A green, fast protocol to estimate the accumulation of airborne anthropogenic microfibers in Pittosporum tobira in urban areas: effects of season and rainfall

Introduction

Plastic is considered one of the major environmental persistent pollutants (Adeniran, Ayesu-Koranteng & Shakantu, 2022; Williams & Rangel-Buitrago, 2022). Being elastic, durable, lightweight, waterproof, cheap and easy to manufacture, synthetic polymers have become the protagonists in several markets, as packaging, textiles, construction materials, and industrial equipment. In 2021, world plastic production was estimated at 390 million tons - of which 90% derived from fossil sources - and China, with one third of the global production, was the largest plastic manufacturer in the world. Since the demand for plastics is predictably growing, and its degradation requests from decades to millennia (Li, Tse & Fok, 2016; Mohan et al., 2022; Ward et al., 2019), the amount of plastic waste is destinated to increase as well (Rednikin et al., 2024). Once produced, plastics undergo ageing and degradation, and break down in smaller fragments, forming meso- micro and nanoplastics which are added to primary micro- and nanoplastics, produced as such with various uses, e.g., chemical abrasives (Rednikin et al., 2024).

Another worrying aspect is that plastic products have been found in the most inhospitable places of our planet (Citterich, Giudice & Azzaro, 2023) indicating that plastic contamination is ubiquitous and affects all environmental matrices. Indeed, it is reported that microplastics (MPs) (1 µm to five mm) (Da Costa Filho et al., 2021) and nanoplastics (NPs) (<1 μm) (Jakubowicz, Enebro & Yarahmadi, 2021) are widely underestimated (Cesarini et al., 2023; Taurozzi et al., 2024). Airborne MPs have been low-considered and studied so far in comparison with MPs dispersed in the ocean, sediments, and soil. Fibers are the predominant morphotype in pristine areas (Roblin & Aherne, 2020; Capozzi et al., 2023), and urban environments (Dris et al., 2015), both indoors (Amato-Lourenço et al., 2022; Chen et al., 2022) and outdoors (Wright et al., 2020).

Road traffic emissions (estimated as 7 million kg year⁻¹) (Evangeliou et al., 2020), sea spray (Harb, Pokhrel & Foroutan, 2023), waste incineration, building materials, and synthetic fabrics (Amato-Lourenço et al., 2022) are considered the sources of airborne MPs giving the highest contribution. Their transport, distribution, and deposition is strongly affected by meteorological conditions and human activities (Paramitadevi et al., 2023; Habibi et al., 2022; Pandey et al., 2022).

As for other airborne pollutants, MPs concentrations in urban areas were recently monitored by using passive samplers (Cai et al., 2017; Dris et al., 2016; Leonard et al., 2023; Zhou, Tian & Luo, 2017). However, these devices are expensive and require specific expertise to perform sampling properly. The role of plants as biomonitors of airborne pollutants is grounded on their high surface to mass ratio, and their ability of intercepting particulate matter and related pollutants (Di Palma et al., 2017; Capozzi et al., 2020a). Thus, plant leaves represent a good alternative to passive samplers, and could potentially be used as a biomonitoring system also for airborne anthropogenic microfibers (MFs), for which a simple and reproducible protocol is still missing.

To fill this gap, in previous work we investigated the ability of Pittosporum tobira leaves to intercept and retain airborne MFs and set up a 4-steps sequential extraction procedure (Capozzi et al., 2024). We found that the percentage of particles, mostly fibers, extracted at each step was constant in all samples. Moreover, the first step of the sequential procedure (consisting in a tape tearing on the leaf surface) extracted the largest fraction of MFs, about 75%. Therefore, in the present work we hypothesized that P. tobira leaves could be sensitive enough to distinguish different levels of pollution due to airborne MFs, even by tape tearing alone. Also, given that most MFs are found on the leaf surface, we hypothesized that their accumulation could be influenced by rainfall regime. To test these hypotheses, we carried out a biomonitoring survey along an urban pollution gradient, analyzing Pittosporum tobira leaves sampled in six urban sites during summer (dry) and winter (rainy season) to investigate possible seasonal variation in their accumulation. Leaf traits of samples collected during both summer and winter, were examined by scanning electron microscope (SEM), as well. Finally, to further investigate the effect of pouring rain, P. tobira transplants (i.e., plants grown in pots), here used for the first time, were exposed sheltered, to protect them from the rain, and unsheltered, and their MFs accumulation was compared after heavy rain.

