Differential effect of nano vs. micro-sized plastics on live Chlorella sp. algae in water environment.

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Alzbeta Marcek Chorvatova, Anton Mateasik, Dusan Chorvat This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5289229/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2025 Read the published version in Microplastics and Nanoplastics → Version 1 posted 10 You are reading this latest preprint version Abstract Environmental pollution by micro and nanoplastics (MPs) is becoming an imminent danger for the environment in the 21st century. However, the effect of the MPs of different sizes is still poorly understood. In this contribution, we compare the effect of fluorescently labelled polystyrene (PS) MPs of sizes between 20 nm and 2000 nm. Spectrally-resolved confocal microscopy and fluorescence lifetime imaging was employed to study the interaction of PS MPs with algae Chlorella sp . We observed differential effect between the smaller and the bigger-sized beads. MPs sized 20–500 nm created “corona-like” structures around algae and induced lowering of the chlorophyll fluorescence, indicating an effect on the cell photosynthesis. In addition, the 20 nm MPs induced shortening of the chlorophyll fluorescence lifetimes, pointing to the effect on the chlorophyll molecular environment. However, MPs of bigger sizes, 1000–2000 nm, rather acted as a “nucleus” for clustering of a number of neighbouring algae without affecting the chlorophyll fluorescence. Understanding the interaction of living organisms with MPs of different sizes is crucial to assess the impact of this environmental pollution on live organisms in their natural environment. nanoplastics microplastics chlorophyll fluorescence Chlorella sp. algae confocal microscopy FLIM Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The use and the production of plastics is exponentially increasing every year and, when discarded, the plastics falls apart into micro and later to nanoplastics, polluting both the marine and the freshwater ecosystems [De Sa et al., 2018 ]. Their presence brings a potential health impact on the aquatic microorganisms and also on humans [Li et al., 2023 ]. According to Koelmans et al. [ 2015 , 2015 b], MPs are defined as particles with size lesser than 5 mm [Galgani et al., 2013 ]; pieces of a size smaller than 1 mm are called small microplastics, while particles < 100 nm are termed as nanoplastics. MPs are often found in marine phytoplankton aggregates [Long et al., 2015 ]. MPs of different types and sizes have now been identified in the seawater worldwide, inducing adverse effects on most aquatic animals [Wright et al., 2013 ; Della Torre et al., 2014 ; Ivar do Sul et al., 2014]. MPs have variable origins and conditions to which they are exposed and, consequently, can be described by a wide range of different properties. Particle size is currently one of the most used forms for the MPs classification and the key feature related to the transport and aquatic morphology [Shamskhany et al., 2021 ]. Nano-sized particles mostly come by airborn influx, while bigger sizes are considered to be more a result of a river and/or an offshore influx (ships, nets). That is why smaller MPs are often gathered at the surface of the water, while the bigger ones can be found in the sediments. Particle size of the MPs is also related to their toxic mechanism [Liu et al., 2020 ]. On one hand, larger sized MPs were shown to have adverse effects by blocking the light transport and affecting photosynthesis. On the other hand, smaller ones were capable of destroying the cell wall by adsorbing onto the algae surface. In other study, nanoplastics were also shown to interact with microalgae, inhibiting the microalgal photosynthesis [Bhattacharya et al. 2010 ]. Nano-sized particles are expected to have more a chemical effect, while micro-sized ones have more a physical effect [Liu et al., 2020 ]. At the same time, the effect of differential sizes is unclear. PS in one of the plastics commonly detected in the water reservoirs; PS is, together with polyethylene (PE), one of the most abundantly used plastic polymers in the world. It is therefore frequently found as part of MPs identified at sea [Barnes et al., 2009 ; Browne et al., 2010 ; Hidalgo-Ruz et al., 2012 ]. For that reason, in the present study, our goal was to evaluate the effect of PS MPs sized from 20 nm to 2000 nm on the endogenous chlorophyll fluorescence of the sweet water green algae Chlorella sp. , employed as a model organism, using the time- and the spectrally-resolved microscopy methods. Material and methods Preparation of MPs MPs of different sizes were purchased commercially as a FluoSpheres® size kit #2 (F8888, Invitrogen by Thermo Fisher Scientific), carboxylate coupling surface, labelled with yellow-green fluorescence (Ex/Em 505/515). We have compared MPs of six sizes with nominal bead diameters of 0.02, 0.1, 0.2, 0.5, 1.0, 2.0 µm. MPs were added to cells in concentration 2 µL/mL (2% solids in the original solution) and cultivated between 7–21 days before use. Algae and solutions We have employed green algae Chlorella sp. from the University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences collection of green algae, previously isolated from the main drinking water supply. Algae were cultivated in the Hoagland cultivation medium containing: NaNO 3 ; CaCl 2 . 2 H 2 O; MgSO 4 . 7H 2 O; K 2 HPO 4 . 3 H 2 O; KH 2 PO 4 ; NaCl; H 3 BO 3 ; FeSO 4 .7H 2 O; H 2 SO 4 ; ZnSO 4 . 7H 2 O; MnCl 2 .4H 2 O; (NH 4 )6Mo7O24.4H 2 O; CuSO 4 .5H 2 O; Co(NO 3 )2. 6H 2 O; EDTA, KOH (all from Lachema, Czech Republic), as specified previously [Marcek Chorvatova et al., 2020 ]. Instrumentation Confocal imaging Fluorescence was measured with Laser scanning confocal microscopy (LSCM) imaging with Axiovert 200 LSM 510 Meta (Carl Zeiss, Germany), equipped with objective C-Apochromat 40x, 1.2 NA, as described previously [Marcek Chorvatova et al., 2020 ]. Chlorophyll fluorescence was excited with the 450 nm laser line (Kvant, Slovakia) and detected using a spectral META detector. Maximal laser power density reached 1580 Wm -2 for sample excitation with 450 nm laser line. Channel 1 was recorded with BP 500–550 nm filter and served to monitor the fluorescently-labelled MPs. Channel 2, using BP 650–710 nm filter, was employed to record the endogenous red chlorophyll fluorescence of the algae, peaking at 680 nm [Waller et al., 2017 ]. To better visualise the distribution of MPs only, zoomed image was recorded with BP 480–520 nm. Spectrally-resolved images of the red algae fluorescence in the presence or in the absence of MPs were taken across 7 spectral channels covering the spectral region from 638 and 713 nm taken with 10.7 nm steps for the red chlorophyll fluorescence. Fluorescence Lifetime Imaging Microscopy (FLIM) Fluorescence lifetime imaging microscopy (FLIM) was employed to gather images by time-correlated single photon counting (TCSPC) technique, using a 445 nm picosecond laser diode (BDS-SM-445-FBC, Becker&Hickl, Germany), as described previously [Marcek Chorvatova et al., 2020 ]. The laser beam was reflected to the sample through an epifluorescence path of the inverted microscope Axiovert 200 LSM 510 Meta (Carl Zeiss, Germany) with C-Apochromat 40x, 1.2 NA. The emitted fluorescence was separated from laser excitation using BP 700 ± 20 nm. Detection was done by PMC-100-20 photomultiplier (Becker&Hickl, Germany) with SPC-830 TCSPC board. Data analysis Confocal images were analyzed by ZEN 2011 software (Zeiss, Germany), or by home-made procedures. Fluorescence intensity from confocal images at Fig. 1 was analysed using image segmentation method, where only fluorescence intensities recorded from the fluorescing algae (without surrounding background), were measured. Red fluorescence spectra at Fig. 2 were evaluated as a mean fluorescence of all fluorescing algae in the sample. FLIM images at Fig. 3 were processed using proprietary software package SPCImage 8.4 NG (Becker&Hickl, Germany), or a custom-made software. Results were visualized as a map and as a distribution of calculated fluorescence lifetimes for each recorded cell. Intensities are presented as the mean and the standard error of the mean. Decay curves were fitted with a one-exponential fitting model with plausible χ 2 using image segmentation and home-made lifetime analysis, as described previously [Marcek Chorvatova et al., 2020 ]. Images were generated by Origin 6.0 Professional. Statistical comparison was done using one-way Anova, with p < 0.05 considered as significant. Results Our aim was to evaluate the interaction between MPs of different sizes and algae Chlorella sp. MPs ranged from micrometric size (2 µm) down to nanometers (20 nm). Chlorella sp. is a unicellular alga of size 10–30 µm; MPs were therefore chosen in order to be smaller than the average cell size. We have recorded a short-term exposure (7–21 days) of MPs to algae, in the aim to discern the early events. Evaluation of the distribution of MPs and of their interaction with Chlorella sp The presence of MPs and their distribution was evaluated based on their fluorescence. The MPs fluorescence was mostly present in the spectral range 480–520 nm (data not illustrated). For the MPs in the lower size range (20–200 nm), a single peak with the maximum at 520 nm was observed; the MPs in the bigger size range (500–2000 nm) presented larger intensity with maximum that remained at 520 nm, but with an additional fluorescence shoulder at 550 nm. At this spectral range, the MPs fluorescence therefore did not interact with the red chlorophyll fluorescence of the Chlorella sp. algae. To evaluate the interaction of MPs with Chlorella sp. algae, spectrally-resolved confocal microscopy following excitation at 450 nm at two emission windows was used (Fig. 1 A, composite image at channel 4). In these studies, the emission window of 500–550 nm of the channel 1 was employed to record the green fluorescence of MPs, while the emission window of 650–710 nm of the channel 2 was designed to record the red chlorophyll fluorescence of the algae (Fig. 1 A). Consequently, the presence of the MPs was discerned based on their blue/green fluorescence, whereas the algae were identified by their red endogenous fluorescence. In these settings, in control conditions, in the absence of MPs, algae exhibited only red endogenous fluorescence of chloroplasts, no green fluorescence was recorded (Fig. 1 B). In the presence of both, algae and MPs (Fig. 1 C), we noted a differential interaction of micro vs. nanoplastics: nano and small MPs (20–500 nm) encircled the algae in a “corona”-like structures, whereas larger MPs (1000–2000 nm) rather created a “nuclei” for clustering the neighbouring algae. For better visualisation of the distribution of MPs, an image with BP 480–520 nm was also recorded (Fig. 1 B and 1 C, in the inset, visualised in blue for better contrast). Effect of MPs on the red chlorophyll fluorescence of Chlorella sp. To study the effect of the MPs on the Chlorella sp ., we first compared the red chlorophyll fluorescence intensities recorded at the Fig. 1 B-C. We observed that the presence of MPs lead to a decrease in the algae chlorophyll fluorescence for the small-sized MPs (20–500 nm), although for MPs of 200 nm, this decrease did not reach significance (Fig. 1 D). Larger MPs (1000–2000 nm) have no significant effect on the algae chlorophyll fluorescence. This result indicates that the size of MPs affects the capacity of individual cells to cope with their presence and thus with photosynthesis. Next, we evaluated the effect of the MPs on the red chlorophyll fluorescence spectra of the Chlorella sp . by recording the confocal images at separate fluorescence wavelengths between 638 nm and 713 nm using a 10.7 nm step (Fig. 2 A). The red fluorescence peaked at 680 nm (Fig. 2 A and 2 D), as expected for the fluorescence of chlorophyll a [Govindjee, 1967 ]. Gathered data corresponded to our previous recordings of the endogenous fluorescence in the algae Chlorella sp. [Marcek Chorvatova et al., 2020 ]. Under these conditions, we observed no significant effect of the MPs on the spectral shape of the red fluorescence, as illustrated after the spectral normalization (Fig. 2 D). This result indicates that the MPs did not modify the photosystem II system, or stimulate the photosystem I and thus that the algae still have a good capacity to maintain the photosystem II system in the presence of MPs, regardless of their size. FLIM images, recorded by TCSPC, served to evaluate the fluorescence lifetimes following excitation by 445 nm picoseconds laser with emission between 700 ± 20 nm (Fig. 3 A, left, between 250–300 ps). In this setting, the MPs exhibit no fluorescence and only fluorescence of chlorophylls is recorded. For that reason, in order to better visualise the presence of MPs, the confocal image of the same regions was also taken (Fig. 3 A, right). A custom-made approach, allowing automatization of the procedure in varying experimental conditions, was used for analysis, as described in [Marcek Chorvatova et al., 2020 ]. We noted a significant decrease in the chlorophyll fluorescence lifetime for MPs of 20 nm size (Fig. 3 C). This indicate alteration in the algae chlorophylls, most likely related to the close encircling (the “corona” like structures) of the algae cell by these small-sized nanoplastics. We noted no significant change in the measured chlorophyll fluorescence lifetime of algae for the plastics with particle size above 100 nm (Fig. 3 B-C). Interestingly, FLIM recordings uncovered an unexpected capability to visualise the MPs of sizes larger than 500 nm (arrows at Fig. 4 ). Indeed, in this FLIM setting, MPs of larger sizes can be visualised non-fluorescently and this observation may help to evaluate the presence of these MPs in the water environment in the presence of algae. Discussion In this contribution, we evaluated the interaction of MPs of different sizes with photosynthetic algae Chlorella sp . Endogenous fluorescence intensity and fluorescence lifetimes of chlorophylls were recorded. We demonstrated differential distribution of nano- vs. micro-sized PS plastics after up to 3 week exposure. Smaller-sized MPs (20–500 nm) resulted in “corona-like” distribution of MPs around algae and decrease in the red chlorophyll fluorescence, indicating their effect on the algae photosynthesis. MPs sized above 1000 nm rather acted as nuclei for clustering the algae, without