# Shape matters: Microplastic fibers disrupt soil carbon cycling via shape-driven physical disturbance

Serra-Willow Buchanan, Pedro Madeira Antunes, Marie Sauvadet, Ryan Prosser, Kari Dunfield

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Serra-Willow Buchanan, Pedro Madeira Antunes, Marie Sauvadet, Ryan Prosser, Kari Dunfield. Shape matters: Microplastic fibers disrupt soil carbon cycling via shape-driven physical disturbance. Environmental Toxicology and Chemistry, 2025, 10.1093/etojnl/vgaf247. hal-05307594

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**Shape matters: Microplastic fibers disrupt soil carbon cycling via shape-driven physical disturbance**

Serra-Willow Buchanan*<fnref n="1" />, Pedro Madeira Antunes<fnref n="2" />, Marie Sauvadet<fnref n="3" />,<fnref n="4" />, Ryan Prosser<fnref n="1" />, Kari Dunfield<fnref n="1" />

**Data Availability Statement:** Soil physicochemical and respiration (CO2) data are available in the Agri-environmental Research Data Repository of the University of Guelph, accession https://doi.org/10.5683/SP3/PUELDE

**Funding:** This work was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) entitled “Plastics science for a cleaner future” ALLRP 558595-20. This work was also supported by NSERC Discovery Grants to PMA and KED.

**Conflict of interest:** The authors have no conflict of interest.

<footnote n="1">¹ School of Environmental Sciences, University of Guelph, Ontario, Canada</footnote>

<footnote n="2">² Department of Biology, Algoma University, Sault Ste. Marie, Ontario, Canada</footnote>

<footnote n="3">³ Centre de coopération internationale en recherche agronomique pour le développement, Eco&Sols mixed research unit, F-34398 Montpellier, France</footnote>

<footnote n="4">⁴ Eco&Sols, Montpellier university, Centre de coopération internationale en recherche agronomique pour le développement, Institut national de recherche pour l'agriculture, l'alimentation et l'environnement, Institut Agro, Institut de recherche pour le développement, Montpellier, France</footnote>

<footnote>*Corresponding author: Serra-Willow Buchanan</footnote>

<footnote>Email: serrawil@uoguelph.ca tel: (647) 463-8661</footnote>
**Acknowledgements:** The authors would like to thank R. Farrell and his laboratory team at the University of Saskatchewan for assistance with isotopic gas analysis.

**Disclaimer:** The peer review for this article was managed by the Editorial Board without the involvement of Ryan Prosser.
Shape matters: Microplastic fibers disrupt soil carbon cycling via shape-driven physical disturbance

Abstract

Soil microplastic (MP) pollution is an emerging concern with uncertain carbon (C) cycling impacts. Among MPs, fiber-shaped particles may exert distinct effects due to their high surface area and dissimilarity from natural soil structures. This study aimed to assess the physical and chemical mechanisms by which MP addition alters soil C dynamics, especially through their impacts on priming effect. We assessed two common MP polymers and shapes —low-density polyethylene beads and acrylic fibers—at two addition rates (0.1% and 1% w/w), paired with inert glass particle controls mimicking the same MP shapes and a negative control with soil only.

Natural abundance δ¹³C isotopic analysis distinguished MP-derived and soil-derived CO₂ emissions over a 63-day incubation period. Fiber-shaped additions of either glass or MPs reduced total CO₂ emissions by 25%, whereas bead-shaped additions had no measurable effect.

Plastic-derived CO₂ constituted a minor but detectable portion of total emissions, with similar decomposition rates across polymer types. Surprisingly, higher MP concentrations decomposed up to 10 times slower than lower concentrations, suggesting that MPs may clump together, reducing the surface area available for microbial colonization, thereby slowing down breakdown.

Our results show that fiber-shaped MPs significantly disrupt soil C cycling, mainly through physical disturbance, while chemical effects (i.e., presence/absence of plastics) are secondary.

Finally, soil priming effect increased with the amount of MPs added, raising additional concerns about the consequences of rising soil plastic contaminations worldwide.

**Key words:** ¹³C-CO₂, microplastic, polyethylene bead, acrylic fiber, priming effect
# 1 Introduction

Soil microplastic (MP) contamination (i.e., imparted by plastics < 5mm in one dimension) is increasingly recognized as a significant and widespread form of pollution, with estimated concentrations in terrestrial environments surpassing those of the ocean surface by one to two orders of magnitude (Kedzierski et al., 2023). Microplastics can act as pollutants that cause toxicity (De Souza Machado, Kloas, et al., 2018) and, more broadly, serve as an agent of global change by altering soil physical, chemical and biological properties (Rillig & Lehmann, 2020; Y. Zhang et al., 2022). Agricultural soils are particularly prone to MP contamination due to the widespread use of plastics in farming materials and the application of biosolids and composts, which often contain MPs (Fakour et al., 2021; Sa'adu & Farsang, 2023), especially microfibers shed from textiles during laundering or waste disposal (Koutnik et al., 2021; Letwin et al., 2024). Because of these amendments, long-term modeling suggests MP concentrations in fertilized agricultural soils may increase substantially over the next century (Meizoso-Regueira et al., 2024). The presence of MP can disrupt soil functioning as they interact both directly and indirectly with soil biota (Boots et al., 2019; De Souza Machado, Lau, et al., 2018; Hodson et al., 2017; Jacques & Prosser, 2021). The extent of these alterations varies especially with the amount and shapes of the MP particles.

