2025 Volume 5 Pages 76-82
Microplastics are ubiquitous environmental contaminants that have significant adverse effects on ecosystems and human health. Lichens, recognized as reliable pollution bioindicators, have been proposed as effective monitors of atmospheric microplastic contamination. This study investigated the dynamics of microplastic deposition and accumulation in lichen thalli using Ramalina celastri as a model organism. Lichen thalli were exposed to controlled suspensions of microfibers across three size categories (<1, 1, and 5 mm) over a 4-week period. Microplastic particles were extracted using a novel sodium hypochlorite oxidation technique. The results revealed a distinct differentiation between the deposited microfibers, which correlated with the environmental deposition rates, and the accumulated microfibers, which showed temporal increases and greater retention of smaller particles. These findings challenge the existing assumptions in microplastic research that extracted particles solely represent accumulated material, emphasizing the need for refined terminology. To address this, we propose the term “retained” to encompass deposited and accumulated particles. The sodium hypochlorite oxidation technique provides a cost-effective and safer alternative to traditional wet peroxide oxidation, enhancing its feasibility for researchers in resource-limited settings. Furthermore, the use of NaCl and oil extraction proved effective in isolating microplastics across varying densities. This work underscores the importance of precise definitions and robust methodologies in the study of microplastic bioindicators, contributing to a more comprehensive understanding of microplastic dynamics in lichens and their utility in environmental monitoring.
Microplastics have become a global issue, contaminating various environments (Cole et al., 2011; Dissanayake et al., 2022), and posing problems to diverse biological communities (Agathokleous et al., 2021), including human populations (Blackburn and Green, 2022). Consequently, the ability of lichens to be reliable bioindicators of this type of contamination was assessed (Loppi et al., 2021), given that lichens have already been recognized as bioindicators of environmental pollution, especially in atmospheric studies (Conti and Cecchetti, 2001). Lichens are effective monitors of environmental variations in microplastic pollution in the atmosphere (Loppi et al., 2021). Following Loppi et al. (2021), a series of related articles were published in a relatively short period of time (Jafarova et al., 2022; Capozzi et al., 2023; Jafarova et al., 2023; Taurozzi et al., 2024). The methodology for extracting microplastics from the thalli was similar in all related articles: (1) cleaning of the material (under a microscope or with washes), (2) oxidation of the material, (3) filtration, and (4) counting and polymer identification. The methodology can involve either active biomonitoring (with transplants) or passive (extracting thalli directly from their habitats and/or sites). In some cases (Capozzi et al., 2023; Taurozzi et al., 2024), in step (1), the material was washed and/or visually inspected (with a stereomicroscope) to remove deposited surface microplastics (particularly microfibers [MFs]) that are not accumulated in the thallus (although not clearly specified in the text). Here, we define the terms “Deposition” and “Accumulation.” Deposition is the process of transferring atmospheric pollutants toward terrestrial and aquatic ecosystems (Amodio et al., 2014), in this case, to the surface of lichenized fungi. Accumulation, “bioaccumulation” as suggested by Taurozzi et al. (2024), is defined as the increase in contaminant concentrations in organisms following uptake from the ambient environmental medium (Borgå, 2008). This methodology makes several implicit assumptions: (i) all microplastics deposited on the thalli (not accumulated) can be observable (by means of optical equipment, considering the size detection limit), and subsequently extracted; (ii) washes are effective in removing deposited microplastics; (iii) the cleaning process (either i and/or ii) leaves only accumulated microplastics (which will then be extracted). In fact, Loppi et al. (2021) refer to all microplastics extracted by digestion from the thalli as accumulated. Because the source of microplastics is environmental and thus uncontrollable, verifying the aforementioned assumptions poses a challenge for the authors.
Therefore, the objective of this study was to measure and differentiate between deposited and accumulated microplastics through a controlled experiment. Through this experiment, we endeavor to assess the feasibility of the aforementioned assumptions. For these experiments, we used a species of fruticose lichen with a wide global distribution, Ramalina celastri (Sprengel) Krog & Swinscow.
Complete thalli were collected from R. celastri populations growing in the experimental fields of the National University of Luján (Luján, Buenos Aires, Argentina). A total of 19 g of wet weight was collected.