affecting algae photosynthesis. Plastic particles are ubiquitous in the aquatic environment: they have been detected worldwide [Waller et al., 2017 ]. MPs have adverse effects on several aquatic animals [Wright et al., 2013 ; Della Torre et al., 2014 ; Ivar do Sul et al., 2014] and also threaten humans, primarily from the point of view of the potential danger of chronic exposure to their chemical toxicity [Wright et al., 2017]. Nevertheless, the interactions of living organisms with MPs are still poorly understood. We have tested the distribution of differently sized MPs of the PS origin, labelled with blue/green fluorescence, together with their effect on the endogenous red chlorophyll fluorescence of the algae. Fluorescence labelling is helpful for evaluation of the presence of MPs and allowed to discern their differential distributions. To study the effect of the MPs on the algae fluorescence, we have employed recording of the endogenous fluorescence of the algae in the red spectal region. We previously demonstrated the use of the endogenous fluorescence for biosensing of the presence of nanoparticles in live algae [Marcek Chorvatova et al., 2020b ]. The red fluorescence with maximum at 680 nm was assigned to the chlorophyll fluorescence in our previous studies [Marcek Chorvatova et al., 2020 ]. Chlorophyll fluorescence is a very useful endogenous probe that is sensitive to variations in the functional state of the algae, as well as of plants [Govindjee, 1967 ; Govindjee, 2004 ]. This fluorescence is heterogeneous, but its major emission band (680–685 nm) and its vibrational satellite (720–735) nm originate mostly from pigments of the photosystem II antenna complexes, namely chlorophylls [Govindjee, 2004 ]. This fluorescence can therefore also be employed for testing the responsiveness of algae to changing water quality, Endogenous fluorescence recorded in living Chlorella sp. algae cells under our experimental conditions is in agreement with the assumption that most of its chlorophyll a fluorescence is derived from photosystem II, with the emission bands at 680–685 nm [Govindjee, 2004 ]. In the presence of the MPs, the spectral shape of the recorded fluorescence remained unchanged, indicating no modification of the photosystem II / photosystem I. We have observed a decrease in the chlorophyll fluorescence for the smaller-sized MPs. On the other hand, we did not observe any effect on the chlorophyll fluorescence with the larger sized MPs. Other authors demonstrated that larger-sized MPs caused adverse effects by blocking the light transport and affecting the photosynthesis, while smaller ones destroyed the cell wall by adsorbing onto the algae surface [Bhattacharya et al., 2010 ]. The reason behind the lack of the effect of the large MPs (above 1000 nm) in our study can be related to their lower numbers, when compared to MPs below 500 nm (see Fig. 1 ). The concentration of the MPs was chosen in relation to the environmental pollution concentrations (between 100 and 1,000 µ g/L) [Jin et al., 2022 ; Chen et al., 2022 ]. With 2% of solids in the sample, taking into consideration PS density of 1.250 g/cm 3 [omnexus web], 2 µ l/ml corresponds to MPs concentration of about 500 µ g/L. However, this means much lower number of MPs of higher sizes when compared to those of lower sizes, as illustrated at Fig. 1 . Many algae cells thus remained without direct interaction with the MPs above 1000 nm, at a difference to MPs smaller than 500 nm. In the future, the effect of higher concentrations of the bigger particles, together with longer exposures (in terms of months) needs to be evaluated. FLIM recording is an advanced imaging technique that allows to record changes of molecular parameters non-invasively and directly in living cell systems. Fluorescence lifetimes depend on several factors, including pH, temperature and/or oxygenation, but are independent on the fluorescence intensity and/or photobleaching [reviewed in Berezin MY and Achilefu S, 2010 and Chorvatova A and Chorvat D. Jr 2014]. This parameter is therefore particularly useful for study of the cell’s responses to changes their environment. We previously demonstrated that under our experimental conditions, the dominant fluorescence lifetime of the endogenous chlorophyll fluorescence reached, in control conditions, between 0.4–0.45 ns [Marcek Chorvatova et al., 2020 ] and this was also observed in this study. Observed decrease in the fluorescence lifetime in the presence of the MPs sized 20 nm indicate that encircling of the cells in these conditions impact on the red endogenous fluorescence at the molecular levels. Recorded change in the fluorescence lifetimes points to a modification in the molecular structure of the chlorophylls, while the decrease in the chlorophyll fluorescence without change in the fluorescence lifetimes for the MPs of sizes between 100–500 nm rather points to the decrease in the number of the active molecules in these conditions. Further work is needed to understand the interaction between MPs and living organisms in details. Nevertheless, performed experimentation acknowledges the employment of the time-resolved chlorophyll fluorescence in biosensing. Gathered measurements confirmed differential interaction of nano vs. micro-sized MPs with algae. Nano-sized particles are expected to have more a chemical effect, while micro-sized plastics have more a physical effect [Liu et al., 2020 ] and the differences in the observed distributions in our study confirm these differences Conclusions In conclusion, our data revealed a differential distribution of the nano vs. microplastics with the Chlorella sp. algae. The nanoplastics of the 20 nm size grouped around the individual algae cell in a corona-like formation, affecting the algae chlorophylls, as evidenced by shortening of the chlorophyll fluorescence lifetimes. MPs of smaller sizes (less or equal than 500 nm) surrounded the algae, while lowering the chlorophyll fluorescence. Larger sized MPs (1000–2000 nm) rather acted as a “nucleus” for a group of algae, but without affecting the cell chlorophyll fluorescence. Such a differential effect can help to comprehend the role that nano vs. microplastics play in the aquatic environment. Declarations Availability of data Data are available at request in an Omero repository at http://microscopy.mlc.sk/omero. Competing Interest None beyond declared funding and affiliations. Funding Funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project ENVIROBIOM N o 09I03-03-V04-00689 and by the LASERLAB-EUROPE (grant agreement no. 871124, European Union’s Horizon 2020 research and innovation programme). Author’s contributions A.M.C. contributed to conceptualization, investigation, writing- original draft preparation, writing- reviewing and editing software. A. M. contributed to data curation and data analysis. D. C. contributed to methodology, validation and visualization of data. Acknowledgements The authors thank M. Valica from FPV UCM for helping with the cultivation of Chlorella sp. Conflict of Interest. 