter, allowing them to start the ingestion. As Lievens et al. (2023a) have shown that the size of the mouth opening does not exceed 110 µm until 17 DAH, this also explains why the 90 – 106 µm spherical particles can only be ingested by larvae in the last few days of the rearing period, and so the number of particles does not exceed 20 in larval guts. In the case of 106 – 150 µm irregularly shaped particles, the larger particles whose size exceed the mouth opening capacity of BSF larvae are certainly not ingested, which explains why they are not recorded in the larval guts. These large particles are therefore not an obstacle to the future use in animal feed of BSF larvae reared on food waste streams containing microplastics.

Calculation of bioaccumulation factors at 17 DAH indicates that there is no bioaccumulation of microplastics during the rearing experiment, whatever the size and the shape of the microplastics. Although there is no bioaccumulation, the risks linked to the presence of microplastics in BSF larvae, which could be used as animal feed, should not be overlooked in the light of some recent studies, which nevertheless involve other animal species and therefore cannot be directly extrapolated to the present study. For example, chronic exposure to high-density polyethylene microplastics through feeding alter nutrient metabolism, intestinal histopathology as well as microbiota community of yellow perch (Perca flavescens) (Lu et al., 2022). A study on chickens also showed that exposure to microplastics significantly affects chickens' skeletal muscle and liver physiological function and reduces meat quality (Chen et al., 2023). In addition, these authors have shown that the smaller the diameter of microplastics, the more they are deposited in chicken muscles, which can then be consumed by humans (Chen et al., 2023). As a result, the smaller the microplastics, the more likely they are to be transferred along the food chain to humans.

## 4.3. Benefits of a starvation period

The addition of a three-day starvation period at the end of rearing appears to be a highly effective option for limiting microplastic contamination of larvae, since a reduction of over 90 % in the number of
particles in the gut is observed for all the sizes and shapes of poly-
ethylene microplastics studied. However, this starvation resulted in an
average larval weight reduction of 30.5 %. Therefore, the systematic
implementation of such a starvation period, or possibly a period of
larvae feeding on uncontaminated substrate, would efficiently reduce
the safety risks at the cost of a lower apparent yield. To ensure that
everyone benefits from increased safety, standards of quality re-
quirements for larval production should reflect a commitment to pro-
ducing safer, and ultimately more valuable, larvae.

4.4. *Ingestion and egestion kinetics model*

The model developed in this study could in future provide an inter-
esting option for studying the concentration of microplastics in BSF
larvae, while avoiding the need for animal experimentation, which
raises ethical and animal welfare issues. The accumulation of micro-
plastics has been studied in other animals, especially aquatic organisms,
and the knowledge from those studies was used as a common starting
point for modeling before adapting to the specificities of larvae. This
initial approach led to mixed results: previously observed models proved
efficient for the egestion but not for the rearing phase, leading to the
current new developments. Although extrapolation of this model with
confidence would require further validation, it is already possible to
attempt to identify potential configurations of interest. Following this
line of reasoning, extrapolation for irregular particles can be achieved by
considering a linear evolution for kᵢ and log₁₀(κ) and a constant value for
kₑ, set at the mean of our results. Microplastic bioaccumulation in larvae
(BAF > 1) should then occur at 17 DAH for mean particle sizes below 37
µm. With a linear extrapolation for kₑ, this value decreases to 28 µm. It
would thus be of interest to examine potential alterations in ingestion
behaviour in a region around those size values, by exploring, for
instance, the fate of microparticles having a diameter of 25 to 40 µm.
These preliminary results suggest that regulatory changes will be needed
in the coming years, as currently only plastic impurities > 2 mm are
regulated in the EU food waste treatment sectors, whereas it is the
smallest particles that appear to pose the greatest risks.