EXPERIMENTAL DESIGNThe experiment was conducted in an experimental field where the thalli were extracted. Wet weight thalli (19 g) were distributed among 38 nylon bags (mesh pore size 1 mm). Each bag contained 0.5 g of thalli. Two bags served as controls (with lichens but without exposure to MFs). The mesh size of the bags allowed the passage of sunlight while reducing the loss of MFs into the environment. Furthermore, it aimed to minimize cross-contamination between the bags. The bags were hung on a forest line for 4 weeks (Fig. 1). At the start of the experiment and once a week thereafter, the thalli within each bag were exposed to 45 mL of a MF suspension in distilled water (~0.38 MF/mL) using a wide-mouth sprayer. This dosage was derived from the determination of local environmental MF deposition (1.09 MF week−1 cm−2, see section “Estimation of the Environmental Deposition of MFs”) and the nylon bag surface area (16.62 cm2). Each bag was opened once a week to expose its content to 17 MF. Refer to the section “Estimation of the Environmental Deposition of MFs” for more information. These suspensions contained MFs of different sizes corresponding to three size categories: 5, 1, and <1 mm. A total of 12 bags were exposed to each MF size category. Each week, three bags of thalli exposed to each MF size category were removed for further processing and extraction. MFs were derived from cuts with a scalpel made under a stereoscopic microscope from fluorescent orange polyester thread (Magma brand). We employed Fourier transform infrared (FTIR) spectroscopy to determine the polymeric identity of the fibers using a Shimadzu IR Prestige 21 instrument (Fig. 1). To avoid sample contamination, we used antimagnetic metal tweezers.
The thalli collected from each bag were subjected to two consecutive processes. With the aim of releasing the MF deposited (MFD) from the thalli, these thalli were submerged in distilled water (100 mL) and sonicated for 15 min (35 kHz) in glass bottles (previously washed). The MFD are fibers that are loosely held on the thalli surface. Subsequently, the thalli were carefully removed to avoid dragging the MFD, and the wash water was filtered through cellulose filters (30 μm pore size). The thalli were then oxidized using the sodium hypochlorite method (Enders et al., 2017; Razeghi et al., 2022) to obtain the MF accumulated (MFA). The MFA are fibers that are either strongly retained in the thalli surface or incorporated into the thalli material. This method, originally applied to soils with different concentrations of organic matter (Bottone et al., 2022), has recently been used with biological material (Hagelskjær et al., 2023). The thalli were submerged in glass bottles with 50 mL of sodium hypochlorite (55 g/L). The containers were then sealed and vigorously shaken for 30 s, after which they were allowed to stand for 24 h. The solution formed in the bottles was transferred to centrifuge tubes and centrifuged at 9000 rpm for 15 min at 20°C. The supernatant was filtered through 30 μm cellulose filters. A volume of 50 mL of NaCl (sat.) and 3 mL of commercial sunflower oil (Cocinero® brand) were added to the remaining pellet, and the mixture was centrifuged again and the supernatant filtered. This process was repeated twice with the remaining pellet. All the solutions used were previously filtered. All filter papers resulting from each methodological process were stored in aluminum paper envelopes until further counting. Fiber counting was performed using a stereoscopic microscope (Zeiss®) in a dark room using an ultraviolet (UV) lamp (325 nm) to exploit the fluorescent properties of the polyester (Fig. 1).
STATISTICAL ANALYSISA two-factor analysis of variance (ANOVA) model was used to analyze the effect of MF size (<1, 1, and 5 mm) and exposure time (week 1, week 2, week 3, and week 4) on the number of MFD (MFD g−1) and MFA (MFA g−1). The Tukey test was used to compare the means between different levels of the factors.
ESTIMATION OF THE ENVIRONMENTAL DEPOSITION OF MFsA sampling of environmental MFs was conducted in September 2023. The sampling area comprised two fruit production plots within the experimental field of the National University of Luján. In each plot, four passive collectors (modified polypropylene bottles, volume 2.5 L) were placed. The average surface area of the open section of the collectors was 80.66 cm2. The collectors were suspended with rope on fruit trees in each plot at a height of 1 m above ground level. The collectors were emptied and washed twice during a 15-day period (at day 5 and day 10 after their suspension on the plot). The collectors’ contents were stored in glass jars with metal lids. Once in the laboratory, the jar contents were filtered (cellulose filters, 30 microns pore size) under vacuum conditions to accelerate the filtration process. The number of plastic MFs deposited on the filters was counted under a stereoscopic microscope (Zeiss®). As environmental MFs are not necessarily fluorescent under UV light, the qualitative identification criteria used in previous studies (Loppi et al., 2021, Gomez et al., 2023) and the hot needle method (Hidalgo-Ruz et al., 2012) were employed. The average number of MFs per collector (± Standard Deviation) was 58.56±47.87. Based on the count and the surface area of the collector containers, the average deposition was determined to be 1.09 MF week−1 cm−2.