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Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2025 Read the published version in Microplastics and Nanoplastics → Version 1 posted Editorial decision: Revision requested 27 Nov, 2024 Reviews received at journal 27 Nov, 2024 Reviewers agreed at journal 19 Nov, 2024 Reviews received at journal 18 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviewers invited by journal 26 Oct, 2024 Editor assigned by journal 21 Oct, 2024 Submission checks completed at journal 21 Oct, 2024 First submitted to journal 18 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5289229","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372622529,"identity":"5653f166-6333-4ff7-b76b-f8c3e5d288ad","order_by":0,"name":"Alzbeta Marcek Chorvatova","email":"data:image/png;base64,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","orcid":"","institution":"Slovak Centre of Scientific and Technical Information","correspondingAuthor":true,"prefix":"","firstName":"Alzbeta","middleName":"Marcek","lastName":"Chorvatova","suffix":""},{"id":372622530,"identity":"a844fffc-3744-42a7-b133-0a91eb4e4c0e","order_by":1,"name":"Anton Mateasik","email":"","orcid":"","institution":"Slovak Centre of Scientific and Technical Information","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Mateasik","suffix":""},{"id":372622531,"identity":"255cfee3-a295-400b-bd4f-d8dfb5952126","order_by":2,"name":"Dusan Chorvat","email":"","orcid":"","institution":"Slovak Centre of Scientific and Technical Information","correspondingAuthor":false,"prefix":"","firstName":"Dusan","middleName":"","lastName":"Chorvat","suffix":""}],"badges":[],"createdAt":"2024-10-18 11:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5289229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5289229/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s43591-025-00111-2","type":"published","date":"2025-02-10T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68719186,"identity":"f563817e-8152-48b1-992b-8c23acbd363e","added_by":"auto","created_at":"2024-11-11 10:32:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12234123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLaser scanning confocal microscopy (LSCM) fluorescence imaging of MPs in the presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlorella sp.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e algae.\u003c/strong\u003e Images are taken following excitation by 450 nm laser, A) channel 1: BP 500-550 nm (“the green fluorescence” to visualise the MPs), channel 2: BP 650-710 nm (“the red fluorescence” to visualise algae chlorophyll fluorescence), channel 3: transmission image, channel 4: overlay of channels 1-3 (example of MPs 20 nm); scale 20 mm. B) overlay of channels 1-3 in control conditions and C) overlay of channels 1-3 in the presence of MPs of different sizes. In the insets, zoomed image with BP 480-520 nm to better visualise the distribution of MPs (in blue for better contrast); scale 20 mm\u003cstrong\u003e. \u003c/strong\u003eC) Red fluorescence intensity calculated per cell (n=4500-6500 cells from 26-31 images per experimental condition).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5289229/v1/85b4af7fbd4c4bcc2ddedc30.png"},{"id":68720681,"identity":"a3f3e5d6-4862-4791-8b10-74fe53264e0b","added_by":"auto","created_at":"2024-11-11 10:40:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3664798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpectrally-resolved imaging of the red chlorophyll fluorescence of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlorella sp.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e algae.\u003c/strong\u003e A) Confocal images recorded at separate fluorescence wavelength in the spectral region between 638-713 nm with a 10.7 nm step following excitation with 450 nm laser. B) Spectrally-resolved image of the endogenous chlorophyll fluorescence of the \u003cem\u003eChlorella sp.\u003c/em\u003e algae, created as composite image from images at A in the control condition; scale 20 mm. C) Spectrally-resolved image of the algae endogenous red chlorophyll fluorescence in the presence of MPs of different sizes. D) Normalized spectra calculated from the spectrally-resolved images in the recorded conditions (n=11 images per experimental condition).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5289229/v1/99311ea79e0ababc180ece65.png"},{"id":68719187,"identity":"82e4845f-75cc-4d93-9e4f-7dd829f85ae5","added_by":"auto","created_at":"2024-11-11 10:32:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8567492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFLIM images of endogenous fluorescence of chlorophylls in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChlorella sp.\u003c/strong\u003e\u003c/em\u003e In A, (left) FLIM image gathered following excitation by 445 nm ps laser, BP 700±20 nm; lifetime distribution is shown for the range of 250-300 ps (red-blue), in A (right) LSCM image following excitation at 450 nm, overlay image under conditions described at Fig. 1 (Ch. 4); scale 20 mm. Images in control conditions (A), or in the presence of MPs of different sizes (B). C) Fluorescence lifetime distribution recorded in the FLIM images of the algae chlorophyll fluorescence dependent on the MPs size (n=30-60 cells from 10-20 images per experimental condition).\u003c/p\u003e","description":"","filename":"Fig3new.png","url":"https://assets-eu.researchsquare.com/files/rs-5289229/v1/b2c65d4d56c982d56f58c657.png"},{"id":68719184,"identity":"eafcb75b-6249-4e7b-8c78-92e18caa7734","added_by":"auto","created_at":"2024-11-11 10:32:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1519349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFLIM intensity images of MPs of different sizes. \u003c/strong\u003eGrey-scale FLIM intensity images of endogenous fluorescence of chlorophylls in \u003cem\u003eChlorella sp.\u003c/em\u003e, arrows point to an example of MPs particles of the size 500, 1000 and 2000 nm, respectively.\u003c/p\u003e","description":"","filename":"Fig4new.png","url":"https://assets-eu.researchsquare.com/files/rs-5289229/v1/26c593a4a6757e08e879d4df.png"},{"id":76487593,"identity":"59be522d-0c21-47a0-8b11-4dd99457a699","added_by":"auto","created_at":"2025-02-17 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23721662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5289229/v1/5c39a5cc-285c-4985-8f63-c3db49f1668d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Differential effect of nano vs. micro-sized plastics on live Chlorella sp. algae in water environment.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use and the production of plastics is exponentially increasing every year and, when discarded, the plastics falls apart into micro and later to nanoplastics, polluting both the marine and the freshwater ecosystems [De Sa et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e]. Their presence brings a potential health impact on the aquatic microorganisms and also on humans [Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to Koelmans et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003eb], MPs are defined as particles with size lesser than 5 mm [Galgani et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e]; pieces of a size smaller than 1 mm are called small microplastics, while particles\u0026thinsp;\u0026lt;\u0026thinsp;100 nm are termed as nanoplastics. MPs are often found in marine phytoplankton aggregates [Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e]. MPs of different types and sizes have now been identified in the seawater worldwide, inducing adverse effects on most aquatic animals [Wright et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Della Torre et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ivar do Sul et al., 2014]. MPs have variable origins and conditions to which they are exposed and, consequently, can be described by a wide range of different properties.