Soil Sampling, Biochar, MPs, and Earthworm Preparation

Soil samples (0–20 cm depth) were collected from a sandy-loam vertisol (USDA soil classification) under a decade-long maize (Zea mays L.) and wheat (Triticum aestivum L.) rotation at the Sustainable Agro-Ecology Experimental Station of Henan University, Kaifeng, China (34° 39′ N, 114° 14′ E, 76 m a.s.l). The soil, derived from Yellow River alluvium, comprised 68.6% sand, 19.7% silt, and 11.7% clay. Visible plant debris and any fragments were removed. Samples were air-dried at 25 ± 2°C for 14 days, homogenized, and sieved through a 2-mm stainless steel mesh prior to use. The biochar used in this study was derived from wheat straw via pyrolysis at 550 °C. Polyvinyl chloride (PVC) microplastics (MPs; <0.5 mm diameter) were sourced from Wangda Plastic Co. (Dongguan, China). The primary properties of the soil, biochar, and MPs are presented in Table 1.

The epigeic earthworm Eisenia fetida was selected for this investigation due to its prevalence in regional agroecosystems and standardized use in ecotoxicological assays [26-27]. The earthworms were carefully washed, dried on filter paper, and weighed prior to their introduction into each experimental pot. Detailed information on the earthworms is provided in Table 2.

Experimental Design and Setup

A full-factorial mesocosm experiment (2 × 2 × 2 design) was conducted to assess the individual and interactive effects of PVC-MPs (MPs: 0% [M0] or 1% w/w [M10]), biochar (0% [B−] or 1% w/w [B+]), and earthworms (0 [E−] or 15 individuals pot⁻¹ [E+]). Each treatment combination was replicated four times (n = 32 pots total). Mesocosms consisted of 6 L polypropylene pots filled with 7.0 kg of air-dried sandy-loam vertisol (sieved to 2 mm), homogenized with MPs and/or biochar. Earthworms (4.8–5.0 g individual⁻¹) were surface-sterilized with clean water, rinsed, and acclimated for 48 h prior to introduction. Pots were maintained at 20 ± 2°C under 60% water-holding capacity for 21 days, with fleece-lined bases to prevent earthworm escape.

Greenhouse Gas Measurement

GHG fluxes were measured at 1, 2, 3, 4, 6, 7, 10, 13, and 21 days using static chambers (50 cm height × 40 cm diameter) sealed to pots via water-filled grooves. Gas samples (20 mL) were collected at 0 and 120 min using gas-tight syringes, stored in pre-evacuated vials, and analyzed within 24 h via gas chromatography (Agilent 7890B). CO₂ and CH₄ were quantified using flame ionization detection (FID; 250°C detector temperature) and N₂O using electron capture detection (ECD; 350°C). The GWP for a 100-year time horizon, including climate–carbon feedbacks, was calculated using a radiative forcing potential relative to CO₂ of 34 for CH₄ and 298 for N₂O.

Soil Analysis

Following a 21-day incubation period, soil samples were analyzed. Soil pH was measured in a 1:2.5 (w/v) soil-deionized water suspension using a calibrated pH meter (Orion Star A329, Thermo Scientific). Dissolved organic carbon (DOC) and total nitrogen (TN) soil extracts (2 M KCl, 1:5 w/v) were shaken (200 rpm, 25°C, 1 h), centrifuged (4,000 × g, 15 min), and filtered (0.45 μm GF/C membranes). DOC and TN concentrations were quantified via high-temperature combustion (Shimadzu TOC-VCSH/TNM-1; detection limits: 0.1 mg C L^-1, 0.01 mg N L^-1). Mineral nitrogen: NH₄⁺-N and NO₃^--N in filtered extracts were analyzed colorimetrically using a Flow Injection Analyzer (Lachat QuikChem 8500; detection limits: 0.01 mg N L^-1). The chloroform fumigation-extraction method determined the soil microbial biomass carbon (MBC) and nitrogen (MBN). Briefly, fumigated (24 h, CHCl₃ vapor) and non-fumigated soils were extracted with 0.5 M K₂SO₄ (1:4 w/v). MBC and MBN were calculated as [(fumigated ‒ non-fumigated C or N) / 0.45 and 0.54, respectively].

Data Analysis and Statistics

Three-way ANOVA with Tukey’s HSD post-hoc tests (α = 0.05) assessed main and interactive effects using the SPSS 27 package. Data met normality (Shapiro-Wilk) and homogeneity (Levene’s test) assumptions; non-normal datasets were log-transformed. Treatment effects were quantified as percentage changes relative to controls (95% confidence intervals).

Earthworm Survival and Soil Biogeochemical Properties

The survival of earthworms across all treatments was not optimistic, although they remained alive throughout the incubation period (Table 2). E+M0 treatment especially exhibited the lowest earthworm survival rate of 53% compared to the initial state. Notably, the incorporation of microplastics or biochar increased survival by 24-33% (p < 0.05), likely due to MPs improving soil aggregation (enhancing habitat structure) and biochar buffering moisture fluctuations. Despite these improvements, individual earthworm biomass declined uniformly by 35-41% (p > 0.05), suggesting physiological stress from MP exposure, potentially via oxidative damage or gut obstruction.

