Potential impacts of two types of microplastics on Solanum lycopersicum L. and arbuscular mycorrhizal fungi

: Microplastics (MPs) in agricultural soils are a major concern. Relatively few studies have been carried out to explore its potential impacts on crops and soil microbiota. The study investigates the effects of two different types of MPs (microfiber, MFB and microfilms, and MFL) on the growth of Solanum lycopersicum L. (tomato) and Arbuscular Mycorrhizal Fungi (AMF). A pot experiment was conducted with MFB and MFL concentrations viz ., 0.4, 2.4, 4.4, 6.4, and 8.4% (w/w) and control without MPs (n = 6). Plant height was measured weekly for 13 wk before plants were harvested destructively. Growth increment, relative growth rate (RGR), and root: shoot ratio (RSR) were calculated. Leaf chlorophyll levels were measured. AMF spores were quantified using the wet sieving and decanting method. Plants showed a concentration-dependent decline in growth, regardless of the MP type. RSR demonstrated a decline with increasing MP concentration. Compared to the control, plants declined the allocation of shoot biomass by 40-74% and root by 6-75%. Plants in MFBs reported a higher chlorophyll content than in MFLs (F = 18.33; p ≤ 0.000). MPs significantly reduced AMF spore abundance in soils, indicating adverse effects on the soil microbiota. The study concluded that MPs impose negative effects on both vegetative growth of tomato and AMF spores. Further studies are imperative to explore the link between MPs and, crop growth and AMF.


INTRODUCTION
Plastics have become the most versatile raw material in the packaging industry due to certain desirable characteristics such as low cost, lightness, effective malleability, and durability (Payne et al., 2019;Ncube et al., 2021). Sri Lanka annually imports a total of 260,000 metric tons of plastic-based raw material and finished products (Palugaswewa, 2018), and the bulk of them eventually end up in landfill sites due to lack of recycling strategies. With time, plastics can break down into smaller particles through various physical, biological, and mechanical processes (Wright and Kelly, 2017;Mai et al., 2018;Mao et al., 2020). Plastic particles smaller than 5 mm in size are termed 'microplastics' (MPs) and are considered an emerging contaminant in the environment (Lehtiniemi et al., 2018;Guo et al., 2020). Once disintegrated into smaller particles, they will remain in the environment for a long period of time causing more or less obscured penalties on the environment and its organisms (Sebille et al., 2015;Liu et al., 2018;. Soil is considered a major sink for MPs, at times even larger than the marine environment (Blasing and Amelung, 2018;Hurley and Nizzetto, 2018).
In comparison to other terrestrial habitats, agricultural lands have become a major sink for MPs. Microplastics pollute agricultural soils in countless ways. Plastic-based materials used in mulching and tunneling, fertilizer packaging, and water pipes are some of the major sources of MPs in agricultural soils (Steinmetz et al., 2016;Brodhagen et al., 2017;Zhang and Liu, 2018). Due to tire wear of machinery used for tilling and ploughing also leads to contamination of agricultural soils with MPs. Microplastics also enter agricultural lands via sewage sludge that is used to fertilize agricultural fields. Plastic encapsulated slow-release fertilizers, plastic-coated seeds (to protect them from pathogenic microorganisms and other predators), and poorly-processed compost is also considered as sources of MP contaminants in farming soils (Katsumi et al., 2021;Withanage et al., 2021).
Plastic residues differ in size (macro, meso, micro, and nano), shape (fragment, fiber, films, beads, etc.), and polymer type (polyethylene, polypropylene, polystyrene, etc.) (Shim et al., 2018). Once entered the soil environment, MPs can either directly or indirectly impact plant-soil health, and eventually their performance and productivity. During the production process of plastics, many additives and heavy metals are added in order to improve the quality of the final product (Salvaggio et al., 2019). Also, due to some unique structural features, plastics take up xenobiotics from their immediate surroundings . Microplastics and their associated toxic substances could affect plant roots and their associated rhizosphere organisms, thus causing a detrimental influence on plant growth Qi et al., 2020). In contrast, others noted the ability of MPs residues to reduce the bioavailability of toxic metals by acting as an adsorbent (Zhou et al., 2020). In addition to these direct toxic effects, MPs can also alter soil physicochemical properties Wan et al., 2019), which in turn converts the habitat inhospitable for plants and microbes (Huang et al., 2019;Qi et al., 2020;Zhou et al., 2021). In particular, microfibers and microfilms are known to cause substantial impacts on the chemical and physical characteristics of soils . Microplastics tend to alter pH, soil aggregation, bulk density, and water retention ability Wan et al., 2019;Xu et al., 2020). These modified soil habitat conditions not only affect plants but also microbial functions, which in turn affect biogeochemical cycles (Kuzyakov and Xu, 2013).
However, due to the high heterogeneity of MPs in terms of the polymer type, size, shape, and concentrations, their impacts on plants, soil microbes, and nutrient cycling processes remain vague and highly unpredictable together with their less-known underlying mechanisms Zhou et al., 2021). As studies noted some clear evidence to show varying impacts of MPs on soil physicochemical properties and microorganisms , it is unjustifiable to think that plant growth is untouched by MP contamination. Available studies note that impacts of MP contamination are species-specific Judy et al., 2019;Rillig et al., 2019), highlighting the need for further studies to evaluate impacts of MPs on different crops and soil biota (Qi et al., 2018;Zhang et al., 2020). Therefore, the main objective of the present study is to evaluate the effects of two types of MPs (microfibers and microfilms) on the growth of Solanum lycopersicum L. (tomato) and the abundance of arbuscular mycorrhizal fungi (AMF) in soil.