\u003c/p\u003e \u003cp\u003eParticle size is currently one of the most used forms for the MPs classification and the key feature related to the transport and aquatic morphology [Shamskhany et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e]. Nano-sized particles mostly come by airborn influx, while bigger sizes are considered to be more a result of a river and/or an offshore influx (ships, nets). That is why smaller MPs are often gathered at the surface of the water, while the bigger ones can be found in the sediments. Particle size of the MPs is also related to their toxic mechanism [Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. On one hand, larger sized MPs were shown to have adverse effects by blocking the light transport and affecting photosynthesis. On the other hand, smaller ones were capable of destroying the cell wall by adsorbing onto the algae surface. In other study, nanoplastics were also shown to interact with microalgae, inhibiting the microalgal photosynthesis [Bhattacharya et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e]. Nano-sized particles are expected to have more a chemical effect, while micro-sized ones have more a physical effect [Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. At the same time, the effect of differential sizes is unclear.\u003c/p\u003e \u003cp\u003ePS in one of the plastics commonly detected in the water reservoirs; PS is, together with polyethylene (PE), one of the most abundantly used plastic polymers in the world. It is therefore frequently found as part of MPs identified at sea [Barnes et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Browne et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hidalgo-Ruz et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e]. For that reason, in the present study, our goal was to evaluate the effect of PS MPs sized from 20 nm to 2000 nm on the endogenous chlorophyll fluorescence of the sweet water green algae \u003cem\u003eChlorella sp.\u003c/em\u003e, employed as a model organism, using the time- and the spectrally-resolved microscopy methods.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of MPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMPs of different sizes were purchased commercially as a FluoSpheres\u0026reg; size kit #2 (F8888, Invitrogen by Thermo Fisher Scientific), carboxylate coupling surface, labelled with yellow-green fluorescence (Ex/Em 505/515). We have compared MPs of six sizes with nominal bead diameters of 0.02, 0.1, 0.2, 0.5, 1.0, 2.0 \u0026micro;m. MPs were added to cells in concentration 2 \u0026micro;L/mL (2% solids in the original solution) and cultivated between 7\u0026ndash;21 days before use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlgae and solutions\u003c/h3\u003e\n\u003cp\u003eWe have employed green algae \u003cem\u003eChlorella sp.\u003c/em\u003e from the University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences collection of green algae, previously isolated from the main drinking water supply. Algae were cultivated in the Hoagland cultivation medium containing: NaNO\u003csub\u003e3\u003c/sub\u003e; CaCl\u003csub\u003e2\u003c/sub\u003e. 2 H\u003csub\u003e2\u003c/sub\u003eO; MgSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. 3 H\u003csub\u003e2\u003c/sub\u003eO; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; NaCl; H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e; FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO; H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e; ZnSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO; MnCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO; (NH\u003csub\u003e4\u003c/sub\u003e)6Mo7O24.4H\u003csub\u003e2\u003c/sub\u003eO; CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO; Co(NO\u003csub\u003e3\u003c/sub\u003e)2. 6H\u003csub\u003e2\u003c/sub\u003eO; EDTA, KOH (all from Lachema, Czech Republic), as specified previously [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eInstrumentation\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConfocal imaging\u003c/h2\u003e \u003cp\u003eFluorescence was measured with Laser scanning confocal microscopy (LSCM) imaging with Axiovert 200 LSM 510 Meta (Carl Zeiss, Germany), equipped with objective C-Apochromat 40x, 1.2 NA, as described previously [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. Chlorophyll fluorescence was excited with the 450 nm laser line (Kvant, Slovakia) and detected using a spectral META detector. Maximal laser power density reached 1580 Wm\u003csup\u003e-2\u003c/sup\u003e for sample excitation with 450 nm laser line. Channel 1 was recorded with BP 500\u0026ndash;550 nm filter and served to monitor the fluorescently-labelled MPs. Channel 2, using BP 650\u0026ndash;710 nm filter, was employed to record the endogenous red chlorophyll fluorescence of the algae, peaking at 680 nm [Waller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e]. To better visualise the distribution of MPs only, zoomed image was recorded with BP 480\u0026ndash;520 nm. Spectrally-resolved images of the red algae fluorescence in the presence or in the absence of MPs were taken across 7 spectral channels covering the spectral region from 638 and 713 nm taken with 10.7 nm steps for the red chlorophyll fluorescence.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFluorescence Lifetime Imaging Microscopy (FLIM)\u003c/h3\u003e\n\u003cp\u003eFluorescence lifetime imaging microscopy (FLIM) was employed to gather images by time-correlated single photon counting (TCSPC) technique, using a 445 nm picosecond laser diode (BDS-SM-445-FBC, Becker\u0026amp;Hickl, Germany), as described previously [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. The laser beam was reflected to the sample through an epifluorescence path of the inverted microscope Axiovert 200 LSM 510 Meta (Carl Zeiss, Germany) with C-Apochromat 40x, 1.2 NA. The emitted fluorescence was separated from laser excitation using BP 700\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nm. Detection was done by PMC-100-20 photomultiplier (Becker\u0026amp;Hickl, Germany) with SPC-830 TCSPC board.