Fluorescent Microplastics Research Articles

Trophic transfer of microplastics from producer (Lemna minuta) to primary consumer (Cataclysta lemnata) in a freshwater food chain

Contamination by microplastics (0.1 μm–5 mm plastic fragments) is currently one of the major threats to the conservation of aquatic and terrestrial ecosystems worldwide. Growing awareness on this issue has led to an increase in studies on the effects of microplastics on freshwater organisms, although there are still few investigations on possible transfer of this contaminant along water trophic chains from producers to primary consumers. In this study, aquatic herbivorous larvae of the moth Cataclysta lemnata were fed on microplastic-free (control) and contaminated (MPs treatment) Lemna minuta fronds. For treatments, Lemna fronds were grown in mineral water enriched with fluorescent microparticles of poly(styrene-co-methyl methacrylate) (MPs, 100 mg/L) and then fed to the larvae as a food source. Microplastics effects on larvae were tested at 0, 7, 14 and 21 days of exposure, corresponding to sensitive phases of the insect life cycle. Contaminant impact was assessed based on some parameters such as viability, larva body size/weight, feeding alterations and regularity of the insect life cycle. Using scanning electron and fluorescence microscopy, the presence of microplastics in the larvae was verified. The finding of fluorescent microplastics in both the intestinal lumen and excrement samples showed that larvae ingested contaminated Lemna fronds. In addition, larvae fed contaminated fronds were strongly affected by the presence of microplastic contaminant over time, showing high mortality (90 %) and total inability to complete the life cycle after 21 days by failing to reach the winged adult phase. In control tests, survival rates were higher than in treatments, and 50 % of the larvae managed to pupate and emerge as moths, reaching the adult phase. The results show that there was a trophic transfer of microplastics from producer to primary consumer along a freshwater food chain, generating negative effects on the life cycle of this aquatic herbivore.

Assessing phytotoxicity of microplastics on aquatic plants using fluorescent microplastics.

Although studies on microplastics are increasing every year, still very little is known about their toxicity. Especially for plant species, even studies of uptake of microplastics are only few, not to mention phytotoxicity of microplastics. Therefore, we conducted a pilot study on the phytotoxicity of 1-μm-sized fluorescent microplastics (FMPs) on the free-floating aquatic plants Spirodela polyrhiza and Salvinia natans and the emergent aquatic plant Phragmites australis using 0.1% and 0.01% FMP treatment. Furthermore, uptake of FMPs by plants was verified by detecting fluorescence of FMPs by laser. A free-floating aquatic plant S. polyrhiza and emergent aquatic plant P. australis showed significantly decreased harvested biomass after 3weeks indicating phytotoxicity of FMPs, but S. natans did not show any differences of harvested biomass or chlorophyll contents among treatments. Detection of fluorescence from plant leaves provided evidence of active FMPs uptake by plants. The emission spectra of plant leaves in 0.1% FMP treatment showed similar peaks to those of free fluorescent microplastics, providing a firm evidence of FMPs uptake by plants. This study is one of the pioneering studies to explore fluorescent microplastic uptake and toxicity in aquatic plants and therefore provides a baseline for further studies.

Correlative Light, Electron Microscopy and Raman Spectroscopy Workflow To Detect and Observe Microplastic Interactions with Whole Jellyfish.

Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 μm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).

Time-course distribution of fluorescent microplastics in target tissues of mussels and polychaetes