Materials & Methods

Results

Site comparison and seasonal variations

The two-way ANOVA analysis revealed statistically significant main effects for both independent variables, as well as a significant interaction:

• Main effect of land use type: significant main effect was found for land use type, F(5, 24) = 79.00, p < .001, indicating that the mean number of MFs differed significantly across the sites.

• Main effect of season: significant main effect was found for season, F(1, 24) = 241.00, p < .001. Overall, the mean microfiber count was higher during the summer than the winter season.

• Interaction effect: the two-way interaction between land use type and season was also statistically significant, F(5, 24) = 24.00, p < .001, indicating that the effect of the season on MF abundance is not uniform across all land use types.

To deconstruct the significant interaction, a Tukey’s honestly significant difference (HSD) post-hoc test was conducted. The results of the pairwise comparisons are illustrated in Fig. 1 (p < .05).

A comparison among sites based on the number of MFs removed by tape tearing (Fig. 1) showed that the highest number of MFs was collected at the industrial site, followed by the urban sites 1 and 2 and the three green sites. Significant differences between sites, even of the same typology (i.e., urban or green), were more pronounced in summer than in winter, where a more homogeneous MFs accumulation occurred. At each site, MFs accumulation was significantly higher in summer than in winter, apart from the green sites 1 and urban 3.

As for the length of MFs, it ranged between 50 µm to 12 mm over all sites, with the highest length found in leaf samples from industrial site. For the analysis, MFs having a length >5 mm were not considered. A comparison between the length of MFs found on the leaves collected at the two seasons (Table 2) showed that those observed in summer were significantly longer than MFs found on winter leaves.

Table 2:

MFs length at each site and season.

Site →	Industrial	Urban1	Urban2	Urban3	Green1	Green2
Season ↓	Number of MFs
	Length (Mean ± SD)
Summer	160	92	125	84	48	54
	0.64 ± 1.3*	0.76 ± 0.58*	0.99 ± 1.10*	0.93 ± 1.07*	0.93 ± 0.94	0.7 ± 0.72
Winter	71	47	30	60	53	24
	0.35 ± 0.39	0.56 ± 0.43	0.43 ± 0.29	0.58 ± 0.72	0.62 ± 0.55	0.5 ± 0.22
*Mann Whitney’s U test (p < 0.05)	p = 0.02	p = 0.02	p = 0.002	p = 0.001	p= 0.06	p= 0.75

DOI: 10.7717/peerj.20558/table-2

Notes:

Comparison according to Mann–Whitney U test (p < 0.05). Asterisks on the median values indicate significantly longer fibers.

Accumulation of MFs in transplants

Water washing of the transplants carried out before exposure significantly decreased the number of MFs; we found indeed, 20.44 ± 2.07 number of MFs in pre-washing samples vs. 9.11 ± 1.05 in post-washing ones (t-test, p < 0.05). The accumulation of MFs in transplanted P. tobira were significantly different between plants unsheltered and sheltered to the rainfall (Fig. 2); the exposure to heavy rain determined, in fact, a significant loss of MFs. Ten days exposure resulted in an adequate exposure time to ensure a clear accumulation on the leaves of P. tobira; no significant difference was found depending on the different exposure times (i.e., 10 and 15 days) before a heavy rain event (Fig. 2).