Controls showed no MFD or MFA present in the samples. Regarding MFD g−1, the ANOVA did not find significant effects of MF size (F [2, 24]=0.87, p=0.43) or exposure time (F [3, 24]=1.16, p=0.35). Moreover, no significant interaction was found between MF size and exposure time (F [6, 24]=0.81, p=0.57).
In terms of MFA g−1, the ANOVA revealed a significant effect of MF size (F [2, 24]=10.15, p<0.01). Tukey’s post-hoc tests showed that smaller MF (<1 mm) accumulated in greater quantity than those of 1 mm (p<0.01, Fig. 2) and 5 mm (p<0.01, Fig. 2). Exposure time had a marginally significant effect on the amount of MFA g−1 (F [3, 24]=3.08, p<0.05), indicating a trend toward increased accumulation over time. No significant interaction was observed between MF size and exposure time (F [6, 24]=1.73, p>0.15).
After sonication and before oxidation, all observed thalli showed MF, which was clearly visible under UV light (Fig. 1).
Here, we present relevant findings within the context of using lichenized fungi as bioindicators of environmental pollution with microplastics. In this study, we propose, experiment, and test the differentiation between microplastic fibers recovered from washing (deposition) and those recovered through digestion (accumulation). The MFD and MFA exhibited distinct responses to fiber size and exposure time. On the one hand, lichenized fungi demonstrated consistent deposition of MFs despite varying exposure times to a constant environmental deposition rate of 17 MF/week. Within a brief period (4 weeks), the MFD exhibited responsiveness to environmental deposition, as previously proposed by Gomez et al. (2023) for plastic microspheres applied to other lichen species. They proposed that the intricate structural complexity of lichen thalli enhances their capacity to “trap” or intercept microplastics. This trend holds true across different sizes of the MFs used. On the other hand, the MFA displayed noticeable increases over time and size, indicating a clear divergence in the dynamics of deposited versus accumulated microplastics (Fig. 2). These results align with findings from other researchers (Jafarova et al., 2022; Capozzi et al., 2023; Jafarova et al., 2023; Taurozzi et al., 2024), highlighting the efficacy of microplastic accumulation in lichen thalli as a robust bioindicator of environmental contamination. However, a challenge emerged following the sonication and washing process of the thalli, where fluorescent fibers adhered to all treated samples, posing a significant obstacle.
These fibers were not removed by the cleaning applied to the thallus (applying intensive methods such as sonication) and were difficult to observe without using UV light. This observation led us to reflect on the fact that the fibers that were not removed by this cleaning process were part of the “accumulation” in the thalli but belonged to a different category. Despite the publications achieving their study objectives, this phenomenon was not taken into account by the works published so far on the topic, which may imply that the microplastics collected in these articles were not all “accumulated.” This necessitates a critical examination of terminologies such as “accumulation” and “deposition” in microplastic pollution studies.
In most scientific papers, where the capacity of lichens as bioindicators of microplastic contamination is analyzed, the “accumulation” of microplastics in the thalli or bodies of these organisms is studied, either implicitly or explicitly (with some exceptions). The definition of accumulation in these cases has limited applicability. Let us begin by presenting the definitions. Deposition is the process of transferring atmospheric pollutants toward terrestrial and aquatic ecosystems (Amodio et al., 2014), in this case, to the surface of lichenized fungi. Accumulation, “bioaccumulation” as suggested by Taurozzi et al. (2024), is defined as the increase in contaminant concentrations in organisms following uptake from the ambient environmental medium (Borgå, 2008). When speaking of an increase in contaminant concentrations (in this case MFs), the compartment of the living material is always compared with the environment. MFs must be “within” the thalli to be considered accumulated. This distinction is not made in the published works so far on lichens as bioindicators of microplastic pollution. The main authors cited in this topic tend to assume that all material extracted from the lichen thallus is accumulated (e.g., Loppi et al., 2021). However, as shown in this work, it is important to distinguish the different dynamics between the accumulated and deposited microplastic material. Accumulated particles would indicate a history of prolonged accumulation over time, whereas deposited fibers, in principle, would have a relatively shorter temporal presence on the thalli compared with those particles accumulated within the thalli. We suggest that future research should concentrate on the internal structure of lichen thalli, dissecting them (e.g., sections with a microtome) to observe the distribution and efficient accumulation of microplastics (fibers or other forms) within the thalli. Alternatively, it is important to emphasize that dislodging the MFs from the thalli (part of the MFD) is challenging even after vigorous washing or intense sonication processes, such as those employed