The majority of the plastic produced in the last century is accumulated in the environment, leading to an exacerbated contamination of marine environments due to transport from land to the ocean. In the ocean, mechanical abrasion, oxidation, and photodegradation degrade large plastics into microplastics (MPs) - 0.1 μm to 5 mm (EFSA, 2016) which are transported through water currents reaching the water surface, water column, and sediments. Further, they can be accumulated by aquatic and benthic species, entering the trophic chain and becoming a potential threat to humans. In the present research, we aimed to decipher the accumulation and distribution time-courses between different organs or target tissues of organisms inhabiting coastal areas such as mussels Mytilus galloprovincialis and polychaetes Hediste diversicolor. Both were exposed in microcosm experiments to fluorescent polystyrene MPs (1 μm) which were spiked at two doses (103 and 105 particles/mL) for 1, 4, 24, and 72 h. Mussels and polychaetes were digested with 10% KOH and filtered to quantify the number of MPs incorporated. Different anatomical parts of the body were selected and processed for cryosectioning and posterior microscopic localisation of MPs. Both species accumulate MPs spiked in water column, mainly after exposure to the highest dose. In mussels, particles were found in distinct parts of the digestive tract (stomach, digestive diverticula, ducts) and gills. Even if the majority of MPs were localised in the lumen of the digestive tract, in some cases, were inside the digestive epithelium. The identification of MPs and their internalization in the digestive system was studied using Raman spectroscopy. A decreasing trend with time regarding MPs number in the digestive tract (stomach) of mussels was observed while the opposite was recorded for polychaetes and sediments. The combination of microscopical observations of frozen sections and Raman, appeared to be accurate methodologies to address MPs abundances and to reveal their localisation in different organs. This work has enabled to understand the distribution and fate of MPs in different environmental compartments and it could contribute to gain knowledge about their impact after ingestion by coastal organisms.

A Novel Approach to Bio-Friendly Microplastic Extraction with Ascidians

Microplastic pollution in water is now recognized as a devastating problem by many organizations, such as the National Oceanic and Atmospheric Administration, with recent studies estimating that the average American consumes around 52,000 of these plastic, toxic particles a year. A successful solution for the extraction of microplastics from oceans must be feasible to be implemented on a large scale and bio-friendly to not further disrupt the environment. To this end, the efficacy of using filter feeders (Ascidians) as biofilters to reduce microplastic pollution was explored. The efficacy of this filtration method was evaluated by adding ascidians to saltwater tanks contaminated with microplastics (experimental group) and comparing the water’s plastic concentration over time against a control. Water samples were then systematically tested with a fluorescence-activating microscope and fluorescent scanner. Fluorescent microplastics were used which allowed for the collection of both quantitative and qualitative data. The samples from the experimental group demonstrated a 24.7% (29.64mg) reduction in microplastics within the first day and a 94.7% (113.64mg) decrease by day 4. The control group showed negligible deviation in microplastic concentration. It is concluded that the Ascidians filtered microplastics from water through their natural feeding and respiratory process. We extrapolate that a 1m x 1m x 1m cage of Ascidians would filter approximately 300g of microplastics every day. This research demonstrates that microplastic filtration with invertebrate filter feeders is an effective and feasible option for extracting microplastics from polluted water.

Size Effects of Microplastics on Embryos and Observation of Toxicity Kinetics in Larvae of Grass Carp (Ctenopharyngodon idella).

Microplastics have caused great concern in recent years. However, few studies have compared the toxicity of different sizes of microplastics in fishes, especially commercial fishes, which are more related to human health. In the present study, we revealed the effects of varying sizes of microplastics on grass carp embryos and larvae using scanning electron microscopy (SEM) and fluorescence imaging. Embryos were exposed to 80 nm and 8 μm microplastics at concentrations of 5, 15, and 45 mg/L. Toxicity kinetics of various sizes of fluorescent microplastics were analyzed through microscopic observation in the larvae. Results found that nanoplastics could not penetrate the embryo’s chorionic membrane, instead they conglutinated or aggregated on the chorion. Our results are the first to explore the defense mechanisms of commercial fish embryos against microplastics. Larvae were prone to ingesting their own excrement, resulting in microplastic flocculants winding around their mouth. For the first time, it was found that excreted microplastics could be reconsumed by fish and reaccumulated in the oral cavity. Microplastics of a certain size (1 μm) could be accumulated in the nasal cavity. We speculate that the presence of a special groove structure in the nasal cavity of grass carp larvae may manage to seize the microplastics with a particular size. As far as we know, this is the first report of microplastics being found in the nasal passages of fish. Fluorescence images clearly recorded the toxicity kinetics of microplastics in herbivorous fish.