Experimental setup
A pot experiment was conducted with Solanum lycopersicum (Family-Solanaceae) as the test plant. Tomatoes are known to form symbiotic associations with AMF (Subramanian et al., 2006). Microfibers (MFB) and microfilms (MFL) were obtained from polyethylene terephthalate (PET or polythene) and Low-Density Polyethylene (LDPE) sheets, respectively. They were cut into pieces using a sharp blade and sifted through a 5 mm steel sieve to ensure the particle size of the end product (< 5 mm). Different weights of MFB and MFL were mixed with 1,500 g of the air-dried potting mixture (garden soil: sand: coir in 1:1:1 ratio) separately in order to achieve the required concentrations viz., 0.4, 2.4, 4.4, 6.4, and 8.4% (w/w as a percentage). The concentration range of MPs used in the study was determined based on the MP levels observed in soil (Ng et al., 2018;Corradini et al., 2019;Meng et al., 2021). However, higher doses were also included in order to magnify the potential effects, which otherwise be unnoticed. NPK (nitrogen, phosphorus, and potassium) fertilizer was added to the potting mixture as recommended for tomatoes by the Department of Agriculture, Sri Lanka (3 g of urea, 14 g of Triple Super Phosphate, and 3 g of Muriate of Potash per pot). Two oneweek-old seedlings, raised from seeds, were transplanted in each pot at a distance of 6 cm apart from each other. Control was maintained without MPs. Six replicates were used for each treatment, and together with the control, totaling 66 pots (5 concentrations × 2 types of MPs × 6 replicates + 6 control pots). Pots were labeled and arranged in a randomized complete block design (RCBD) in the glasshouse at the Department of Botany, Faculty of Science, University of Peradeniya, Sri Lanka. Pots were watered as required and were randomly repositioned regularly to evade any position effect on the growth of seedlings.

Growth parameters
Plant height was measured weekly for a period of 13 wk before plants were destructively harvested. Fresh and dry weight measurements of the shoot and root samples (after oven-drying samples at 70 ℃ to constant weight) were recorded. Relative growth increment (as a % of the initial height; Equation 1), relative growth rate (RGR; Equation 2), and root: shoot ratio (RSR; Equation 3) were calculated.
Chlorophyll levels of mature leaves were quantified spectrophotometrically using randomly plucked leaves at the time of the destructive harvest. Chlorophyll was extracted using 1.0 g fresh leaf samples in 80% acetone and 0.5 g of MgCO 3 and absorbance values (A) was measured spectrophotometrically at 645 nm and 663 nm wavelengths (Kamble et al., 2015). Chlorophyll contents (Chl-a and Chl-b) in mg per g of fresh plant material were quantified using the following formula (Equations 4 and 5).