Petroleum-based plastic polymers contain a high amount of carbon (C), typically around 80% (Rillig, 2018), representing a significant source of C in soils, which can alter microbial community composition and activity. More specifically, soil MP pollution can potentially impact the processing and storage of soil organic C (SOC), by increasing the rate of SOC mineralization by microorganisms, a phenomenon known as the priming effect (PE; Fontaine et al., 2003; Kuzyakov, 2010; Kuzyakov et al., 2000). While MP-shaped fibers may amplify PE to a greater
extent than MPs of other shapes due to their larger volume-surface area ratio, the relative
contribution of physical vs. chemical mechanisms in mediating SOC responses to MPs remains
poorly understood.

Microplastics can alter soil physical structure by increasing its overall porosity, which
may enhance O₂ diffusion, stimulating microbial activity and CO₂ emissions (Rillig, Hoffmann,
et al., 2021; Rillig, Leifheit, et al., 2021). According to the shape dissimilarity hypothesis, fibers
- with their elongated, irregular morphology and high surface - differ more drastically from
natural soil particle shapes than spherical or fragment-shaped MPs. Fibers also present a much
greater surface area-to-mass ratio than spherical MPs (Kataoka et al., 2024). And so, fibers of
equal mass can expose more surface area than beads, enhancing their potential to disrupt soil
pore networks and interact with microbial communities. As a result, fibers are likely to exert
stronger effects on soil physical structure and associated biogeochemical processes such as C
cycling (Rillig et al., 2019). Although MPs are highly resistant to microbial degradation, they are
not entirely inert and can also influence soil chemical properties. Studies have reported changes
in soil pH (Gao et al., 2021; Lozano et al., 2021; W. Zhou et al., 2023) and dissolved organic C
(DOC) concentrations (H. Liu et al., 2017; Sun et al., 2022) following MP additions. On one
hand, MP can act as a source of external C within soils, marked by increased soil DOC (Gao et
al., 2021; H. Liu et al., 2017). This increase may result in a negative PE (i.e., reduced SOC loss
as CO₂ with MP addition) due to a temporary shift from microbial utilization of SOC to plastic-
derived C– a phenomenon explained by the ‘preferential utilization of substrate theory’
(Kuzyakov & Bol, 2006). Plastic particles themselves could also act as a medium for the
adsorption of DOC, causing a dilution effect within soils (Rillig, Leifheit, et al., 2021). This
dilution could lead to persistence of organic C on the adsorbed surfaces of the MPs, resulting in
an overall reduction in soil C mineralization. On the other hand, positive priming effects (i.e., increased SOC loss as CO₂ with MP addition) could result from various microbially-mediated pathways. For example, elevated DOC due to the breakdown of plastic might lead to an overall rise in both microbial biomass and enzymatic activities. Once the microorganisms deplete the DOC from plastic, the microbial communities could transition back to SOC at a faster rate. Alternatively, the high C:N ratio of MP could lead to positive PE due to ‘microbial N-mining’ if soil N availability is low (Chen et al., 2014). The impact of MP on SOC dynamics remains poorly understood, as it can be challenging to determine plastic degradation and mineralization rates within soils accurately.

Assessing MP degradation in soils presents several methodological challenges, particularly in distinguishing true mineralization from physical fragmentation. To address this limitation, natural abundance of stable C isotopes offers a more precise method to follow real MP mineralization within soils. This approach eliminates errors associated with fragmentation and mass loss (Rohrbach et al., 2024), providing a clearer distinction between actual microbial degradation and mere disintegration. Additionally, researchers can track MP-derived CO₂ emissions and distinguish these from soil-derived CO₂ emissions, enabling a better understanding of how plastic decomposition influences soil PE. This method has been used successfully to assess biodegradable MP decomposition (Guliyev et al., 2023; G. Zhang et al., 2023), and offers potential to extend to petroleum-based polymers, which break down much more slowly within environments.