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eConfocal images were analyzed by ZEN 2011 software (Zeiss, Germany), or by home-made procedures. Fluorescence intensity from confocal images at Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e was analysed using image segmentation method, where only fluorescence intensities recorded from the fluorescing algae (without surrounding background), were measured. Red fluorescence spectra at Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e were evaluated as a mean fluorescence of all fluorescing algae in the sample. FLIM images at Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e were processed using proprietary software package SPCImage 8.4 NG (Becker\u0026amp;Hickl, Germany), or a custom-made software. Results were visualized as a map and as a distribution of calculated fluorescence lifetimes for each recorded cell. Intensities are presented as the mean and the standard error of the mean. Decay curves were fitted with a one-exponential fitting model with plausible χ\u003csup\u003e2\u003c/sup\u003e using image segmentation and home-made lifetime analysis, as described previously [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. Images were generated by Origin 6.0 Professional. Statistical comparison was done using one-way Anova, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOur aim was to evaluate the interaction between MPs of different sizes and algae \u003cem\u003eChlorella sp.\u003c/em\u003e MPs ranged from micrometric size (2 \u0026micro;m) down to nanometers (20 nm). \u003cem\u003eChlorella sp.\u003c/em\u003e is a unicellular alga of size 10\u0026ndash;30 \u0026micro;m; MPs were therefore chosen in order to be smaller than the average cell size. We have recorded a short-term exposure (7\u0026ndash;21 days) of MPs to algae, in the aim to discern the early events.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the distribution of MPs and of their interaction with\u003c/b\u003e \u003cb\u003eChlorella sp\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe presence of MPs and their distribution was evaluated based on their fluorescence. The MPs fluorescence was mostly present in the spectral range 480\u0026ndash;520 nm (data not illustrated). For the MPs in the lower size range (20\u0026ndash;200 nm), a single peak with the maximum at 520 nm was observed; the MPs in the bigger size range (500\u0026ndash;2000 nm) presented larger intensity with maximum that remained at 520 nm, but with an additional fluorescence shoulder at 550 nm. At this spectral range, the MPs fluorescence therefore did not interact with the red chlorophyll fluorescence of the \u003cem\u003eChlorella sp.\u003c/em\u003e algae.\u003c/p\u003e \u003cp\u003eTo evaluate the interaction of MPs with \u003cem\u003eChlorella sp.\u003c/em\u003e algae, spectrally-resolved confocal microscopy following excitation at 450 nm at two emission windows was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, composite image at channel 4). In these studies, the emission window of 500\u0026ndash;550 nm of the channel 1 was employed to record the green fluorescence of MPs, while the emission window of 650\u0026ndash;710 nm of the channel 2 was designed to record the red chlorophyll fluorescence of the algae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consequently, the presence of the MPs was discerned based on their blue/green fluorescence, whereas the algae were identified by their red endogenous fluorescence. In these settings, in control conditions, in the absence of MPs, algae exhibited only red endogenous fluorescence of chloroplasts, no green fluorescence was recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the presence of both, algae and MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), we noted a differential interaction of micro vs. nanoplastics: nano and small MPs (20\u0026ndash;500 nm) encircled the algae in a \u0026ldquo;corona\u0026rdquo;-like structures, whereas larger MPs (1000\u0026ndash;2000 nm) rather created a \u0026ldquo;nuclei\u0026rdquo; for clustering the neighbouring algae. For better visualisation of the distribution of MPs, an image with BP 480\u0026ndash;520 nm was also recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, in the inset, visualised in blue for better contrast).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of MPs on the red chlorophyll fluorescence of\u003c/b\u003e \u003cb\u003eChlorella sp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo study the effect of the MPs on the \u003cem\u003eChlorella sp\u003c/em\u003e., we first compared the red chlorophyll fluorescence intensities recorded at the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C. We observed that the presence of MPs lead to a decrease in the algae chlorophyll fluorescence for the small-sized MPs (20\u0026ndash;500 nm), although for MPs of 200 nm, this decrease did not reach significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Larger MPs (1000\u0026ndash;2000 nm) have no significant effect on the algae chlorophyll fluorescence. This result indicates that the size of MPs affects the capacity of individual cells to cope with their presence and thus with photosynthesis.\u003c/p\u003e \u003cp\u003eNext, we evaluated the effect of the MPs on the red chlorophyll fluorescence spectra of the \u003cem\u003eChlorella sp\u003c/em\u003e. by recording the confocal images at separate fluorescence wavelengths between 638 nm and 713 nm using a 10.7 nm step (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The red fluorescence peaked at 680 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), as expected for the fluorescence of chlorophyll a [Govindjee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1967\u003c/span\u003e]. Gathered data corresponded to our previous recordings of the endogenous fluorescence in the algae \u003cem\u003eChlorella sp.\u003c/em\u003e [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. Under these conditions, we observed no significant effect of the MPs on the spectral shape of the red fluorescence, as illustrated after the spectral normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This result indicates that the MPs did not modify the photosystem II system, or stimulate the photosystem I and thus that the algae still have a good capacity to maintain the photosystem II system in the presence of MPs, regardless of their size.\u003c/p\u003e \u003cp\u003eFLIM images, recorded by TCSPC, served to evaluate the fluorescence lifetimes following excitation by 445 nm picoseconds laser with emission between 700\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, left, between 250\u0026ndash;300 ps). In this setting, the MPs exhibit no fluorescence and only fluorescence of chlorophylls is recorded. For that reason, in order to better visualise the presence of MPs, the confocal image of the same regions was also taken (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, right). A custom-made approach, allowing automatization of the procedure in varying experimental conditions, was used for analysis, as described in [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. We noted a significant decrease in the chlorophyll fluorescence lifetime for MPs of 20 nm size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This indicate alteration in the algae chlorophylls, most likely related to the close encircling (the \u0026ldquo;corona\u0026rdquo; like structures) of the algae cell by these small-sized nanoplastics. We noted no significant change in the measured chlorophyll fluorescence lifetime of algae for the plastics with particle size above 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003eInterestingly, FLIM recordings uncovered an unexpected capability to visualise the MPs of sizes larger than 500 nm (arrows at Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Indeed, in this FLIM setting, MPs of larger sizes can be visualised non-fluorescently and this observation may help to evaluate the presence of these MPs in the water environment in the presence of algae.