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish.

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

Preparation of biological samples for microplastic identification by Nile Red

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 μm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94–100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97–100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

Bioaccumulation and reproductive effects of fluorescent microplastics in medaka fish

This study was performed to elucidate the uptake and bioaccumulation of polystyrene microplastics (PS-MPs) in Japanese medaka (freshwater fish) and Java medaka (marine fish), and to assess its impacts on the survival, reproduction, and gene expression of Japanese medaka. Both species were exposed to 2-μm fluorescent PS-MPs (107 beads/L) for 3 weeks. The bioaccumulation factor of PS-MPs for Java medaka was calculated at about 4 × 102, higher than that for Japanese medaka (about 1 × 102). The exposure had no significant effects on the survival and reproduction of Japanese medaka. The mRNA sequencing analysis showed that the expression of a few genes involved in the cell adhesion, xenobiotic metabolic process, brain development, and other functions in medaka intestines significantly changed after exposure. These results suggest that virgin PS-MPs can potentially accumulate in medaka intestines, but has limited toxicity to Japanese medaka at the concentration up to 107 beads/L.

Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages

Plastics are persistent synthetic polymers that accumulate as waste in the marine environment. Microplastics (MPs, <5 mm) can be found either as microbeads in body care and some industrial products or as plastic debris through degradation. Plastic microbeads (1–5 μm, fluorescent, Cospheric) were used to characterise the MP ingestion and determine their potential harmful effects on both the swimming behaviour and development of oyster D-larvae (Crassostrea gigas). For 24 h, embryos were first exposed to MPs at a temperature of 24 °C. In addition, 3 day-old D-larvae were exposed to the same temperature for 1, 3 and 5 h. Three concentrations of MPs were used: 0.1, 1 and 10 mg MP. L−1. After a 24-h period of embryonic exposure, we noted that MP agglomerates were stuck to the D-larvae coat and locomotor eyelashes. We also observed a significant increase in severe malformations and developmental arrests for larvae exposed to MPs ranging from 1 mg MP. L−1. In terms of swimming behaviour, the maximum speed recorded was lower for larvae exposed at 0.1 and 1 mg MP. L−1. After an acute exposure to MPs, particles were found in the digestive tract of 3 dpf (days post fertilisation) D-larvae. After 1-h exposure, the concentrations tested (0.1, 1 and 10 mg MP. L−1) resulted in respectively 38%, 86% and 98%. The larvae swimming behaviour was recorded and analysed. Unlike the results observed at the embryo-larval stage, 3-dpf larvae showed significant impacts with no dose-response effect.

Major factors influencing the quantification of Nile Red stained microplastics and improved automatic quantification (MP-VAT 2.0)

Automated count of Nile Red fluorescent microplastics allows fast and reliable quantification. However, factors involving staining, digital camera conditions and settings introduce variability to the results. The objective of this paper is to identify and propose solutions to these factors and improve on the previous MP-VAT script. While removal of digital sensor defects had little influence on results and staining can be reduced to 5 min, Nile Red concentrations cannot be reduced <0.01 mg mL−1, the 470 nm LED lantern emission must be >1600 lx, and photographic conditions should be maintained as stable as possible ideally improving the filter membrane area and using the recommended settings of 2 s, ISO100, F5.6. It was also found that Nile Red can be removed from microplastics using acetone or hydrogen peroxide with iron. More importantly, both particles and fluorescent are lost with time and thus quantification should be conducted within a week. Finally, MP-VAT 2.0 was developed to remove unselected areas and to identify only red particles, excluding white reflections from quantification. This updated version of MP-VAT produced improved recovery rates of 98.2 ± 6.9 for spiked samples and 95.9 ± 10.3 on actual environmental samples, presenting a cheap and reliable complementary method for microplastic identification.