In this study, we investigate how MP shape influences soil carbon cycling by testing two commonly found MPs in soils: low-density polyethylene beads and acrylic fibers. To isolate physical effects from chemical ones, we use glass particles, mimicking the same size and shape
as the MPs, as inert controls. Our aim is to disentangle the physical versus chemical
contributions of MPs to soil carbon cycling dynamics. We hypothesize that fiber-shaped MPs
will influence soil CO₂ emissions and soil PE more strongly than bead-shaped MPs, consistent
with the shape dissimilarity hypothesis, and that these contrasts between MPs shapes will be
stronger for the higher levels of MPs contamination.

## 2 Methods

### 2.1 Soil and microplastics’ initial properties

Soil used in incubations was collected in the spring of 2018 from the topsoil (0-10 cm
layer) of a farm field under corn-soybean rotation, with corn in rotation at the time of soil
collection, in the Township of Blandford-Blenheim, Oxford County, Ontario, Canada (43°18′N
80°33′W). The soil within this area is a Grey Brown Luvisol (Mozuraitis & Hagarty, 1996), with
a sandy loam texture. Soil was sieved at 2 mm to remove rocks and plant debris and stored at 4°C
until the beginning of the incubation experiment. A subsample of soil was dried for 48h at 105°C
while low-density polyethylene MP beads (Cospheric LLC, CA, United States) and acrylic MP
fibers (Soft Classic™ Solid Yarn by Loops & Threads) were freeze-dried using liquid nitrogen.
Both soil and MPs were then ground to a fine powder using a Retsch ball mill (Retsch,
Düsseldorf, Germany) and analyzed in triplicate for total C and δ¹³C content on a 4010
Elemental Analyzer (Costech Instruments, Italy) coupled to a Delta Plus XL (Thermo-Finnigan,
Germany) continuous flow isotope ratio mass spectrometer (CFIRMS; See table S1 for details).
The variation of natural ¹³C isotope abundance between C₄ crop cultivated soil and petroleum-
based MPs was used to assess C allocation within this experiment (Balesdent et al., 1987).
## 2.2 Incubation design and setup

Incubations consisted of adding 150 g equivalent dry weight of soil to 1 L mason jars, which were affixed with rubber septa to allow for headspace gas analysis by syringe. Soil was adjusted to 60% water-holding capacity and pre-incubated at 20 °C for one week before adding MPs or glass amendments to stabilize microbial activity (Creamer et al., 2015; Sauvadet et al., 2018; Zhao & Zhang, 2018). Clear (colorless) low density polyethylene beads (Cospheric LLC, CA, United States) and glass beads (Cole Safety Products, USA), were manually sieved and categorized between 850-1000 µm in diameter. Additionally, white acrylic fibers (Soft Classic™ Solid Yarn by Loops & Threads) and glass fibers (LECO corporation glass wool, Borosilicate fibers) were manually cut between 4000 and 5000 µm in length. All beads and fibers were sterilized with UV light for 15 minutes before incubation. Incubation treatments consisted of two polymer types (polyethylene beads and acrylic fibers) paired with their respective glass controls mimicking size and shape (Glass beads and Glass fibers, respectively). Microplastics or glass were added at two concentrations to simulate low (0.1% w/w) and high (1.0% w/w) levels of contamination (see Table 1). These concentrations corresponded to 858.8 and 8588.0 mg C. kg⁻¹ dry soil added as polyethylene beads, and 658.8 and 6588.5 mg C. kg⁻¹ dry soil added as acrylic fibers, in low and high incubations, respectively. Long-term modeling predicts that microplastic concentrations in fertilized agricultural soils could reach up to 0.67% w/w within 100 years under business-as-usual conditions (Meizoso-Regueira et al., 2024), supporting the relevance of the 0.1% and 1.0% experimental additions used here. Glass controls were included to distinguish between the physical impacts caused by the presence of the MPs and the chemical impacts of their degradation since glass lacks a source of degradable C. This resulted in 9 treatments (see Table 1).
Each treatment consisted of 6 replicate incubations. Polyethylene, acrylic, and glass beads or fibers were mixed into the incubation jars using a glass stirring rod and metal scoop. To ensure consistency among incubation modalities, negative control treatments (soil without glass or MP addition) received the same amount of disturbance through mixing.

## 2.3 Gas and isotopic analysis

Gas samples were collected from the headspace of the jar at 1, 3, 5, 10, 13, 21, 35, and 63 days after the initiation of incubation, using a gastight syringe for sampling. They were then transferred to a 12 mL exetainer vial until analysis (Labco, Ceredigion, UK). Jars were opened for 20 minutes after sampling to allow for reoxygenation, and moisture was readjusted with deionized water to maintain 60% water holding capacity with a fine misting glass spray bottle. Sampling dates were chosen as a compromise to maintain oxic conditions throughout the incubation, consistent with the work by Bertrand et al. (2007), with similar timelapses observed in work by Chao et al. (2019) and Shahbaz et al. (2017). Gas samples were assessed for CO2 concentration using a Scion Instruments 456-GC and δ¹³C on a Picarro G2201-i at the University of Saskatchewan.