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this contribution, we evaluated the interaction of MPs of different sizes with photosynthetic algae \u003cem\u003eChlorella sp\u003c/em\u003e. Endogenous fluorescence intensity and fluorescence lifetimes of chlorophylls were recorded. We demonstrated differential distribution of nano- vs. micro-sized PS plastics after up to 3 week exposure. Smaller-sized MPs (20\u0026ndash;500 nm) resulted in \u0026ldquo;corona-like\u0026rdquo; distribution of MPs around algae and decrease in the red chlorophyll fluorescence, indicating their effect on the algae photosynthesis. MPs sized above 1000 nm rather acted as nuclei for clustering the algae, without affecting algae photosynthesis.\u003c/p\u003e \u003cp\u003ePlastic particles are ubiquitous in the aquatic environment: they have been detected worldwide [Waller et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e]. MPs have adverse effects on several aquatic animals [Wright et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Della Torre et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ivar do Sul et al., 2014] and also threaten humans, primarily from the point of view of the potential danger of chronic exposure to their chemical toxicity [Wright et al., 2017]. Nevertheless, the interactions of living organisms with MPs are still poorly understood. We have tested the distribution of differently sized MPs of the PS origin, labelled with blue/green fluorescence, together with their effect on the endogenous red chlorophyll fluorescence of the algae. Fluorescence labelling is helpful for evaluation of the presence of MPs and allowed to discern their differential distributions.\u003c/p\u003e \u003cp\u003eTo study the effect of the MPs on the algae fluorescence, we have employed recording of the endogenous fluorescence of the algae in the red spectal region. We previously demonstrated the use of the endogenous fluorescence for biosensing of the presence of nanoparticles in live algae [Marcek Chorvatova et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e]. The red fluorescence with maximum at 680 nm was assigned to the chlorophyll fluorescence in our previous studies [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. Chlorophyll fluorescence is a very useful endogenous probe that is sensitive to variations in the functional state of the algae, as well as of plants [Govindjee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Govindjee, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e]. This fluorescence is heterogeneous, but its major emission band (680\u0026ndash;685 nm) and its vibrational satellite (720\u0026ndash;735) nm originate mostly from pigments of the photosystem II antenna complexes, namely chlorophylls [Govindjee, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e]. This fluorescence can therefore also be employed for testing the responsiveness of algae to changing water quality, Endogenous fluorescence recorded in living \u003cem\u003eChlorella sp.\u003c/em\u003e algae cells under our experimental conditions is in agreement with the assumption that most of its chlorophyll a fluorescence is derived from photosystem II, with the emission bands at 680\u0026ndash;685 nm [Govindjee, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e]. In the presence of the MPs, the spectral shape of the recorded fluorescence remained unchanged, indicating no modification of the photosystem II / photosystem I.\u003c/p\u003e \u003cp\u003eWe have observed a decrease in the chlorophyll fluorescence for the smaller-sized MPs. On the other hand, we did not observe any effect on the chlorophyll fluorescence with the larger sized MPs. Other authors demonstrated that larger-sized MPs caused adverse effects by blocking the light transport and affecting the photosynthesis, while smaller ones destroyed the cell wall by adsorbing onto the algae surface [Bhattacharya et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e]. The reason behind the lack of the effect of the large MPs (above 1000 nm) in our study can be related to their lower numbers, when compared to MPs below 500 nm (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The concentration of the MPs was chosen in relation to the environmental pollution concentrations (between 100 and 1,000 \u003cem\u003e\u0026micro;\u003c/em\u003eg/L) [Jin et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e]. With 2% of solids in the sample, taking into consideration PS density of 1.250 g/cm\u003csup\u003e3\u003c/sup\u003e [omnexus web], 2 \u003cem\u003e\u0026micro;\u003c/em\u003el/ml corresponds to MPs concentration of about 500 \u003cem\u003e\u0026micro;\u003c/em\u003eg/L. However, this means much lower number of MPs of higher sizes when compared to those of lower sizes, as illustrated at Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Many algae cells thus remained without direct interaction with the MPs above 1000 nm, at a difference to MPs smaller than 500 nm. In the future, the effect of higher concentrations of the bigger particles, together with longer exposures (in terms of months) needs to be evaluated.\u003c/p\u003e \u003cp\u003eFLIM recording is an advanced imaging technique that allows to record changes of molecular parameters non-invasively and directly in living cell systems. Fluorescence lifetimes depend on several factors, including pH, temperature and/or oxygenation, but are independent on the fluorescence intensity and/or photobleaching [reviewed in Berezin MY and Achilefu S, 2010 and Chorvatova A and Chorvat D. Jr 2014]. This parameter is therefore particularly useful for study of the cell\u0026rsquo;s responses to changes their environment. We previously demonstrated that under our experimental conditions, the dominant fluorescence lifetime of the endogenous chlorophyll fluorescence reached, in control conditions, between 0.4\u0026ndash;0.45 ns [Marcek Chorvatova et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e] and this was also observed in this study. Observed decrease in the fluorescence lifetime in the presence of the MPs sized 20 nm indicate that encircling of the cells in these conditions impact on the red endogenous fluorescence at the molecular levels. Recorded change in the fluorescence lifetimes points to a modification in the molecular structure of the chlorophylls, while the decrease in the chlorophyll fluorescence without change in the fluorescence lifetimes for the MPs of sizes between 100\u0026ndash;500 nm rather points to the decrease in the number of the active molecules in these conditions. Further work is needed to understand the interaction between MPs and living organisms in details. Nevertheless, performed experimentation acknowledges the employment of the time-resolved chlorophyll fluorescence in biosensing.