Soil pH exhibited dual regulation: earthworms reduced pH by 1.9% (8.54 vs 8.38; p < 0.01) through acidifying excretions (e.g., ammonium), while biochar increased it by 1.8% (8.39 vs 8.54; p < 0.001) via its alkaline ash content (Table 3). This pH modulation influenced microbial community composition, favoring nitrifying bacteria under biochar-amended conditions. MPs reduced NO₃⁻-N by 29.0% (p < 0.01), likely by adsorbing NO₃⁻ on hydrophobic surfaces, whereas earthworms increased NO₃⁻-N by 58.7% (p < 0.001) via enhanced mineralization of organic N (Table 3). The MP × E interaction amplified NH₄⁺-N depletion (p < 0.05), reflecting earthworm-driven nitrification of NH₄⁺-N in MP-aerated soils. MPs elevated DOC by 60.3% (p < 0.001) and DOC:TN by 45.8% (p < 0.01) (Table 3), directly correlating with CO₂ emissions. Biochar increased DOC by 22.1% (p < 0.05), likely through leaching of labile C fractions. Microbial biomass responded divergently: MPs and biochar synergistically increased MBC by 28.4% (p < 0.01) but reduced MBN by 19.7% (p < 0.05), elevating MBC:MBN by 32.6% (p < 0.01). This shift suggests N-limitation for microbial growth, suppressing nitrifier activity and N₂O production.

Greenhouse Gas Flux Dynamics

The three greenhouse gases exhibited distinct temporal flux dynamics (Fig. 1). CO₂ fluxes declined sharply within 132 hours, followed by slight fluctuations at low rates until the end of the incubation. Adding earthworms and biochar significantly enhanced CO₂ emissions. Across all treatments, N₂O flux emissions peaked in the first 4 days (~84 h) of incubation and then declined steadily thereafter. Some negative N₂O fluxes were observed between 300 and 516 hours. In contrast, CH₄ emissions were relatively variable and did not exhibit a clear pattern. Furthermore, CH₄ emissions were negative for all the treatments, indicating net CH₄ oxidation.

A substantial amount of CO₂ emissions occurred in the initial stage and then decreased steadily. This is likely due to the addition of MPs and biochar providing an external input of available C for heterotrophic consumption of labile C, while exhaustion of labile C thereafter leads to a steady decrease [28]. Furthermore, earthworms generally lead to higher CO₂ emissions, as their activity in soil facilitates the availability of more oxygen for CO₂ production and release [29]. Additionally, biochar amendment resulted in higher CO₂ emissions than the control in the initial stages (Fig. 1c), primarily due to the increased soil organic C mineralization induced by its priming effect [30]. N₂O emission fluxes generally followed a similar trend but exhibited varied responses to the three factors during the first 10 days of the incubation. These results are likely attributed to two distinct processes: 1) adding MPs or biochar enhancing soil C/N ratio and promoting ammonia oxidation [31], and 2) earthworm activity accelerating the availability of soil mineral N, which is associated with soil nitrification and denitrification processes [32]. CH₄ fluxes were almost negative for all the treatments, which means the sandy-loam alkaline soil here has an intense capacity for CH₄ oxidation [33].

Effect of MPs on GHG Emissions

Adding MPs exerted significant yet inconsistent effects on the three greenhouse gas emissions (Fig. 2, Table 4). Specifically, it significantly increased CO₂ emissions and GWP while decreasing N₂O and CH₄ emissions. The different effects of MPs on these three GHG emissions may stem from various potential reasons. Firstly, adding MPs improves soil porosity, aggregation, and aeration [34, 35], facilitating the diffusion of O₂ and CO₂ from the soil [36]. It is known that N₂O production primarily depends on denitrification under anaerobic conditions and nitrification under aerobic conditions [37]. Soil O₂ increase can reduce microbial denitrification, decreasing soil N₂O emissions. Additionally, the increased soil O₂ content also accelerates CH₄ oxidation and restrains soil CH₄ emission [38]. Secondly, the soil C:N ratio is a crucial indicator for assessing soil GHG emissions, particularly in relation to N₂O emissions [39]. Previous studies have demonstrated a robust inverse correlation between the soil C/N ratio and N₂O emission [40], consistent with our findings. Furthermore, 22%-46% of the DOC in MPs can be utilized by organisms or microorganisms in the carbon cycle as energy or nutrients [41], correlating with the increase in CO₂ emission and the decrease in N₂O emission. Thirdly, a higher MBC:MBN ratio primarily impeded soil nitrification, subsequently reducing N₂O emission [42]. A recent global analysis revealed a negative correlation between the MBC:MBN ratio and soil N₂O emissions [43]. Furthermore, previous research discovered a strong correlation between soil N₂O emissions and NO₃^- content in spatial and temporal models [44]. This suggests that a reduction in NO₃^- levels would decrease inorganic nitrogen availability for microbial denitrification, ultimately leading to a decrease in N₂O emissions as an intermediate product.