Quantification of AMF spore abundance
After 13 wk, soil samples were collected from the rooting zone of three randomly selected pots to quantify AMF spore counts. Only two treatments were chosen to quantify AMF spores viz., 8.4% MFB, 8.4% MFL, and the control (without MP). Soil samples were kept in a refrigerator until they were processed. Arbuscular mycorrhizal fungal spores were extracted using the wet sieving and decanting method (Pacioni, 1992). From each soil sample, a 100 g sub-sample was used to extract spores into different size categories with the help of steel sieves of different mesh sizes viz., 500, 125, 63, and 45 μm (Brundrett et al., 1996). The extracted spores on filter papers (Whatman GF/A) were then counted using a compound light microscope (OLYMPUS CX 12). From different morphotypes of spores, semi-permanent slides were prepared in Polyvinyl alcohol-Lacto-glycerol (PVLG) with Melzer's reagent to observe detailed spore characteristics using descriptions given in INVAM (https:// invam.wvu.edu/). Photographs of AMF spores were taken using a compound microscope with a built-in camera (Optika vision Lite 2.13).

Data analysis
Growth parameters (RGR, root and shoot biomass, and RSR) and Chlorophyll contents (Chl-a and Chl-b) were analyzed using two-factor ANOVA in Minitab 17.0. Arbuscular mycorrhizal spore counts were analyzed using General Linear Model (GLM) using microplastic type (MFB and MFL), concentrations (0.0 and 8.4%), and size categories (45, 63, 125, and 500 µm) as factors. Tukey's test was used for pairwise comparisons at a significance level of less than 0.05. Before using ANOVA, the data were tested for normality of distribution using the Anderson-Darling test.

Growth of tomato
In comparison to plants grown in control pots (without MPs), tomato seedlings grown with MPs demonstrated a concentration-dependent growth decline, irrespective of the type of MPs (Figures 1 and 2).
The relative growth rate (RGR) also showed a decreasing trend along the concentration gradient of MPs ( Figure 2). In particular, plants grown at higher concentrations of MPs (6.4 and 8.4%) displayed a significant reduction in RGR in comparison to that of the control (without MPs) (Figure 2).

Biomass allocation
No significant differences were observed in RSR between the two MP types (ANOVA: F = 0.40; p = 0.529). However, concentration-dependent differences were observed, with RSRs gradually declining from the lowest to the highest concentration of MPs, with some significant differences between them (Figure 3, ANOVA: F = 4.33, p = 0.002).
In comparison to plants grown without MPs (control), the plants grown with the lowest concentration of MPs (0.4%) showed a notable rise in the RSR (approximately 53% and 62% increase in MFB and MFL, respectively), followed by a concentration-dependent gradual decline in RSR with the increasing MP concentrations (Figure 3). This sharp rise in RSR is mainly due to the significant reduction in biomass allocation to above-ground parts than to belowground parts (55 and 40% decline in shoot biomass in comparison to a 6 and 22% decline of root biomass in MFL and MFB, respectively) ( Figure 4). However, in plants grown at the highest concentration of MPs (8.4%), both shoot and root biomasses were plunged by 67 and 69% with MFB and 74 and 75% with MFL, respectively (in comparison to the shoot and root biomass of control plants). The results indicate a considerable reduction in the biomass allocation to above-ground parts (shoot) in comparison to below-ground parts (roots) in presence of MPs (Figure 4).

Chlorophyll pigments
Exposure to MFB and MFL over a period of 13 wk was reflected by statistically significant differences between Chl a and Chl b levels across their concentrations (Chl a: F = 3.59, p = 0.015; Chl b: F = 3.98, p = 0.009) and types (Chl a: F = 10.9, p = 0.003; Chl b: F = 17.16, p ≤ 0.000). Chlorophyll b levels were always higher than Chl a irrespective of the type of MP or their concentrations. Both chlorophyll pigments showed statistical differences across the concentration gradient of MPs, though not in a consistent manner. However, both Chl pigments were significantly higher in plants grown with MFBs compared to that of MFLs.