## 2.4 Soil properties

At the end of the 63-day experiment, a subsample of soil from each jar was air-dried and sieved at 2 mm for analysis of soil pH with a glass electrode, using a 1:2.5 (w/v) soil to deionized water ratio. A second subsample of fresh soil was collected for DOC analysis immediately after collection, following procedures outlined by Jones and Willet (2006). Briefly, DOC was extracted at 1:5 soil: water (w/w). Approximately 5 g of fresh soil was added to 25 ml of deionized water and shaken for 60 min, followed by 20 min in a centrifuge. The supernatants were filtered through 0.45 mm cellulose-acetate membranes and kept frozen until DOC (mg
kg⁻¹) was determined using the “NPOC” method on a Shimadzu TOC-LCPH Total Organic Carbon and Nitrogen Analyzer. Finally, fresh soil was extracted with 2 M KCl and analyzed using the Seal AQ2 discrete analyzer (SEAL Analytical, US) at the Agriculture and Food Laboratories at the University of Guelph for ammonium (N-NH₄) and nitrate (N-NO₃; mg N kg⁻¹ dry soil). These values were combined to assess overall inorganic N within soils after 63 days of incubation.

## 2.5 Calculations of total and ¹³C-CO₂ emissions

For each sampling date, CO₂ (R; mg C kg⁻¹ soil) efflux was calculated according to Chao et al. (2019).

$$C_{Total} = \frac{C \times V \times M \times 273.15}{22.4 \times (273.15 + T) \times W \times t} \quad (1)$$

where C is the total measured C-CO₂ value (ppm); V is the volume of space (excluding soil) within 1L mason jar (0.83L); M is the molecular weight of C (12 g mol⁻¹); T is the temperature at which jars were kept constant (20°C); W is the dry weight of soil (150 g); and t is the accumulation time in days between sampling periods. Finally, the standard molar volume of an ideal gas at 1 bar and 273.15 K (L mol⁻¹) is 22.4 L.

The amount of CO₂ derived directly from the mineralization of MP (polyethylene or acrylic; mg C kg⁻¹ soil) was calculated as:

$$C_{MP} = C_{Total} \frac{\delta C - \delta S}{\delta MP - \delta S} \quad (2)$$

where C<sub>Total</sub> represents total C-CO₂ from the MP treatment (equation 1), δC is the δ¹³C value of the CO₂ emitted from treatments amended with polyethylene or acrylic, δS is the δ¹³C value of CO₂ emitted from control treatments (soil only) and δMP is the δ¹³C values of solid polyethylene or acrylic, which was assessed before the incubation (Table S1).

Soil-derived mineralization (mg C kg⁻¹ soil) from MP treatments was then calculated as:
$$C_{SOM} = C_{Total} - C_{MP} \quad (3)$$

where C<sub>Total</sub> is the total amount of C-CO₂ from MP treatments (equation 1), and C<sub>MP</sub> is the amount of CO₂ derived directly from the mineralization of MP treatments (equation 2). We attributed all C-CO₂ emissions from the glass-amended treatments to soil-derived mineralization since glass lacks a source of degradable C. Because our incubations were conducted in the dark—thereby excluding photodegradation pathways—and involved recalcitrant polymers, we assume isotopic fractionation during CO₂ evolution to be negligible. This assumption is supported by recent studies showing that even under UV-induced weathering, which degrades plastics much faster than microbial processes, changes in δ¹³C of residual plastics remain relatively minor (typically <2‰), indicating minimal fractionation during early-stage degradation (Birch et al., 2021).

Soil PE was calculated as the difference between soil-derived mineralization in MP addition treatments (C<sub>SOM</sub>, equation 3) and the corresponding control (C<sub>CON</sub>). When assessing PE using traditional methods, we used the negative control (soil only) treatment (total_PE), while to assess the chemical effect of MPs, we used the paired glass treatment (GB-H, GB-L and GF-H, GF-L) to account for and remove priming induced by physical changes to the soil (MP_PE):

$$PE = C_{SOM} - C_{CON} \quad (4)$$

## 2.6 Statistical analysis

Statistical analyses were performed using R statistical analysis software (R Foundation for Statistical Computing, Vienna, Austria). Before analysis, data were log transformed where necessary to obtain normality of the residual variance (Total CO₂, Plastic-derived CO₂, DOC). A one-way Analysis of Variance (ANOVA) was conducted to assess differences in cumulative, plastic-derived and soil-derived CO₂ emissions and plastic decomposition (%) among MP
treatments. A one-way ANOVA was also used to assess soil physicochemical properties between control, MP and glass treatments after 63 days. We used a bootstrapping approach with 5,000 iterations to evaluate and visualize the impact of MP and glass control treatments on total CO₂, generating unpaired mean differences and 95% confidence intervals with the "dabestr" package (Ho et al., 2019). The estimation method was applied to assess the effects of polymer type and addition rate on total CO₂ emissions. Principal component analysis (PCA) was conducted to explore multivariate patterns and trade-offs among soil physiochemical properties. A permutation-based multivariate analysis of variance (PERMANOVA) was conducted to assess the effects of material type, addition rate and their interaction on the response matrix using the Euclidean distance metric using the 'vegan' R package (Oksanen et al., 2001). Significance was tested with 999 permutations.