in this study (15 min at 35 Hz, Fig. 1). Therefore, it is evident that a more effective way to distinguish between “accumulated” and “deposited” microplastic material in lichen thalli could be through microtomy techniques. However, the extraction of microplastics from lichen thalli and their use as bioindicators do not necessarily lose their scientific value. In this case, it is only a matter of redefinition. We propose that, despite the impossibility of differentiating between accumulated and deposited microplastics, the term “retained” be used. In fact, a gradation associated with “retention” could be used, for example, loosely and strongly retained. In the context of ecotoxicology studies, retained contaminants are distinguished from accumulated and deposited ones and can be defined as those that retain contaminants through adsorption, redox reactions, and complexation, among other factors (Sarkar et al., 2021). In this context, we are not referring to contaminants accumulated within the thallus but rather to contaminants “trapped” by the thallus through the phenomenon of interception and subsequent interaction between the thallus surface and the contaminants (in this case, MFs). Although the reasons behind the retention of MFs in the thalli are unknown, it is necessary to alter the terminology employed in related scientific studies, as the microplastic particles extracted from lichen thalli cannot unequivocally be assigned to the category of “accumulated.”
Finally, we want to briefly address some methodological considerations applied in this study. Here, we implemented an alternative approach to the oxidation process commonly employed in the extraction of microplastics from lichen thalli, as observed in all other related scientific publications (Loppi et al., 2021; Jafarova et al., 2022; Capozzi et al., 2023; Jafarova et al., 2023; Taurozzi et al., 2024). The conventional method typically involves a wet peroxide reaction. In contrast, a more cost-effective and straightforward method involving sodium hypochlorite oxidation was used for the lichen material. This technique is widely used for microplastic extraction across various matrices (e.g., Bottone et al., 2022) and has recently been incorporated in bryophyte studies (Hagelskjær et al., 2023). The use of sodium hypochlorite offers not only cost efficiency but also enhanced safety (hypochlorite is less irritating than peroxide). Unlike the wet peroxide reaction, which exhibits relative instability at 70°C–75°C and may result in material loss owing to spillage when there is no equipment that allows greater control of the procedure, sodium hypochlorite oxidation is a more stable and secure process as it is conducted at room temperature with a less irritating reagent. Monteiro et al. (2022) compared this oxidation method with other more commonly used methods. Their results showed that sodium hypochlorite oxidation was able to efficiently digest the organic matter in organic-rich freshwater samples, and the overall microplastics extraction efficiency was above 94% with no significant modification of the polymer structure. Moreover, sodium hypochlorite is a more accessible and simpler option for researchers in developing nations than the wet peroxide reaction, which requires a greater array of instruments and reagents. Additionally, we employed extraction with NaCl and oil, a technique known for its effectiveness in extracting microplastics of varying densities (e.g., Crichton et al., 2017).
We introduce a novel differentiation between microplastic deposition, retention, and accumulation, shedding light on their distinct dynamics in lichen thalli. Our findings reveal that the MFD exhibit consistent responses to the environmental deposition rates, whereas the MFA display noticeable increases over time and size. This distinction underscores the need for a critical reevaluation of terms such as “accumulation” and “deposition” in microplastic pollution studies. We propose the use of the term “microplastic retention,” which reflects the nature of the methodologies generally applied and avoids the ambiguity of the “accumulated/deposited” dichotomy. Furthermore, our study advocates for future research focusing on the internal structure of lichen thalli to better understand the distribution and efficient accumulation of microplastics within them. Overall, our study suggests the need for a paradigm shift in the terminology and methodology employed in the study of microplastic pollution, emphasizing the importance of accurate categorization in advancing scientific understanding and environmental management practices. Methodologically, we suggest an alternative approach involving sodium hypochlorite oxidation for microplastic extraction, which offers cost efficiency and enhanced safety compared with conventional methods. Furthermore, the extraction with NaCl and oil was effective in extracting microplastics of varying densities.
We would like to thank the National University of Luján for providing the infrastructure to conduct the experiments outlined in this scientific work. This research was funded by a grant from the National University of Luján (Subsidized Institutional Research Projects of the Basic Sciences Department [PI^2+] 2022. Financing entity: Basic Sciences Department, National University of Luján, Luján, Buenos Aires, Argentina).