SEM observations

The comparison between summer and winter leaves (Fig. 3) highlighted that the first ones (a, b, and c) showed numerous glandular hairs on the upper surface, which is also characterized by irregular ridges, and sunken stomata on the lower surface, showing residual, very small epicuticular waxes (white arrows). Winter leaves instead, did not show glandular hairs, but abundant epicuticular waxes, rod-shaped on the upper surface (d, d’), and in the form of tiny scales on the lower surface (e, f). Stick-shaped epicuticular waxes were found on the upper surface of the winter leaves, and had an irregular distribution, with an average number ± SD of 16 ± 2 µm⁻² in areas densely covered, and 2.5 ± 1.8 µm⁻² in areas with reduced coverage (see Fig. S1). Waxy structures were 0.05 to 0.5 µm in length. Glandular hairs observed on the upper surface of summer leaves were 20.6 ± 2.2*50 µm⁻² in number (average ± SD) and 1 to 5 µm in length.

Discussion

In the present work we demonstrated that the leaves of Pittosporum tobira can be a useful biomonitor of airborne MFs in urban environment, being capable of distinguishing among the different land uses, with the highest accumulation in the industrial site and lowest in urban parks. In a previous work (Capozzi et al., 2024) we set-up a four-steps sequential extraction technique and found that the largest fraction of MFs, about 75%, was extracted by the first step, consisting in a tape tearing; therefore, we suggested this single step as a fast, and reproducible method to quantify the level of airborne MFs. In the present work we highlighted that the extraction procedure is reliable in both dry and wet seasons, despite the different morphological traits of the leaves. Notably, this single step extraction protocol could distinguish different levels of anthropogenic impact due to airborne MFs, even in sites within few kilometers from each other.

Our results only partly agree with the previous literature on plant biomonitoring. Leonard et al. (2023) did not find significant differences in MPs accumulation depending on the land use; however, they considered some plants species having different ability to intercept and retain MPs, without comparing the same species at the different sites, which could provide unclear results, since each species shows peculiar pollutants uptake and retention capacity due to different morphological traits (Capozzi et al., 2020b; Corada et al., 2021; Prigioniero et al., 2023). The same authors (Leonard et al., 2023) hypothesized a limited impact of the land use on MPs accumulation due to long-distance transport of these pollutants. Although long-range transport could have a homogenizing effect on the accumulation of MPs masking the differences due to the land uses, the results of the present study highlight that other factors can also influence the retention of these pollutants by acting on both the accumulation and the release capacity of the biomonitor. Specifically, our results suggest that the continuous supply of MFs from sources close to the monitored sites, as for industrial and urban sites, can enhance accumulation in the biomonitor, counteracting the homogenizing effect of long-distance transport. The longest MFs were found in the industrial site, which supports this hypothesis; in fact, the proximity to pollution sources, would ensure a high, continuous contribution in terms of number of longer MFs, without a parallel increase in their degradation.

In agreement with our results, Jafarova et al. (2023), using Robinia pseudoacacia leaflets as biomonitors of airborne MPs, found an interesting relation between accumulation and land use. In fact, a high number of microfibers, but a limited number of tire wear particles were found in urban parks, whereas an opposite trend was observed along roadside. Also including cryptogams for literature comparison, the same authors found that lichen bags exposed in Milan accumulated a higher number of MFs in the city center and periphery in comparison to city parks outside the city center (Jafarova et al., 2022). Moreover, Loppi et al. (2021) found a MPs accumulation gradient in the native lichen Flavoparmelia caperata sampled at different distances from a landfill damping, with the highest accumulation near to the landfill and the lowest at 1,500 m distance.

The higher accumulation of MPs during the dry season (Enyoh et al., 2019; Qian, Chong Guo & Yong Ming, 2017; Zhao et al., 2015), as well as the lower accumulation in the wet season (Zhang et al., 2020) are in line with the previous literature reports (Jahandari, 2023). Moreover, since the length of MFs accumulated in summer was significantly longer than in winter, it is likely that rainfall can induce the loss of the longest MFs. Anyhow, heavy rainfall decreases the level of MPs accumulation, as here specifically highlighted by P. tobira transplants. Transplant experiment also evidenced that a vigorous wash before exposure is recommended to lower the number of MFs, increasing transplant sensitivity; further, a couple of weeks is an exposure time long enough to ensure an accumulation of MPs significantly higher than pre-exposure content.