# 3 Results

## 3.1 Impact of physical disturbance on soil CO₂ emissions

To assess the baseline disturbance effect of additive shape, we compared cumulative CO₂ emissions between the negative control (soil only), inert glass treatments (GB and GF) and the MP additions (polyethylene beads and acrylic fibers; Figure 1). The addition of fibers to soil, either glass or acrylic, significantly reduced cumulative total CO₂ emissions compared to the negative control (Figure 1b), translating to a 25.38% decrease in the GF-L addition treatment, a 22.40% decrease in the GF-H addition treatment, a 24.32% decrease in the acrylic-L addition treatment and a 14.24% decrease in the acrylic-H addition treatment. In contrast, the addition of any bead-shaped materials, glass or polyethylene, did not significantly change cumulative total CO₂ emissions compared the negative control treatment (soil only; Figure 1b).
## 3.2 Plastic-derived CO₂

Cumulative plastic-derived CO₂ emissions after 63 days of incubation showed no significant differences between MP treatments (F = 0.52, *p* = 0.68; Table 2). However, the percentage of total MP C decomposed relative to the initial amount of C added over 63 days was significantly different among treatments (F = 5.59, *p* < 0.01; Table 2), with lower overall decomposition at the higher addition rates compared to the low addition treatments. For polyethylene beads, C decomposition was 8.47 ± 3.67% at the low addition rate and only 0.68 ± 0.29% at the high addition rate. Similarly, for acrylic fibers, C decomposed at 11.59 ± 2.55% at the low addition rate and 1.53 ± 0.37% at the high addition rate.

## 3.3 Alterations to soil physicochemical properties after MP incubation

PERMANOVA results indicated a significant effect of addition rate (none, low, or high) on soil physicochemical properties (F = 10.74, *p* < 0.01), explaining 17% of the variation. However, the effect of material type (polyethylene, acrylic, glass beads or fibers) was not significant (F = 2.04, *p* = 0.09), nor was the interaction addition rate and material type (F = 0.79, *p* = 0.57; Figure S1). Post hoc pairwise comparisons revealed significant differences between the low and high addition treatments (F = 11.48, *p* < 0.01) and between the control and high addition treatments (F = 8.96, *p* = 0.003). However, there was no significant difference between the control and low addition treatments (F = 1.08, *p* = 0.34).

A one-way ANOVA revealed that MP treatment significantly altered several soil physicochemical properties (Figure 2). Notably, acrylic-H (6.98 ± 0.01), polyethylene-H (7.02 ± 0.02) and GB-H (7.00 ± 0.02) treatments significantly lowered pH compared to the control (7.19; F = 4.84, *p* < 0.001), with acrylic-H having the strongest effect (Figure 2a). Microplastic treatment also had a significant effect on DOC levels (F = 2.78, *p* = 0.01), driven by the acrylic-
L treatment, which had significantly higher DOC than other fiber treatments (Figure 2b).

However, DOC in acrylic-L did not differ from the control, and this treatment exhibited high variability. Lastly, inorganic N remained unchanged in amended treatments compared to the control, but a significant difference was observed between GB-H (76.6 ± 5.31 mg N kg⁻¹ dry soil) and acrylic-L (42.54 ± 11.45 mg N kg⁻¹ dry soil; Figure 2c).

## 3.4 Microplastic induced priming effects

Relative PE was calculated using two methods to account for physical disturbance impacts. The difference between soil-derived CO₂ mineralization in MP treatments and (1) the average CO₂ from the negative control (soil only; total_PE, Figure 3a) or (2) the average CO₂ from paired glass treatments (MP_PE, Figure 3b).

Using the negative control, both polyethylene and acrylic treatments resulted in cumulative negative PE (Figure 3a). Acrylic fiber treatments (both low and high addition rates) exhibited significantly greater negative PE compared to polyethylene bead treatments (F = 34.54, *p* < 0.01; Figure 3a). By the end of the 63-day incubation, acrylic-L and acrylic-H treatments resulted in a negative PE of -126.07 ± 15.08 mg C kg⁻¹ soil, and -82.17 ± 11.05 mg C kg⁻¹ soil, respectively.