AMF abundance
Tomato plants grown in soils without MPs (control) showed a significantly higher total spore count of AMF (1,356) compared to soils with microfibers (952) and microfilms (822). Irrespective of the MP type, AMF spore density was significantly higher in soils without MPs than with MPs (Table 1). The smallest and the largest spore sizes (45 μm and 500 μm, respectively) were notably more prevalent in soils without MPs than in soils with MFBs and MFLs (Table1; Figure 5).

DISCUSSION
Despite to the emerging nature of MPs as an environmental pollutant, the potential impacts of MPs on plants and microbiota and their underlying mechanisms are not welldocumented (Qi et al., 2018;Rillig et al., 2019;Khalid et al., 2020). However, some evidence is starting to appear proving that MPs impact plants and crops in either direct or indirect manner. Rillig et al. (2019) recognized some possible mechanistic pathways through which MPs can influence plant growth based on the species, type, and concentration of MPs. In agreement, the findings of the present study revealed that MPs impose adverse effects on the overall growth of S. lycopersicum plants. However, contrary to previous evidence, the type of MP (MFB and MFL) showed no distinct effect on the growth of tomatoes. Machado et al. (2019) observed a similar growth decline in spring onions (Allium fistulosum) with different types of MPs. As shown in other studies Zhang and Liu, 2018;Wan et al., 2019), the inherent toxicities of MPs or else their ability to alter soil physicochemical properties seem to have influenced tomato plants in a detrimental manner.
In addition to overall growth parameters (height increments and RGR), root to shoot ratio (RSR) provides an insight into a plant's ability to perform across different environmental gradients (Rogers et al., 2019;Liu et al., 2021). According to the optimal partitioning theory, plants tend to allocate relatively more biomass to organs in oder  to assist in absorbing the most limiting resources (Mao et al., 2012). The present study demonstrated a concentrationdependent decrease in RSR in tomato plants. Along the MP concentration gradient of 0.4% to 8.4%, the RSR declined approximately by 40% and 42% with MFB and MFL, respectively. Even at low concentrations of MPs, the biomass allocation to shoot was notably declined in comparison to the root allocation, irrespective of the MP type. This is perhaps due to the fact that seedlings tend to invest more biomass into roots in order to optimize water uptake at a cost of the shoot allocation. Microplastics tend to increase soil aggregation and decrease soil bulk density thus leading to better soil aeration that may favor root penetration, while increasing soil dryness (Wan et al., 2019;Khalid et al., 2020). Previous studies also noted somewhat a similar effect on root and shoot growth when wheat seedlings are exposed to microplastics (Qi et al., 2018). Meng et al. (2021) observed that while biodegradable MPs strongly reduced root and shoot allocations, lowdensity polyethylene (LDPE) has inflicted no effect in a study carried out with a narrower concentration gradient (0.5 to 2.5%) and a smaller-sized (< than 1 mm) MP residues. In contrast,  observed no effect on the root and shoot growth of maize when plants are exposed to high-density polyethylene (HDPE), further confirming the unpredictable nature of MPs depending on the crop, polymer type, and concentration. Machado et al. (2019) too observed an increased allocation of biomass to roots of spring onion, especially with polyester particles, but with a caution of oversimplification of plant responses due to different properties of MPs (size, chemical composition, etc.). With the increase in the concentration of MPs, both root and shoot allocations were declined in a parallel manner, thus confirming an overall growth reduction in tomato plants as a result of the contaminant.
The growth of terrestrial plants is primarily influenced by edaphic factors such as drought, nutrient deficiencies, and mineral toxicities (Lynch, 2007). Microplastics are known to cause varying effects on soil physicochemical properties, soil microbes, and their activities (Fei et al., 2020;Zhou et al., 2021), with an ultimate influence on the soil fertility status (Kasirajan and Ngouajio, 2012). In addition to indirect effects of MPs on the growth of plants, the toxic effects of MPs may also influence plant growth in a detrimental manner. Such phytotoxic effects could be accredited to the presence of additives and pollutants  that are weakly bound to polymer molecules thus easily leach into soils (Bolan et al., 2020). Therefore, the interactive effects due to MP-driven edaphic changes and toxic effects may eventually cause plants to perform poorly. Also, MPs have the potential to degrade further into nano plastics (NPs), with the ability of plants to absorb and accumulate them in the plant body causing even more damage to plants (Navarro et al., 2008).