In contrast with the general trend, the seasonal variation in the accumulation and retention of MFs was not observed in two sites, Urban 3 and Green 1; this result could depend on the structure of the vegetation in the two sites, in which P. tobira plants used for leaf sampling were protected by the tree canopies, thus avoiding the MFs washout observed in the wet season in the other sites. In agreement with this result, it is known that the canopy of plants in urban environments can also exert a filter effect against contaminants dispersed in the atmosphere, improving air quality of the cities (McPherson et al., 2011); this has been demonstrated for several pollutants and plant species (Beckett, Freer-Smith & Taylor, 2000; De Souza et al., 2022; Oyana et al., 2017; Wannaz et al., 2013). More generally, the lack of seasonal variability in MFs accumulation at the Urban 3 and Green 1 sites suggests that the geomorphology of the territory, as well as the presence of densely wooded areas, could play a relevant role in the MFs interception and retention ability by P. tobira, affecting the balance between their accumulation and release.

Seasonal differences in the accumulation of MFs found in the other sites could partly depend on the different traits observed in the mature leaves in the two seasons; in fact, in the wet season leaves are covered with epicuticular waxes scale-shaped on the lower surface and stick-shaped on the upper surface. In summer instead, leaves develop on the upper surface numerous glandular hairs, which could play a role in facilitating MFs adhesion. Furthermore, despite the presence of glandular hairs only on the upper surface of the leaf, the exudates of these glands could play a role in the adhesion of the MFs on both surfaces, given the shoot architecture, with the leaves of the different layers in close contact with each other.

Other authors investigated MPs accumulation ability of different plant species (Liu et al., 2020; Perera et al., 2024) and found a noticeable variation in accumulation performance, both within and between species, suggesting that all plants can intercept and retain these pollutants, regardless leaf traits. Our work, instead, evidenced in a single species sampled at the same sites, a different ability of MFs accumulation depending on the different leaf traits observed in summer and winter, indicating that species (and its leaf traits), as well as the season, are important factors to ensure an appropriate sensitivity of the method and reliable results.

All these considerations suggest the need for further work, to precisely focus on the role of the leaves in the retention of MFs and the mechanisms affecting their uptake, retention, and release into the atmosphere. This knowledge is essential to set up a reliable, standardized methodology, which can be applied in different environmental conditions.

Conclusions

In this work we tested the ability of Pittosporum tobira leaves to highlight the level of airborne MFs pollution in six sites with different land uses (industrial, urban, and green), all embedded in the urban context of Naples city. P. tobira leaf samples subjected to a fast, green protocol for MFs extraction, simply consisting in a tape tearing, accumulated a higher number of MFs in the industrial site, followed by urban and green sites, indicating that this species is a suitable biomonitor of airborne MFs. The accumulation of MFs was higher in summer than in winter, likely due to the water-washing effect caused by rainfall occurring in winter, and to the specific leaf traits observed in the different seasons. For the first time, P. tobira transplants were used to estimate the effect of pouring rain on MFs accumulation. This experiment highlighted that heavy rainfall determined a loss of MFs, specifically the longest ones, indicating that biomonitoring of airborne MFs in the wet season could lead to an underestimation of these air pollutants, also decreasing the sensitivity of the method; therefore, the comparison between biomonitoring surveys should be done considering the season. Although further studies are needed to reach a deep knowledge of plants as biomonitors of airborne MFs and set up a standardized methodology, the simple extraction method proposed in the present work could provide a feasible tool for a project of citizen science to increase people awareness of air pollution due to plastic waste.

Supplemental Information