When using paired glass treatments as the control to account for physical disturbance impacts on soil, there was no significant difference in PE between polyethylene bead and acrylic fiber treatments (F = 0.18, *p* = 0.67) or high and low addition rates (F = 2.87, *p* = 0.11), at the end of 63 days (Figure 3b).
# 4 Discussion

## 4.1 Microplastic fiber shape — not polymer chemistry — drives disruption of soil carbon cycling via physical mechanisms

One key finding from our analysis is that fiber-shaped materials, whether glass or plastic, induce substantial physical changes in the soil. The interference of fibers with the micro-scale spatial organization of soil structure appears to be a dominant driver of reduced CO₂ emissions and altered C cycling, independent of chemical effects, highlighting the need to treat particle shape as a key aspect in soil MP pollution. While it has been posited that the addition of MP could increase CO₂ production by improving oxygen supply within soils, our findings did not support this hypothesis. On the contrary, we observed a reduction in total CO₂ emissions in the fiber treatments (glass and acrylic) by approximately 20%, while bead-shaped additions (glass and polyethylene) did not significantly alter cumulative total CO₂ emissions. Recent studies have demonstrated that fibers can reduce soil aggregate stability (De Souza Machado, Lau, et al., 2018; Lehmann et al., 2021) which typically increases soil CO₂ emissions because the breakdown of aggregates exposes previously protected soil organic matter (SOM) to microbial decomposition. However, fibers have also been shown to reduce microbial activity (De Souza Machado, Lau, et al., 2018) compared to non-fiber shaped MP materials, which is consistent with the reduction in CO₂ production we observed. Fiber-shaped additives may disrupt pore connectivity, limiting microbial access to resources, thereby restricting aerobic respiration and reducing CO₂ emissions. Indeed, research has shown the importance of surface area contact between microorganisms and their substrates (Angers & Recous, 1997; Negassa et al., 2015). These findings are consistent with the shape dissimilarity hypothesis suggesting that MP fibers
disrupt soil functioning more than bead-shaped MPs due to their structural incongruity with
natural soil particles (Rillig et al., 2019).

While previous studies have shown that biodegradable MP can modify soil chemistry and biology through degradation byproducts (Guliyev et al., 2023; Rauscher et al., 2023; G. Zhang et al., 2023), our results using conventional petroleum-based polymers show only limited changes in soil chemical properties. Previous studies indicate that conventional MPs can affect DOC through contrasting mechanisms - either serving as an external C source (Gao et al., 2021; H. Liu et al., 2017) or adsorbing DOC, thereby reducing its bioavailability (Rillig, Leifheit, et al., 2021). Similarly, studies have shown that MP can alter soil N cycling by modifying microbial activity, altering ammonium/nitrate sorption, or disrupting aggregate-associated N pools (Liu et al., 2023; Rillig et al., 2021). However, we observed no discernible patterns in DOC or inorganic N alterations across treatments, suggesting minimal chemical alteration of microbial substrate availability or N cycling during the short-term incubation. A small but significant decline in pH was detected in the high MP concentration fiber and bead treatments, though the underlying mechanisms remain unclear. The reductions in cumulative CO₂ observed in fiber treatments were therefore likely driven by physical alterations to soil structure rather than changes in DOC availability. These findings align with the work of Zhang et al. (2023), who proposed that PE depends on MP degradability, suggesting that chemical effects from persistent MPs may be weak or masked by dominant physical mechanisms over short timescales.

## 4.2 Higher MP concentrations reduce degradation efficiency, likely due to surface area limitations

While oil-based polymers are considered recalcitrant, isotopic analysis confirmed measurable degradation over 63 days. This suggests that even small amounts of C lost from
plastic degradation can have long-term effects, as MP persist and degrade in the soil over
decades. This slow breakdown leads to an accumulation of MPs in soils, which could amplify
their effects over longer timescales. Interestingly though, decomposition rates did not scale
proportionally with MP addition levels. The percentage of total C decomposed from high-MP
treatments was significantly lower than in low-MP treatments, meaning that C from low-MP
additions was mineralized more rapidly over the 63-day incubation period. This finding is
surprising, as studies comparing the addition of different amounts of plant residues generally
report similar decomposition rates between different quantities (Buchanan et al., 2024; Dietrich
et al., 2017; Sauvadet et al., 2016; Shahbaz et al., 2017; Wang et al., 2019). At higher MP
concentrations (1% w/w), clumping may have reduced the available surface area for microbial
colonization, thereby limiting physical contact between microbes and MP surfaces (Negassa et
al., 2015). This could partially inhibit microbial degradation processes not only by reducing
access to the MPs themselves but also by disrupting microbial activity more broadly within the
soil matrix. In contrast, within the low addition rate (0.1% w/w), plastic particles may have been
more evenly distributed throughout the soil matrix, maximizing microbial exposure to the MP
surface and enhancing degradation. These findings are novel in suggesting a non-linear dose-
response in MP degradation, driven by physical distribution and accessibility rather than total
MP mass. Based on our findings, we recommend that future studies use larger incubation
systems with improved mixing techniques and longer durations to more accurately capture MP
degradation dynamics. Additionally, future research should investigate how MP concentration
affects surface area availability, potentially using high-precision quantification methods such as
X-ray computed microtomography or scanning electron microscopy.
Crucially, including shape-mimicking glass particles enabled us to isolate the physical disturbance effects of MP, offering insight beyond studies focused solely on MP chemistry. When glass controls were used to correct for morphology, differences in PE across polymer type largely disappeared, and fiber-driven CO₂ suppression could be attributed to changes in the soil structure rather than chemical alterations. This underscores the importance of physical disruption as a dominant mechanism, particularly for fiber-shaped MP. However, MPs can induce toxicity effects that reduce microbial abundance (Sait et al., 2021; Zaborowska et al., 2021) or shift microbial communities toward K-strategists, which thrive in resource-limited environments and decompose recalcitrant organic matter (Chen et al., 2014; X. Liu et al., 2023; Shahbaz et al., 2017). Future research should explore how these microbial shifts interact with plastic degradation dynamics to better understand their implications for soil C cycling.