4.5. *Study limitations and future directions*

Although our study provides relevant insights regarding the impact
of microplastic size and shape on their ingestion and egestion by black
soldier fly larvae during industrial biowaste recycling, it remains a
proof-of-concept study carried out at lab-scale. These results will need to
be validated on a larger scale, such as industrial conditions. Large-scale
validation will also enable the model developed in this study to be
refined in the future. In addition, this study needs to be complemented
by further studies exploring potential biological effects, such as the
impact of microplastics on the larval gut microbiota or on a potential
inflammatory response. It is noteworthy that the protocol optimized in
this study for staining irregular microplastics was effective in tracking
microplastics in the guts of BSF larvae throughout the experiment, while
having a negligible effect on the irregular particle shapes. This protocol
relies on thermal expansion and contraction properties of microplastics
and includes several rinsing steps. Given that Lv et al. (2019) observed
that, based on SEM images, there was no significant change in the sur-
face morphology of microplastics after heat treatment (30 min at 75 °C),
the changes observed in our study could be linked to the rinsing steps
applied. The latter were however of crucial importance to avoid sub-
sequent leaching of the dye into the substrate. In the future, the effect of
the staining protocol could be clarified by using atomic force microscopy
(AFM) measurements to specify the surface roughness of stained
microplastics, which is critical with regard to the carrying effect of
pollutants by microplastics. In the meantime, the results obtained in the
present study should be extrapolated with great caution to even sharper
microplastics that could be found in biowaste used as substrate for BSF
larvae rearing. Finally, an assessment of the microplastic content of the

frass recovered after rearing BSF larvae on biowaste was not carried out
in this study, but will be important in the future. Indeed, this frass could
be used as a fertilizer, but this use must not lead to further contamina-
tion of soils with microplastics, particularly agricultural soils, for which
recent French data have already revealed concentrations of up to 80
particles per kg of dry soil (Palazot et al., 2024).

**5. Conclusion**

As already observed in preliminary studies, this small-scale study
confirmed that, in the case of industrial rearing of BSF larvae on bio-
waste, the presence of microplastics has no impact on their growth,
whatever their size or shape. Moreover, kinetic modelling has shown
that the shape of the particles may slightly modify the kinetic parame-
ters of ingestion without having any final effect on the microplastic
bioaccumulation in larval guts. Although there is no microplastic bio-
accumulation in larvae during rearing (BAF < 0.3), the ingestion of
small particles is significantly higher than that of large particles.
Therefore, while large particles do not seem to be an issue for insect-
based bioconversion, it would be interesting to study the fate of even
smaller plastic particles (< 20 µm) that could be ingested early and
easily by BSF larvae, and potentially transfer through membrane tissue.
In order to better assess the risk associated with the presence of these
plastic particles in larval rearing substrate, future research is required,
not only to study the impact of chronic exposure to successive genera-
tions of larvae, but also to monitor the fate of these particles throughout
the food chain if the larvae are used as feed, and their correlated po-
tential health effects.

Ethics statement

This study complies with relevant institutional, national, and inter-
national guidelines and legislation. No endangered species, or at risk of
extinction, have been used for this research.

CRediT authorship contribution statement

**Christelle Planche:** Writing – original draft, Methodology, Investi-
gation, Formal analysis, Conceptualization. **Siebe Lievens:** Writing –
review & editing, Methodology, Conceptualization. **Tom Van der
Donck:** Writing – review & editing, Investigation. **Jason Sicard:**
Writing – review & editing, Methodology, Investigation, Formal anal-
ysis. **Mik Van Der Borgh:** Writing – review & editing, Supervision,
Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

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

Acknowledgements

This work was supported by the Research Foundation Flanders (grant agreement S008519N, ENTOBIOTA).

Christelle Planche would like to thank INRAE (International Re-
lations Directorate, Metaprogramme Bioeconomy for urban areas
(BETTER), TRANSFORM scientific division, and QuaPA unit) for fund-
ing her 6-month research stay at KU LEUVEN.

**Supporting Information.** Number of spherical and irregularly sha-
ped microplastics found in BSF larval gut depending on their size; Light
and fluorescence microscope images of the three size ranges of irregular
self-produced polyethylene microplastics after Nile Red staining; Parti-
cle size distribution of the three size ranges of irregularly shaped self-
produced polyethylene microplastics after Nile Red staining; Mean
particle size and volume-weighted percentile values of the three size
ranges of irregularly shaped self-produced polyethylene microplastics
after Nile Red staining; SEM images of the self-produced polyethylene microplastics before and after the application of the staining procedure.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2025.114852.

## Data availability

Data will be made available on request.

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