Fig. 1.

Hourly CO₂ (a, b, and c), N₂O (d, e, and f), and CH₄ (g, h, and i) emission fluxes during the 21-day incubation. The error bars indicate the standard errors of means (±SE, n=16).

Effect of Earthworms on GHG Emissions

The presence of earthworms in the soil significantly increased soil CO₂ and N₂O emissions. The presence of earthworms significantly enhanced GWP by 50.9% compared to the control. However, earthworms did not affect CH₄ production (Fig. 2, Table 4). This effect was likely due to the changes in soil properties caused by earthworms and their inherent characteristics. Earthworm respiration contributes significantly to soil respiration [26], accounting for the 42.3% increase in CO₂ emissions observed in our study due to the presence of earthworms. This result is similar to the findings of a 34% increase in CO₂ emissions due to the presence of earthworms [45, 46]. Our results indicated that the presence of earthworms had a strong influence on accelerating N₂O emissions, which is consistent with previous studies [47]. This phenomenon is likely due to the increase in soil NO₃^- content, which provides sufficient substrate for denitrification and thus stimulates soil N₂O emissions [48]. In the current study, the presence of earthworms did not affect CH₄ emissions, a finding that is supported by previous studies [49]. However, there are differing results regarding the effect of earthworms on CH₄ emissions. For instance, aquatic earthworms could reduce CH₄ fluxes by promoting a favorable environment for methanotrophs [50].

Effect of Biochar on GHG Emissions

Biochar amendment had a significant but opposite effect on CO₂ and N₂O emissions while having no effect on CH₄ production (Fig. 2, Table 4). Specifically, biochar amendment significantly increased CO₂ emissions while decreasing N₂O emissions relative to the unamended treatments.

Fig. 2.

Cumulative CO₂ (a, b, and c), N₂O (d, e, and f), and CH₄ (g, h, and i) emissions during the 21-day incubation. The error bars indicate the standard errors of means (±SE, n=16).

Biochar is a highly effective material for regulating soil’s physico-chemical environment, given that GHG emissions are closely correlated with soil physical properties (e.g., pH, porosity, aggregation) and chemical properties (e.g., carbon-nitrogen ratio, microbial carbon) [51]. Our study found that biochar amendment significantly impacted several soil properties, including pH, DOC, and MBC, thereby influencing soil CO₂ and N₂O emissions. Previous studies reported that biochar amendment could increase CO₂ emissions by 22.14% and decrease N₂O emissions by 38% [52, 53]. These findings align closely with our own results, demonstrating a 20.6% increase in CO₂ emissions and a 26.1% decrease in N₂O emissions. Biochar has a carbon content exceeding 90%, and its addition results in higher mineralization of labile C and inorganic C release, which stimulates soil microbial activities, ultimately promoting CO₂ emissions [54]. Furthermore, adding biochar to soil enhances soil aeration, which not only facilitates CO₂ diffusion but also suppresses microbial denitrification, thereby leading to a reduction in N₂O emissions [55]. Additionally, the higher carbon-to-nitrogen ratio in soil resulting from biochar amendment could also restrain N₂O emissions. The increased nitrogen demand by microorganisms elevates the soil carbon-to-nitrogen ratio, a crucial factor affecting nitrification and denitrification processes, ultimately decreasing N₂O emissions [56].

Comprehensive Effects of the Three Factors on GHG Emissions

Our findings revealed few interactive effects of the treatments on soil GHG emissions. Only CO₂ emissions and GWP were significantly influenced by the interaction of MPs and earthworms (Table 4, Fig. 3). Specifically, when earthworms were absent, there was no significant difference in CO₂ emissions with or without MPs. However, the presence of earthworms significantly enhanced CO₂ emissions by 32.7% when MPs were added compared to the treatment without adding MPs (Fig. 3a). This was primarily attributed to the stimulation of earthworms’ respiration by adding MPs, which is linked to soil respiration, resulting in a significant increase in soil CO₂ emissions [26]. Previous research has demonstrated that adding MPs can promote soil aeration, which favors earthworms’ activities such as burrowing, feeding, and excretion [57]. Furthermore, MPs increased the survival rate of earthworms in our study, factors that collectively enhance the respiration of earthworms themselves, ultimately increasing soil CO₂ emission. Similarly, earthworms significantly enhanced GWP by 27.7% when adding MPs (Fig. 3g). This interaction was closely associated with the combined contribution of CO₂, N₂O, and CH₄ emissions under different treatments [58]. However, the absence of earthworms resulted in no significant difference in GWP regardless of adding MPs. Currently, research on the interactive effects of earthworms and MPs on the GWP of agricultural soils is relatively limited, and the underlying mechanisms are complex, warranting further investigation in future studies.

Fig. 2.

Cumulative CO₂ (a, b, and c), N₂O (d, e, and f), and CH₄ (g, h, and i) emissions during the 21-day incubation. The error bars indicate the standard errors of means (±SE, n=16).