\u003c/p\u003e \u003cp\u003eGathered measurements confirmed differential interaction of nano vs. micro-sized MPs with algae. Nano-sized particles are expected to have more a chemical effect, while micro-sized plastics have more a physical effect [Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e] and the differences in the observed distributions in our study confirm these differences\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our data revealed a differential distribution of the nano vs. microplastics with the \u003cem\u003eChlorella sp.\u003c/em\u003e algae. The nanoplastics of the 20 nm size grouped around the individual algae cell in a corona-like formation, affecting the algae chlorophylls, as evidenced by shortening of the chlorophyll fluorescence lifetimes. MPs of smaller sizes (less or equal than 500 nm) surrounded the algae, while lowering the chlorophyll fluorescence. Larger sized MPs (1000\u0026ndash;2000 nm) rather acted as a \u0026ldquo;nucleus\u0026rdquo; for a group of algae, but without affecting the cell chlorophyll fluorescence. Such a differential effect can help to comprehend the role that nano vs. microplastics play in the aquatic environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available at request in an Omero repository at http://microscopy.mlc.sk/omero.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone beyond declared funding and affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project ENVIROBIOM\u0026nbsp;N\u003csup\u003eo\u003c/sup\u003e 09I03-03-V04-00689 and by\u0026nbsp;the LASERLAB-EUROPE (grant agreement no. 871124, European Union\u0026rsquo;s Horizon 2020 research and innovation programme).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.M.C. contributed to conceptualization, investigation, writing- original draft preparation, writing- reviewing and editing software.\u003c/p\u003e\n\u003cp\u003eA. M. contributed to data curation and data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD. C. contributed to\u0026nbsp;methodology, validation and visualization of data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank M. Valica from FPV UCM for helping with the cultivation of \u003cem\u003eChlorella sp.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with an unregulated invertebrate species. No patients participated in the presented research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave their consent for the ms to be published as submitted.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarnes DK, Galgani F, Thompson RC, Barlaz M. Accumulation and fragmentation of plastic debris in global environments. Philos Trans R Soc Lond B Biol Sci. 2009;364:1985\u0026ndash;98. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1098/rstb.2008.0205\u003c/span\u003e\u003cspan address=\"10.1098/rstb.2008.0205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. 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Environ Sci Technol. 2017;51:6634\u0026ndash;57.\u003c/span\u003e \u003cspan\u003e https://\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eomnexus.specialchem.com/polymer-property/density#Density-measurement\u003c/span\u003e\u003cspan address=\"http://omnexus.specialchem.com/polymer-property/density#Density-measurement\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microplastics-and-nanoplastics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mina","sideBox":"Learn more about [Microplastics and Nanoplastics](http://microplastics.springeropen.com)","snPcode":"43591","submissionUrl":"https://submission.nature.com/new-submission/43591/3","title":"Microplastics and Nanoplastics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"nanoplastics, microplastics, chlorophyll fluorescence, Chlorella sp. algae, confocal microscopy, FLIM","lastPublishedDoi":"10.21203/rs.3.rs-5289229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5289229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnvironmental pollution by micro and nanoplastics (MPs) is becoming an imminent danger for the environment in the 21st century. However, the effect of the MPs of different sizes is still poorly understood. In this contribution, we compare the effect of fluorescently labelled polystyrene (PS) MPs of sizes between 20 nm and 2000 nm. Spectrally-resolved confocal microscopy and fluorescence lifetime imaging was employed to study the interaction of PS MPs with algae \u003cem\u003eChlorella sp\u003c/em\u003e. We observed differential effect between the smaller and the bigger-sized beads. MPs sized 20\u0026ndash;500 nm created \u0026ldquo;corona-like\u0026rdquo; structures around algae and induced lowering of the chlorophyll fluorescence, indicating an effect on the cell photosynthesis. In addition, the 20 nm MPs induced shortening of the chlorophyll fluorescence lifetimes, pointing to the effect on the chlorophyll molecular environment. However, MPs of bigger sizes, 1000\u0026ndash;2000 nm, rather acted as a \u0026ldquo;nucleus\u0026rdquo; for clustering of a number of neighbouring algae without affecting the chlorophyll fluorescence. Understanding the interaction of living organisms with MPs of different sizes is crucial to assess the impact of this environmental pollution on live organisms in their natural environment.\u003c/p\u003e","manuscriptTitle":"Differential effect of nano vs. micro-sized plastics on live Chlorella sp. algae in water environment.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 10:32:11","doi":"10.21203/rs.3.rs-5289229/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-28T01:00:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-28T00:48:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207669831586954870369507922314034216346","date":"2024-11-19T19:30:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-19T02:50:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298258848136288466641605845720285298365","date":"2024-10-30T10:29:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214544942558830619631028017089401762376","date":"2024-10-29T20:40:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-26T12:04:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-21T08:22:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-21T08:20:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microplastics and Nanoplastics","date":"2024-10-18T11:42:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microplastics-and-nanoplastics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mina","sideBox":"Learn more about [Microplastics and Nanoplastics](http://microplastics.springeropen.com)","snPcode":"43591","submissionUrl":"https://submission.nature.com/new-submission/43591/3","title":"Microplastics and Nanoplastics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8f81dfc-857f-4912-83e3-df999102bd2d","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-17T16:02:18+00:00","versionOfRecord":{"articleIdentity":"rs-5289229","link":"https://doi.org/10.1186/s43591-025-00111-2","journal":{"identity":"microplastics-and-nanoplastics","isVorOnly":false,"title":"Microplastics and Nanoplastics"},"publishedOn":"2025-02-10 15:57:01","publishedOnDateReadable":"February 10th, 2025"},"versionCreatedAt":"2024-11-11 10:32:11","video":"","vorDoi":"10.1186/s43591-025-00111-2","vorDoiUrl":"https://doi.org/10.1186/s43591-025-00111-2","workflowStages":[]},"version":"v1","identity":"rs-5289229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5289229","identity":"rs-5289229","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}