Low‐cost microplastic visualization in feeding experiments using an ultraviolet light‐emitting flashlight

AbstractMicroplastics are pollutants threatening the health of marine, freshwater and terrestrial organisms. To analyze whether an organism is able to ingest microplastics, the organism is usually fed with expensive fluorescent microbeads and placed under a fluorescence microscope for microplastic detection. However, such equipment cannot be afforded by many laboratories. Therefore, we developed a low‐cost method to study the ingestion and egestion of low‐cost, fluorescent microplastic fragments and fibers by aquatic invertebrates. During our feeding experiments, we exposed the freshwater snail Radix balthica to artificial biofilms containing 15% microplastic fragments and fibers, respectively. We then used an ultraviolet (UV) flashlight to irradiate the microplastics. Hence, we could directly observe the microplastics in the food, during ingestion and egestion as well as in the snail feces. Our method, thereby, allowed us to analyze the snails' behavior (during ingestion and egestion), the microplastics' distribution in the snail feces and the microplastics' shape after the snails' digestion. Furthermore, we observed that the snails ceased to egest microplastics after 76 hr following microplastic ingestion. However, chemical digestion of the snails 6 days after microplastic exposure revealed that microplastics were still present in R. balthica, emphasizing the microplastics' persistence in the animals' bodies. This is the first study combining the usage of low‐cost fluorescent microplastics with an inexpensive UV flashlight for microplastic detection during microplastic ingestion and egestion by living organisms. Our new method is not only cost‐effective, but also fast and can be performed by a wide range of researchers without special previous training.

A new approach for routine quantification of microplastics using Nile Red and automated software (MP-VAT).

Microplastics are widespread contaminants in the environment. However, most identification protocols rely on long and subjective visual counting, which could be improved using staining dyes. Thus, the objective of this work is to identify the best staining dye protocol and create an objective and quick automated counting software for microplastics. Tests were conducted to identify the most appropriate of eight staining dye solutions and of six wavelengths for virgin and weathered synthetic polymers, textile fibers, natural organic matter and filters. Nile Red produced the best results (without interfering in infrared spectra) rendering microplastics fluorescent at 254 nm, but with limited number of fluorescent polymers, and at 470 nm (with orange filter), with fluorescence of plastics as well as natural organic matter (requiring a digestion step). Next, a script was developed in ImageJ for the automatic quantification and characterization in shape (fiber, fragment, particle) and size of fluorescent microplastics, the Microplastics Visual Analysis Tool (MP-VAT). MP-VAT was evaluated, producing recovery rates in the range of 89.0-111.1% in spiked filters under 470 nm. Furthermore, this package is accompanied by a script that sets a scale from a known filter diameter, MP-SCALE, and a script that allows user threshold setting, MP-ACT.

Ecotoxicity and genotoxicity of polystyrene microplastics on higher plant Vicia faba

Nano- and microplastics have been widely spread in environmental matrices, especially in marine and terrestrial systems. In this study, higher plant Vicia faba root tips were exposed to 5 μm and 100 nm with 10, 50 and 100 mg/L polystyrene fluorescent microplastics (PS-MPs) for 48 h. Root length, weight, oxidative stress and genotoxicity of V. faba were assessed to investigate toxic effects of PS-MPs. The results showed that the biomass and catalase (CAT) enzymes activity of V. faba roots decreased under 5 μm PS-MPs whereas superoxide dismutase (SOD) and peroxidase (POD) enzymes activity significantly increased. Under the 100 nm PS-MPs exposure a significant decrease of growth was observed only at the highest concentration (100 mg/L). However, micronucleus (MN) test and antioxidative enzymes activities showed that 100 nm PS-MPs induce higher genotoxic and oxidative damage to V. faba than 5 μm PS-MPs. Furthermore, the laser confocal scanning microscopy (LCSM) demonstrated that 100 nm PS-MPs can accumulate in V. faba root and most probably block cell connections or cell wall pores for transport of nutrients. These findings provide a new insight into the toxic effects of microplastics on V. faba, and further apply to the ecological risk assessment of microplastics on higher plants.