## 5 Conclusions

This study demonstrates the potential of natural abundance stable C isotopes to distinguish MP-derived CO₂ from soil organic C, offering a way to quantify MP degradation in soil. This method is suitable for petroleum-based plastics, using C₄ labeled soil and should be altered for studies assessing biodegradable MP, as most biodegradable plastic is corn-based (a common C₄ plant), and therefore requires a soil with a distinct different isotopic signature to partition C sources (G. Zhang et al., 2023). In this study, only certain types of MP were introduced to the soil, potentially inducing a preferential use of MP as C sources by the microorganisms. However, these findings might vary in natural and managed systems with more readily available C sources, such as labile plant and root material. Therefore, experiments which assess 3-source mixing models (soil, plastic and a plant-based source) may be useful for understanding C-cycling mechanisms in natural environments (J. Zhou et al., 2024). Additionally, analyzing δ¹³C of
residual MPs post-incubation and measuring δ¹³CO₂-C from sterile, MP-only incubations could help quantify isotopic fractionation. We recommend that future studies include direct analysis of residual MPs when recovery is feasible to improve the accuracy of Cₘₚ (equation 2).

These results emphasize the need for future research to explore soil physical disturbances directly under field conditions, particularly across different soil types. Moreover, pairing these approaches with assessments of microbial community composition will be essential to fully understand the ecological consequences of MP contamination in terrestrial systems. Importantly, our findings underscore the role of physical disturbance as a major mechanism through which MPs influence soil C processes. Incorporating inert glass controls allowed us to disentangle these effects, revealing the often-overlooked role of MP shape and structural interference.

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**Figure 1:** Cumulative total CO₂ emissions within negative control soil (soil only), as well as incubations amended with glass and polyethylene beads and glass and acrylic fibers at low (0.1% w/w) and high (1.0% w/w) rates after 63 days of incubation. (a) Raw data distributions for total CO₂ are presented as swarm plots (top row). Data distributions are aligned with corresponding mean and standard deviation (n = 6 for each group). (b) The multi-group estimation plots (bottom row) depict the unpaired mean differences of bead (glass and polyethylene) and fiber
(glass and acrylic) treatment conditions under low and high amendment scenarios. Downward-pointing arrowheads indicate negative effect sizes, and their corresponding confidence intervals (CIs) are presented in black. Neutral effects, illustrated by circles, occur when the CIs overlap the dashed zero line (line of no effect). Asterisks indicate a significant change as compared to the control.

**Figure 2:** Effects of high and low additions of microplastic and glass beads (purple) and fibers (green) on soil physicochemical properties. Mean (± SE) values of (a) soil pH, (b) dissolved organic carbon (DOC), and (c) inorganic nitrogen (Inorganic N) across different treatment groups. Different letters indicate significant differences among treatments based on Tukey's *Honestly Significant Difference test* (*p* < 0.05).

**Figure 3:** Cumulative priming effect (PE) of soil carbon mineralization during a 63-day incubation with microplastic (MP) additions. Priming effect (mg C kg⁻¹ soil day⁻¹) was calculated using a) total CO₂ from control (soil only, total\_PE) or b) total CO₂ from paired glass treatments (MP\_PE). Purple coloured points represent polyethylene bead treatments, while green coloured points represent acrylic fiber treatments at low (L) and high (H) addition rates.