The chlorophyll content is a well-recognized parameter to monitor plant health (Cortarzar et al., 2015). Abiotic stresses such as drought, salinity, and heat cause harmful impacts on the biosynthesis of chlorophyll pigments (Dalal and Tripathy, 2012). In the present study, chlorophyll levels showed no consistent trend across the concentration gradient of MPs. However, tomato plants grown with MFBs showed relatively higher chlorophyll contents (both Chl a and Chl b) compared to those in MFLs. According to , PLA (polylactic acid) decreased the chlorophyll content markedly at higher concentrations, which eventually reflected in the overall growth of plants, whilst no similar effect with polyethylene (PE). In the present study, despite a clear concentrationdependent growth decline in tomato plants regardless of the MP type, the chlorophyll levels showed no parallel decline. Despite minor differences between treatments, the present results indicate that MPs did not influence the photosynthetic capacity of plants severely, the findings that have been endorsed by previous workers (Qi et al., 2018;Pignattelli et al., 2020).
Arbuscular mycorrhizal fungi (AMF) provide numerous benefits to host plant growth (Smith and Read, 2008), with significant implications for sustainable management of agriculture Madawala, 2021). Despite some contradictory evidence regarding the role of AMF in crop growth, studies noted a positive link between the colonization potential of AMF and crop  (Smith and Read, 2008;Srivastava et al., 2017). Despite studies done to explore the effects of MPs on soil's physical and chemical properties, relatively little is known about their biological impacts until recently Horton and Barnes, 2020). Despite certain barriers such as lack of sporulation and parasitism that may lead to a possible underestimation, AMF spore counts provide baseline information to estimate their population in soils (Leal et al., 2017). Therefore, spore counts from field soils are still considered a reliable evidence in comparing AMF populations under different land-use types and treatments (Leal et al., 2017). According to prior studies, any factors responsible for modifying physicochemical features in soil could eventually mete out harmful impacts on the structural and functional diversity of the soil microbiota (Kaushal and Wani, 2016;Awet et al., 2018;Qi et al., 2018;Rillig and Bonkowski, 2018;Wan et al., 2019;Yi et al., 2020). Accordingly, soil pollutants inflict detrimental effects on AMF by reducing their potential to colonize, infect roots, and develop arbuscules and spores (Desalme et al., 2012;Ferrol et al., 2016). The present findings too showed a negative effect of MPs on the abundance of AMF spores.  confirmed that MPs change the abundance, structure, and diversity of AMF communities based on the type of MP and, the size and the concentration of residues (Brodhagen et al., 2017;Khalid et al., 2020). However, the present study suggests no significant difference between the two MP types (MFB and MFL) in their respective influences on the AMF spore abundance. However, the present study did not allow us to test the concentration-dependent effect of MPs on AMF as the comparison is carried out between the highest MP concentration (8.4%) and the control (without MPs).
Microbial communities including AMF play an important role in plant growth. Therefore, MP-driven reduction of the AMF population could directly influence crop growth in an adverse manner. Microplastics are known to alter soil parameters that govern habitat space and conditions for AMF and at the same time influence soil functions such as soil aggregation and nutrient transport (Leifheit et al., 2021). Thus, this evidence indicates that the negative influence of MPs on soil microbes and their functions in soils could be one of the major driving forces behind reduced plant and crop growth. However, further studies are needed to confirm this association.

CONCLUSION
Microplastics have imposed concentration-dependent negative impacts on the vegetative growth of S. lycopersicum plants regardless of the MP type. Arbuscular mycorrhizal fungal spore abundance was declined by the presence of MPs in soils. More studies are imperative to uncover the varying impacts of MPs on plant growth and soil microbiota to understand a potential link between them. Understanding the role of MPs as soil pollutants and their impacts on the soil-plant system is critical in order to introduce remedial measures to minimize risks posed by MPs in agriculture.