Microplastics accumulate on pores in seed capsule and delay germination and root growth of the terrestrial vascular plant Lepidium sativum

The impacts of nano- and microplastics (<100 nm and <5 mm, respectively) on terrestrial systems is to the present largely unexplored. Plastic particles are likely to accumulate in these systems primarily by the application of sewage sludge. The aim of the current study was to investigate the effects of three sizes of plastic particles (50, 500, and 4800 nm) on a terrestrial plant (cress; Lepidium sativum), using a standardized 72 h bioassay. Cress seeds were exposed to five different concentrations of plastics, ranging from 103 to 107 particles mL−1. Germination rate was significantly reduced after 8 h of exposure for all three sizes of plastics, with increased adverse effect with increasing plastic sizes. Seeds exposed to 4800 nm microplastics showed a germination rate decline from 78% in control to 17% in the highest exposure. No difference in germination rate occurred after 24 h of exposure, regardless of the size of the plastic used. Significant differences in root growth were observed after 24 h, but not after 48 or 72 h of exposure. Impacts on germination are likely due to physical blockage of the pores in the seed capsule by microplastics as shown by confocal microscopy of fluorescent microplastics. In later stages, the microplastics particularly accumulated on the root hairs. This is the first detailed study on the effect of nano- and microplastics on a vascular, terrestrial plant, and our results indicate short-term and transient adverse effects.

Influence of Six Digestion Methods on the Determination of Polystyrene Microplastics in Organisms Using the Fluorescence Intensity

Microplastic pollution has become a global environmental problem and is a cause of great concern. To evaluate the biological effects of microplastics, microplastics in organisms need to be accurately quantified. The quantification of microplastics in organisms using the fluorescence intensity is common; the digestion of biological samples is an important pretreatment method. However, the microplastics may be destroyed by digestion, which affects the fluorescence intensity of the microplastics and results in large deviations between measured and true values. In this study, six commonly used digestive agents were studied:KOH, NaOH, H2O2, HNO3, HNO3:HCl, and HNO3:HClO4. The effect of different digestion methods on the fluorescence intensity and surface morphology of microplastics was studied and the most suitable protocol was selected. The results show that, among the six different digestion methods, KOH digestion (100 g·L-1, 60℃) has the least influence on the fluorescence intensity of the microplastics and does not affect their surface morphology. The other five digestion methods lead to different degrees of reduction of the fluorescence intensity of microplastics and damage the microplastics' surface (aggregation, bubbles, scratches, and depressions). In addition, the KOH digestion method was used to extract microplastics from biological samples. The recovery rate was ≥ 96.3%±0.5%, indicating that the KOH digestion method is suitable for fluorescent microplastics in biological samples.

Fate of microplastics in the marine isopod Idotea emarginata.

Plastic pollution is an emerging global threat for marine wildlife. Many species of birds, reptiles, and fishes are directly impaired by plastics as they can get entangled in ropes and drown or they can ingest plastic fragments which, in turn, may clog their stomachs and guts. Microplastics of less than 1 mm can be ingested by small invertebrates, but their fate in the digestive organs and their effects on the animals are yet not well understood. We embedded fluorescent microplastics in artificial agarose-based food and offered the food to marine isopods, Idotea emarginata. The isopods did not distinguish between food with and food without microplastics. Upon ingestion, the microplastics were present in the stomach and in the gut but not in the tubules of the midgut gland which is the principal organ of enzyme-secretion and nutrient resorption. The feces contained the same concentration of microplastics as the food which indicates that no accumulation of microplastics happens during the gut passage. Long-term bioassays of 6 weeks showed no distinct effects of continuous microplastic consumption on mortality, growth, and intermolt duration. I. emarginata are able to prevent intrusion of particles even smaller than 1 μm into the midgut gland which is facilitated by the complex structure of the stomach including a fine filter system. It separates the midgut gland tubules from the stomach and allows only the passage of fluids and chyme. Our results indicate that microplastics, as administered in the experiments, do not clog the digestive organs of isopods and do not have adverse effects on their life history parameters.