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<table><caption>**Table:** Experimental treatments used in this study. GB for glass beads, and GF for glass fibers. Quantity of microplastic (MP) or glass added are indicated by L for low (0.1%) and H for high (1%). Each treatment consisted of 6 replicate incubations (n = 54).</caption><thead><tr><th>Treatment name</th><th>Material type</th><th>Shape</th><th>Concentration (% w/w)</th><th>Purpose / control for</th></tr></thead><tbody><tr><td>Control (soil only)</td><td>None</td><td>None</td><td>0%</td><td>Baseline (soil-only control)</td></tr><tr><td>Polyethylene-L</td><td rowspan="2">Polyethylene microplastic</td><td>Beads</td><td>0.1%</td><td>MP chemical + physical effects</td></tr><tr><td>Polyethylene-H</td><td>Beads</td><td>1.0%</td><td>MP chemical + physical effects</td></tr><tr><td>GB-L</td><td rowspan="2">Glass</td><td>Beads</td><td>0.1%</td><td>Physical control for polyethylene-L</td></tr><tr><td>GB-H</td><td>Beads</td><td>1.0%</td><td>Physical control for polyethylene-H</td></tr><tr><td>Acrylic-L</td><td rowspan="2">Acrylic microplastic</td><td>Fibers</td><td>0.1%</td><td>MP chemical + physical effects</td></tr><tr><td>Acrylic-H</td><td>Fibers</td><td>1.0%</td><td>MP chemical + physical effects</td></tr><tr><td>GF-L</td><td rowspan="2">Glass</td><td>Fibers</td><td>0.1%</td><td>Physical control for acrylic-L</td></tr><tr><td>GF-H</td><td>Fibers</td><td>1.0%</td><td>Physical control for acrylic-H</td></tr></tbody></table>

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<table><caption>**Table 2:** Mean (± SE) of cumulative plastic-derived CO₂ (mg C kg⁻¹ soil) and the percent decomposition of plastic relative to initial quantity of C added (%) after a 63-day incubation experiment from polyethylene bead and acrylic fiber amended incubations, at two levels of addition : low (0.1%w/w) and high (1% w/w).</caption><thead><tr><th rowspan="2"></th><th colspan="2">Polyethylene beads</th><th colspan="2">Acrylic fiber</th><th rowspan="2">F (p-value)</th></tr><tr><th>Low (0.1%w/w)</th><th>High (1.0% w/w)</th><th>Low (0.1% w/w)</th><th>High (1.0% w/w)</th></tr></thead><tbody><tr><td>Plastic-derived CO₂ (mg C kg⁻¹ soil)</td><td>10.91 ± 4.72 <sup>a</sup></td><td>8.70 ± 3.62 <sup>a</sup></td><td>11.45 ± 2.52 <sup>a</sup></td><td>15.12 ± 3.62 <sup>a</sup></td><td>0.52 (0.68)</td></tr><tr><td>Plastic decomposition (%)</td><td>8.47 ± 3.67 <sup>ab</sup></td><td>0.68 ± 0.29 <sup>b</sup></td><td>11.59 ± 2.55 <sup>a</sup></td><td>1.53 ± 0.37 <sup>b</sup></td><td>5.59 (&lt;0.01)</td></tr></tbody></table>

*Note:* Significant differences among treatments represented by a, b at *p* < 0.05
<figure><img src="image_2.png" /><figcaption>Figure 1: Cumulative total CO2 emissions within negative control soil (soil only), as well as incubations amended with glass and polyethylene beads and glass and acrylic fibers at low (0.1% w/w) and high (1.0% w/w) rates after 63 days of incubation. (a) Raw data distributions for total CO2 are presented as swarm plots (top row). Data distributions are aligned with corresponding mean and standard deviation (n = 6 for each group). (b) The multi-group estimation plots (bottom row) depict the unpaired mean differences of bead (glass and polyethylene) and fiber (glass and acrylic) treatment conditions under low and high amendment scenarios. Downward-pointing arrowheads indicate negative effect sizes, and their corresponding confidence intervals (CIs) are presented in black. Neutral effects, illustrated by circles, occur when the CIs overlap the dashed zero line (line of no effect). Asterisks indicate a significant change as compared to the control.</figcaption></figure>

240x140mm (300 x 300 DPI)
<figure><img src="image_3.png" /><figcaption>Figure 2: Effects of high and low additions of microplastic and glass beads (purple) and fibers (green) on soil physicochemical properties. Mean (± SE) values of (a) soil pH, (b) dissolved organic carbon (DOC), and (c) inorganic nitrogen (Inorganic N) across different treatment groups. Different letters indicate significant differences among treatments based on Tukey's Honestly Significant Difference test (p < 0.05).</figcaption></figure>

300x220mm (254 x 254 DPI)

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<figure><img src="image_4.png" /><figcaption>Figure 3: Cumulative priming effect (PE) of soil carbon mineralization during a 63-day incubation with microplastic (MP) additions. Priming effect (mg C kg⁻¹ soil day⁻¹) was calculated using a) total CO₂ from control (soil only, total_PE) or b) total CO₂ from paired glass treatments (MP_PE). Purple coloured points represent polyethylene bead treatments, while green coloured points represent acrylic fiber treatments at low (L) and high (H) addition rates.</figcaption></figure>

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